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    Monitoring winter crop areas during wartime: remote sensing support for Ukraine’s agricultural statistics

    AbstractTimely and transparent agricultural statistics are essential for safeguarding global food security. When the war in Ukraine disrupted agricultural reporting – particularly in Russian-held territories – complementary and/or alternative approaches were needed to produce reliable statistics. We developed a fully remote sensing–based framework to estimate areas of two major crops, wheat and rapeseed, from 2022 to 2025. Using Planet and Sentinel-1/2 imagery, we applied clustering techniques to generate in-season crop type maps that supported stratified random sampling in sample-based area estimation, using remotely interpreted reference data. Our estimates closely matched official statistics in Government-controlled areas (RMSE = 0.138 and 0.32 million hectares (Mha) for wheat and rapeseed, respectively), while filling critical data gaps in Russian-controlled regions. Between 2022 and 2025, wheat area declined from 5.14 ± 0.52 to 4.84 ± 0.25 Mha in Government-controlled areas and from 2.06 ± 0.16 to 1.55 ± 0.09 Mha in Russian-held regions. Rapeseed expanded from 1.16 ± 0.17 to 1.47 ± 0.23 Mha (2022–2025) in Government-controlled territories but collapsed in Russian-held areas, from 0.17 ± 0.01 to 0.05 ± 0.01 Mha. Our findings underscore the critical role of remote sensing in providing timely, transparent, and independent agricultural statistics to support informed food security and market-stabilizing decision-making.

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    IntroductionRussia’s full-scale invasion of Ukraine in February 2022 disrupted grain markets and agricultural statistics alike. The war triggered sharp commodity price fluctuations1 and logistical bottlenecks, while Ukraine’s agricultural exports, though partially sustained through the Black Sea Grain Deal and later via European “Solidarity Lanes” (European Council, 2023; European Commission, 2025), continued under highly uncertain conditions. As one of the world’s major breadbaskets and a key wheat supplier to the Middle East and North Africa, Ukraine plays a crucial role in global food security (European Council, 2025). Amid these shocks, reliable information on Ukraine’s crop production became increasingly critical for maintaining market transparency and global food security2.Reports such as the United States Department of Agriculture World Agricultural Supply and Demand Estimates (USDA WASDE) and Joint Research Centre Monitoring Agricultural ResourceS (JRC MARS) bulletins publish production estimates to increase market transparency and actively inform trade decisions2,3,4. Before the war, large-scale commercial farms, which produced about 85% of Ukraine’s wheat, were required to report planted and harvested areas, as well as production, to the State Statistics Service of Ukraine (SSSU). The remaining 15% was estimated from sample surveys of small producers (SSSU, 2024). However, from March 3rd, 2022 to August 18th, 2025 Law №2115-IX suspended mandatory reporting for these agricultural statistics due to martial law (Government of Ukraine, 2022, Government of Ukraine, 2025). While many farmers continued to provide data voluntarily, surveys of smallholder producers were halted for security reasons, creating national-level gaps in official statistics for Government-held areas. Data flow from Russian-controlled territories ceased abruptly from the onset of the war to the present. In 2023, 2024, and 2025, USDA stopped reporting on Russian-controlled territories except for Crimea, where agro-statistics are sourced from Rosstat, the Russian statistical agency (USDA, 2022; 2023; 2024; 2025). The statistical data blackout in Russian-held territories required alternative, remotely actionable solutions for compensating the loss of reporting-based agricultural production estimates.Several studies used remote sensing data to estimate crop production losses in Ukraine. Dai et al. 5 mapped grid-level crop abandonment rates, then proportionally adjusted pre-war crop production numbers based on abandoned area rates. He et al. 6 and Chen et al. 7 first assessed changes between pre- and during the war vegetation index dynamics and then applied change ratios to estimate lost agricultural productivity over abandoned and non-abandoned croplands in Ukraine. Jia et al. 8 used remote sensing data to map winter cereals and rapeseed and to estimate corresponding yields. They subsequently estimated production for season 2022 and assessed losses by comparing to pre-war levels. While estimating these losses provided valuable insights, official agencies had an even more critical need for timely information on ongoing agricultural production to contextualize these losses and support food security assessments, policy planning, and recovery efforts.Since the beginning of the war, only Qadir et al. 9, Qadir et al. 10 and Qadir et al. 11 developed an operational, statistically sound, remote sensing-based only framework for generating sunflower planted area estimates in war-torn Ukraine. In this paper, we address the complementary need for an alternative source of reliable winter wheat and rapeseed planted area statistics, given Ukraine’s global importance in the production and export of these crops. Our objectives are twofold: (i) to support and strengthen Governmental statistics, which are inferred from incomplete datasets as compared to pre-war, and to (ii) track planted areas in support of production estimates in Russian-held territories, from where data flow ceased entirely.Quantifying the uncertainty of area estimates is extremely important to official statistics. For instance, the USDA NASS June Agricultural survey aims for a coefficient of variation lower than 2% for maize, soybean, and winter wheat area estimates12. Regardless, most remote sensing-based area estimates in literature do not quantify uncertainty and often use the pixel-counting estimator for area estimation: the multiplication of pixel area by the number of pixels in a map class5,6,8,13,14. Such an estimator is biased because of omission and commission errors in the classification maps and mixed pixels at class boundaries15,16,17,18 and is discouraged for estimating crop area. Instead, maps can be used as a stratification tool in the sampling design to further employ estimators that are unbiased15,16,17,18. Studies by Song et al. 19, Li et al. 20 and King et al. 21 established the feasibility of planted area estimation using a two-stage sampling approach for maize and soybean area estimation in the USA, Argentina, and China. Ideally, once drawn, each sample unit should be visited on the ground to collect the “true” crop type label. However, when ground visits may jeopardize lives22,23, remote sensing imagery interpretation is a safe and cost-efficient alternative for reference data collection so long as it is carried out by experts with a deep and comprehensive understanding of the agricultural landscape. Olsen et al. 24, Kerner et al. 25, Li et al. 26 and Skakun et al. 27 substituted field visits with remote sensing imagery interpretation to collect reference data. They estimated active cropland areas and crop loss based on satellite-based classification maps and stratified random sampling in conflict-affected Sudan, Tigray, Syria, and Ukraine regions.But it is worth emphasizing that, unlike ground surveys, which provide a gold-standard dataset, remote sensing imagery interpretation can suffer from interpreter variability, potentially affecting area estimates and their precision28. Annotation quality ultimately depends on the annotator’s domain knowledge, which, when insufficient, can lead to severely flawed labeling and estimates. For instance, Chen et al. 7 overestimated Ukraine’s winter wheat, rapeseed, sunflower and maize planted areas in 2020 by respectively 35%, 209%, 14% and 72% relative to official statistics (before the war): they communicated “unbiased estimates” (± standard errors) of 8.7 ± 0.4, 3.4 ± 0.4, 7.3 ± 0.5 and 8.9 ± 0.5 million hectares (Mha) for wheat, rapeseed, sunflower and maize, while the State Statistics Service of Ukraine reported 6.5, 1.1, 6.4 and 5.2 Mha respectively for these crops, in 2020 (SSSU, 2024). In every paper relying on annotated data for validation, clear guidelines on how reference data is interpreted from remote sensing imagery should be provided.In this paper, we leveraged the specific phenological and temporal traits of winter crops29 and rapeseeds30,31 to operationally generate wall-to-wall, unsupervised, and in-season crop type maps32, based on Planet satellite data, for each agricultural season from 2022 to 2025. Maps were then used for stratification in a stratified random sampling design for winter crop planted area estimation, separately in Government-controlled and Russian-held territories. After annotating sample units, we estimated areas and uncertainties using a stratified estimator, which is unbiased33. Planted area estimates were compared against SSSU agricultural statistics in Government-controlled territories, reinforcing confidence in numbers for Russian-controlled areas. In the absence of feasible ground data collection, we established reference data through systematic remote sensing imagery annotation. Given past issues with annotation reliability7 and often lacking explanations on photointerpretation with remote sensing imagery13,24,34,35,36,37, we also present a set of guidelines for visually annotating winter cereals, rapeseed, spring and summer crops, and non-cultivated cropland classes in Ukraine to support consistency and accuracy in future assessments. During a time of war, this work provided the only in-season planted areas derived from an unbiased estimator, for winter wheat and rapeseed across all of Ukraine, one of the world’s largest exporters of both commodities (USDA, 2022). This work is part of a collaboration between NASA Harvest (NASA Harvest), the State Statistics Service of Ukraine (SSSU), the Ministry of Agrarian Policy and Food of Ukraine (MAPFU), the Food and Agriculture Organization (FAO), and the European Development and Reconstruction Bank (EBRD). It aimed at operationally generating crop areas, yields, and production estimates, based on remote sensing data, in war-affected Ukraine (FAO Investment Centre).ResultsWinter crop planted area estimatesSince the onset of the full-scale war, we seasonally estimated planted areas and associated standard errors for winter wheat, other winter cereals, and rapeseed, by military control status in Ukraine. We used military control lines as of July 11th, 2022, July 31st, 2023, May 22nd, 2024, and June 17th, 2025 in final area estimates delivered to the SSSU.In Government-controlled territories (Fig. 1), winter wheat planted area decreased by 9% from 2022 to 2023, from 5.14 ± 0.52 Mha to 4.68 ± 0.25 Mha (Here and throughout the paper, uncertainties are represented as one standard error). Wheat planted areas slightly increased in 2024 compared to 2023, with 4.73 ± 0.28 Mha planted, and remained nearly stable in 2025, with 4.84 ± 0.25 Mha planted. In 2022, we recomputed planted area estimates based on the September 26th, 2022, military control line, after significant portions of Sumy, Kharkiv, and Mykolaiv oblasts were reclaimed by Ukrainian forces. Updated estimates showed planted areas of 5.39 ± 0.56 Mha, 0.9 ± 0.09 Mha, and 1.18 ± 0.17 Mha for winter wheat, other winter cereals, and rapeseed, respectively. During the same period, rapeseed planted area increased from 1.16 ± 0.17 Mha in 2022 to 1.96 ± 0.21 Mha in 2023. Following this increase, rapeseed planting returned to a level comparable to 2022, with 1.29 ± 0.15 Mha planted in 2024. In 2025, the rapeseed area increased again to 1.47 ± 0.23 Mha.Fig. 1: Sample-based area estimates and associated standard errors for winter crops in Government-controlled territories.2022 area estimates are reported based on the July 11th military control line.Full size imageIn Russian-controlled territories as of the July 2022 military control line, winter wheat planted area decreased by 10% from 2022 to 2023, dropping from 2.06 ± 0.16 to 1.86 ± 0.09 million hectares (Mha) (Fig. 2). In our September 2022 update, following the reclamation of land by Ukrainian forces, we estimated that 1.80 ± 0.16 Mha were planted to winter wheat, 0.3 ± 0.03 Mha to other winter cereals, and 0.16 ± 0.01 Mha to rapeseed. In 2024, significant disruption in planting led to a one-third decline in winter wheat area compared to 2023, with only 1.39 ± 0.08 Mha planted. Over the same period, rapeseed area fell from 0.17 ± 0.01 Mha in 2022 to 0.14 ± 0.03 Mha in 2023. Planting was severely disrupted in 2024, with rapeseed area declining fourfold to just 0.03 ± 0.01 Mha. In 2025, winter wheat and rapeseed planted areas saw modest recoveries to 1.59 ± 0.1 Mha and to 0.05 ± 0.01 Mha.Fig. 2: Sample-based area estimates and associated standard errors for winter crops in Russian-controlled territories.2022 area estimates are reported based on the July 11th military control line.Full size imageWinter crop maps and accuracy estimatesTrends observed in planted area estimates are also reflected in the maps (Fig. 3). The overall distribution of winter crops remained stable over the three years, except for the absence of cultivation along the front. Notably, a larger area of rapeseed was planted in 2023 across Government-controlled territories, but was negligible in Russian-held areas.Fig. 3: Winter cereal and rapeseed maps for seasons 2022 to 2025 in Ukraine.A 2022 winter cereals and rapeseed map July 11th, 2022 military control line. B 2023 winter cereals and rapeseed maps and July 31st, 2023 military control line. C 2024 winter cereals and rapeseed map and May 22nd, 2024 military control line. D 2025 winter cereals and rapeseed map and June 17th, 2025 military control line. Frontlines: Institute for the Study of War and AEI’s Critical Threats Project. Maps were created based on Planet and Sentinels data in Qgis.Full size imageDetailed yearly confusion matrices and accuracy estimates (area-based using unbiased estimators) can be found in Tables S1 to S4. Across seasons and control zones, winter cereals (including winter wheat) consistently showed higher producer’s accuracy (PA, average = 87.6%) than user’s accuracy (UA, average = 75.3%). UA corresponds to the proportion of elements predicted to class i that rightfully belong to i. PA corresponds to the proportion of elements that truly belong to class i that were mapped to i. Most commission errors (1-UA) arose from confusion with summer crops in 2022–2023 or non-winter crops in 2024–2025, while omission errors (1-PA) reflected limitations of the ESA WorldCereals cropland mask38,39, which excluded some true winter crops as evidenced during reference data collection.In contrast, rapeseed classification was less stable and more sensitive to seasonal and sensor differences. Averaged across all years and control zones, UA was 82% and PA was 72%, with UA typically exceeding PA except in Russian-controlled areas in 2022 and 2025. Omission errors mainly involved confusion with non-crop, summer crop, or winter cereal classes, again linked to the cropland mask. From 2022–2024, reliance on Planet two-week composites introduced temporal inconsistencies that obscured flowering signals. In 2025, use of Sentinel-1 VH polarization composites produced asymmetric outcomes: in Government-controlled territories, UA = 100% and PA = 55 ± 9%, while in Russian-held regions, PA = 100% and UA = 30 ± 7%. Visual inspection confirmed artifacts, especially in Crimea, indicating that while winter cereal mapping remained robust and consistent, rapeseed accuracy was more variable and sensor dependent.DiscussionWhen the Russian full-scale invasion of Ukraine began in February 2022, wheat prices spiked on international markets, triggered by the uncertainty around Ukraine’s production and export capacity to come. However, despite warnings of a “Coming food catastrophe” (The Economist, 2022), “Half of Harvests […] Wiped Out by War” (Bloomberg, 2022), “harvests could be halved […]” (The Guardian, 2022) relayed in media, our results demonstrated that total Ukraine’s wheat harvested area did not drop drastically in 2022 (Becker-Reshef et al., 2022), nor did planted areas in upcoming years. In this situation, timely tracking of crop production, starting with winter crop planted areas, helped keep the market transparent and ultimately participated in lifting initial export licensing on wheat by July 2022 (FAO, 2022). Our results showed that, when aggregated at Ukraine-scale, wheat areas approximately decreased by 3 to 15% (0.19 to 0.92 Mha) compared to the USDA 2017 to 2021 average (USDA PSD-Online). For rapeseed, a cash industrial crop, areas increased by 15 to 48% or between 0.19 and 0.97 Mha more planted, compared to the USDA 5-year pre-war average.Between 2022 and 2025, NASA Harvest supported the SSSU by providing in-season estimates of winter wheat, rapeseed, and other winter cereals planted areas within Government-controlled territories. NASA Harvest’s remote-sensing-based estimates strengthened numbers reported by the SSSU across conflict years. Figure 4 compares NASA Harvest’s planted area estimates and the SSSU final reported areas for winter wheat, rapeseed, and other winter cereals. SSSU statistics reported here are final for the season 2022 to 2024 but may still be revised for season 2025 (report as of September 2025).Fig. 4: SSSU vs. NASA Harvest winter crop areas (Mha) comparison for Government-controlled territories.a Winter wheat; b rapeseed; c other winter crops. Root Mean Squared Error (RMSE) and bias are indicated for each crop. Y-axes have different scales. RMSE is computed as (sqrt{frac{1}{n}{sum }_{i=1}^{n}{({x}_{i}-{y}_{i})}^{2}}) where n is the number of observations, xi and yi are predicted and reference values. RMSE quantifies the average magnitude of the differences between predicted and reference values. Bias is computed as (frac{{sum }_{i=1}^{n}{x}_{i}-{y}_{i}}{n}) and quantifies systematic under- or over-estimation of predictions relative to reference values.Full size imageFor winter wheat, the target commodity in this paper, SSSU’s reported areas, based on voluntary reporting, consistently fell within one standard error of NASA Harvest estimates across 2022 to 2025 seasons (Fig. 4a). The maximum difference between SSSU and NASA Harvest was 0.21 Mha (<5%) in 2023. We found the best alignment in 2024, with a difference of less than one percent, equal to 0.02 Mha. The RMSE of 0.138 Mha and a bias of −0.002 Mha indicate that NASA Harvest estimates closely align with SSSU data, with no evidence of systematic over- or underestimation.As for winter rapeseed, SSSU’s reported areas for rapeseed were within the bounds of one standard error from the NASA Harvest estimates, in seasons 2022 and 2024. The 2023 NASA Harvest planted area estimate was significantly higher than SSSU’s reported area, at 1.96 ± 0.21 Mha compared to SSSU’s 1.39 Mha (Fig. 4b). Results from other data providers converge on estimating a higher rapeseed area than SSSU: USDA reported 1.54 Mha of winter rapeseed in Government-controlled territories (Table 1), and JRC reported a planted area of 1.7 Mha for full Ukraine in their June 2023 MARS bulletin40. Similarly, in 2025, SSSU reports 1.18 Mha planted, to rapeseed while NASA Harvest, USDA (USDA PSD-Online) and JRC41 respectively estimated 1.47 ± 0.23 Mha, 1.27 Mha and 1.311 Mha. Due to those inconsistencies, the RMSE for rapeseed was 0.321 Mha, and the bias was 0.23 Mha, indicating slight misalignment and overestimation of planted areas by NASA Harvest, as compared to SSSU. Some limitations might come from the area estimation process. Small strata – like, in our case, rapeseed with a 3% stratum weight – may be affected by higher standard errors and increased risk of divergence in area estimation. As recommended by Olofsson et al. 33, we systematically oversampled rapeseed to control these phenomena.Table 1 Comparison of NASA Harvest (NH) estimates of winter wheat and rapeseed planted areas for Government-controlled Ukraine (NH Gov. Cont.) and full Ukraine (NH Full) against SSSU and USDA areasFull size tableFor other winter cereals, NASA Harvest estimates aligned well with SSSU’s in 2022, and exceeded SSSUs by less than 0.15 Mha between 2023 and 2025 (Fig. 4c). At the time of writing this paper, SSSU statistics that were made available for season 2025, only covered wheat, rapeseed and barley. The RMSE and bias are comparatively low, respectively 0.1 Mha and −0.055 Mha, indicating slight underestimation of other winter crop areas. The overall agreement between the two data sources, especially for winter wheat, reinforces the validity of our methodology and enables trust in area estimates for the Russian-controlled territories, where ground surveys and statistics are not available.We further cross-compared our winter wheat and rapeseed planted area estimates with USDA’s harvested area reports from 2022 to 2025 (Table 1). USDA combines spring and winter planted areas into one single number, respectively, for wheat and rapeseed. To ensure comparability, we derived winter-to-spring area ratios from SSSU yearly statistics (SSSU, 2024) and applied these to USDA’s reported crop areas. USDA reported harvested areas for all of Ukraine in September 2022 (USDA, 2022). In 2023, 2024, and 2025, USDA discontinued their reporting on Russian-held territories: numbers include only Government-controlled territories plus Crimea, with Crimea’s data sourced from Rosstat, the Russian Statistical Agency (USDA, 2023; 2024; 2025).In 2022, USDA’s harvested area estimates for winter wheat in full Ukraine were 1.82 Mha below NASA Harvest’s planted area estimates, aligning more closely with SSSU and NASA Harvest numbers for Government-controlled territories. We note that after 2022, USDA’s estimates were slightly higher than those from SSSU, but within one standard error of NASA Harvest’s Government-controlled estimates. For rapeseed, USDA and NASA Harvest estimates were closely aligned across seasons, reflecting that major rapeseed production areas are outside Russian-controlled territories. After 2022, only a few data providers, including JRC42 and NASA Harvest, continued monitoring Russian-held territories, demonstrating the importance of Remote Sensing analysis to maintain critical market information and transparency in times of war.Estimating crop planted area using in-season maps for stratification within the sampling design typically requires a large number of sample units, representing significant investments in time and funding43. To optimize ground data collection over extended regions, two-stage area frame and regression estimators are often employed, which involve visiting multiple spatially close points rather than randomly scattered ones19,20,21. However, in our case, ground data collection was prevented by the war in Ukraine, particularly in Russian-controlled territories. We therefore relied on remote sensing imagery interpretation as an alternative, safe, and cost-effective way to collect reference data. Freed from constraints on the spatial distribution of validation points, we implemented a stratified random sampling approach, as outlined in Olofsson et al. 33, to estimate planted areas in Government and Russian-controlled territories. Despite the availability of methods for generating in-season field boundaries in Ukraine44, we preferred using map pixels as spatial assessment units. Using polygons as assessment units would have unnecessarily complexified the area estimation task45 as field boundary delineations are imperfect44, leading to within-polygon heterogeneity and possible omissions of croplands in the dataset. Thus, in our stratified random sampling design, map classes were the strata and map pixels were the sample units. In each stratum sample units were drawn as a simple random sample. Because certain parts of the non-cropland stratum from the map may have been misclassified and actually been cropped, we sampled this stratum in addition to the cultivated strata. We reduced the annotator’s bias28 using clearly defined annotation guidelines, applied on multiple satellite image sources (two-week Planet composites, daily Planet imagery, and Sentinel-2) and vegetation index time-series. Additionally, two to three annotators labeled each sample unit, followed by a review of discrepant labels until a consensus was reached.We acknowledge several limitations in using remote sensing for reference data collection. First, the approach is restricted to crop types or groups that can be reliably identified through visual inspection of satellite imagery time-series. Clear phenological traits, such as rapeseed’s yellow flowering30,31, sunflower’s heliotropic behavior9,10,11, or distinct sowing windows for winter and summer crops (USDA crop calendars), are required. While supervised models can separate summer crops using ground data, certain crop types like soybean and maize remain visually indistinguishable, preventing area estimates without field validation. Second, satellite-based annotation is also time-intensive. Based on our experience, one trained annotator can review ~400 sample units per full workday. Thus, annotating 1600 units with two people requires 2 × 4 workdays per estimation cycle for Ukraine. Despite the effort, this method remains more cost-efficient than field visits. Lastly, reliable annotation depends heavily on domain expertise. Even minor disagreements on the labels should be documented and discussed collectively to improve consistency and understanding. To support training of future annotators, we openly share (i) annotated sample units from 2022–2025, including locations, strata, and class labels, and (ii) a Sentinel-2–based Google Earth Engine tool used for annotation in our workflow (see Section Data Availability).This work emphasizes the pivotal role that satellite-based estimates play in informing agricultural commodity markets and policy. We provide an important and missing piece of information to official statistics by keeping track of the planted area in Russian-held territories. It is also the cornerstone of a continued partnership with the SSSU, MAPFU, FAO, and EBRD, aimed at complementing traditional survey-based statistics with remote sensing-based production estimates in Government-controlled territories (FAO Investment Centre). This paper details the winter crop planted area estimation component of the initiative, which, together with yield estimates derived from the ARYA model46,47, enabled calculation of winter wheat and rapeseed production across Ukraine. We conclude by citing Mr. Taras Vysotskyi, First Deputy Minister of Agrarian Policy and Food of Ukraine:“The timeliness, objectivity, and transparency of the information remain essential factors in shaping a balanced and effective agrarian policy. We acknowledge and appreciate the vital role of NASA Harvest’s solutions and calculations in assessing the real state of agricultural production development in Ukraine, ensuring regional food security, and determining the export potential to support global food security.”MethodsWorkflow overviewFigure 5 outlines the workflow for sample-based crop planted area estimation, further detailed in Sections Satellite data to Remote Sensing imagery interpretation. First, remote sensing imagery from Planet, Sentinel-1, and Sentinel-2 is used to produce an in-season unsupervised map of winter crops (Section Domain knowledge for multi-year crop mapping). This map serves as a stratifier to draw two separate stratified random samples, one for each occupation status. Both samples are annotated through remote sensing imagery interpretation, following rigorous sample unit annotation guidelines. Finally, areas and map accuracies are estimated using the approach described by Olofsson et al. 33, yielding in-season planted area estimates for Government-controlled and Russian-held territories (Section Sample-based crop area estimation and accuracy assessment).Fig. 5Sample based crop planted area estimation workflow.Full size imageSatellite dataFor seasons 2022 to 2024, we used daily, 3-meter spatial resolution, four bands (blue (490 nm), green (565 nm), red (665 nm) and near infra-red (865 nm)) Planet data, composited into two-week periods from February 24th, 2020 onwards, until May 27th, 2024 (Planet, Planet Technical Specifications). Analysis-ready data were transferred from Planet compute environments to Google Cloud Storage and subsequently ingested into Google Earth Engine48 for further processing. For each Planet two-week composite, we computed 14 vegetation indices described in Table S5.For season 2025, we used Sentinel-2 imagery from the 1st of November 2024 to the 31st of May 2025. We masked out invalid pixels using the Scene Classification Layer (SCL). We then computed temporal features named on “S2_[…]” in Table S5.Across seasons, to cope with cloud cover, we used Sentinel-1 synthetic aperture radar imagery ground range detected data, freely available in GEE. We created median VV, VH, and VV/VH backscatter composites for two-week time intervals, matching the Planet data compositing periods.Ancillary dataWe selected the ESA-World Cereals 2021 cropland product for this study. Classification accuracies over Europe are high, with a 96.6% user’s accuracy and a 92.0% producer’s accuracy for their active cropland layer38,39. To account for the climatic gradient across the country, we adapted the USDA-FAS agro-ecological zoning (AEZ) (USDA AEZs) of Ukraine to align with oblast boundaries. The AEZs are highlighted in green in Figure S2.To estimate unbiased planted area per military control status, we used boundaries provided by the Institute for the Study of War and AEI’s Critical Threats Project (ISW and AEI) (Red lines in Figure S1).Domain knowledge for multi-year winter crop mappingDrawing on crop calendar information (USDA crop calendars), and our extensive previous experience in crop monitoring in Ukraine27,29,46,49,50,51, we developed an in-season, deductive, phenology-informed mapping approach that relies on different crop phenological characteristics:In Ukraine, winter crops are typically sown from September until the end of October, followed by a green-up period in November. While some winter crops exhibit clear signals, detecting autumn green-up in remote sensing imagery is not always straightforward. Factors such as management practices52, soil conditions53, cultivar54, and weather conditions55 can influence or delay crop establishment. Additionally, green-up detection using optical sensors can be obstructed by cloud cover56 and snow57. After undergoing vernalization58, winter crops resume vegetative growth in early spring. The peak vegetative stage is typically reached around May, coinciding with approximately 1000 °C of accumulated growing degree days since January 1st49, with crops generally ready for harvest by July. During the flowering phase, rapeseed can be differentiated from winter cereals due to its bright yellow flowers, which are oriented skyward, producing a strong yellow reflectance signal in optical satellite imagery30,31. Summer crops green up starting from mid-May, peak in July–August and get harvest ready from mid-September onward (USDA crop calendars).From 2022 to 2024, we leverage these crop-specific spectro-temporal signals as inputs into a K-means clusterer for mapping winter cereals, rapeseed, summer crops, and non-cultivated cropland along the season. For each mapped crop type and, to account for the climatic gradient in Ukraine, per AEZ, we train and infer a separate unsupervised classifier. We first compute 14 vegetation indices and three SAR features from Planet and Sentinel-1 composites over relevant in-season mapping periods (Table S5). For model training, we randomly generate several thousand samples within cropland and extract the corresponding feature values stacked into a single input vector for each sample. Next, we train a K-means classifier on sampled vegetation index values and SAR features and infer the model at the AEZ level. Finally, we assign each cluster to its corresponding crop type based on Planet two-week imagery composites, vegetation index time series, and precise assignment guidelines presented in the section Remote Sensing imagery interpretation, Figs. 6–11. The classification process starts from the ESA World Cereals cropland layer38,39, within which winter crops are first identified and subsequently subdivided into winter cereals and rapeseed, by end of May. From the remaining cropland area not classified as winter crops, summer crops are then mapped. Any residual cropland that is not attributed to either winter or summer crops is labeled as non-cultivated for the season. Resulting maps are then used as stratifiers to guide our sampling frames and estimate unbiased areas.In 2025, the mapping steps remained consistent compared to previous seasons. Expanding on work by Skakun et al.29 we substituted K-means by a Gaussian Mixture Model trained on four temporal features extracted from Sentinel-2 Normalized Difference Vegetation Index time series and Sentinel-1 VH polarization time series, for mapping winter cereals and rapeseed.Sample-based crop area estimation and accuracy assessmentEach year, we use our in-season crop type map for stratification to generate a stratified random sample for unbiased area estimation across three to five strata. While this paper focuses on winter crop mapping and area estimation, we also map summer crops and classify residual, non-planted, cropland as non-cultivated. We independently assess planted areas in the Government- and Russian-controlled territories.Table 2 presents the sampling designs along with stratum weights (Wi) and number of sample units (ni) for each military control status. The stratum weight is the biased area estimate, derived from the map through pixel counting. Eq. (13) in Olofsson et al.33 can be used for estimating the number of sample units required when targeting overall accuracy as the estimation objective. Sample units should then be allocated proportionally according to stratum weight. However, when sample weights are small, proportional allocation may result in very low sample counts, unless the total sample number is very large. In such cases, Olofsson et al.33 recommend slightly over-sampling small classes. While suboptimal sample allocation may be reflected in standard errors, it will not compromise the unbiasedness of the estimates.Table 2 Sampling designsFull size tableIn the 2022 season, we used the military control line from the 11th of July to separate Government and Russian-controlled territories (Figure S1A) and estimated areas for four strata: winter cereals, rapeseed, summer crops, and non-crops. Non-cultivated fields were integrated into the non-crop stratum due to their low sample weight. In 2023, cropland abandonment became distinctly visible along the frontline, which we set to the 31st of July (Figure S1B). As a result, we estimated unbiased areas for the non-cultivated cropland stratum, regardless of its low sample weights. In 2024 and 2025, we split the area estimation tasks for winter and summer crops. We finalized our planted area estimates for winter cereals, rapeseed, and non-winter crop classes based on the military control status from the 22nd of May 2024 (Figure S1C) and 17th of June 2025 (Figure S1D).Planet 5*5-meter spatial resolution pixels were used as the spatial assessment unit, also called “sample units” in seasons 2022 to 2024. In season 2025, Sentinel-2 10*10-meter pixels were used as sample units. Two to three experienced labelers annotated all sample units to classes corresponding to the strata. Our primary data sources for sample unit annotation are Planet two-week image and NDVI time-series, alongside daily Planet images. Additionally, we used openly available Sentinel-2 optical imagery composited to two-week intervals matching Planet, especially in 2025. Two-week composite time series are generally sufficient to distinguish between winter, spring, and summer crops, non-crop, and non-cultivated cropland classes. However, short-timed events, such as rapeseed flowering could easily be missed on two-week compositing intervals. We thus systematically verify for a rapeseed flowering signal, on daily Planet imagery, all sample units annotated to winter crop. Detailed visual annotation guidelines are provided in Section Remote Sensing imagery interpretation. Shapefiles of sample unit locations, stratum and annotated class values are openly available for seasons 2022 to 2025 on GitHub (see Section Data Availability). Sampled points and class values can be used for training future annotators or supervised machine learning models. Codes to replicate our Sentinel-2 based annotation software on GEE are openly available on GitHub (see Section Data Availability).Following mathematical formulae to calculate unbiased planted areas, users, producers and overall accuracies next to associated standard errors are cited from Olofsson et al. 33. As per Eq. 1, we computed a confusion matrix based on mapped area proportions rather than sample counts. The proportion of area of map class i and of reference class j in the confusion matrix is noted pij. The sample-based estimator (hat{p}) ({hat{p}}_{{ij}}) of pij. (Eq. 1), is used to compute overall, producer’s and user’s accuracy per class (Eqs. 2 – 4).$${hat{p}}_{{ij}}={W}_{i}frac{{n}_{{ij}}}{{n}_{i}}$$
    (1)
    where Wi is the proportion of area mapped as class i, nij is the number of sample units of map class i and of corresponding reference class j and ni are total number of sample units mapped to class i.Overall accuracy (OA) is computed as$${OA}={sum }_{n=1}^{q}{hat{p}}_{{ij}}$$
    (2)
    with q, the number of mapped classes. User’s accuracy of class i, noted Ui, is determined by$${U}_{i}=frac{{p}_{{ii}}}{{p}_{i}.}$$
    (3)
    where pii represents the proportion of area mapped to class i that belongs to reference class i, and pi., the total proportion of area mapped to class i. Producer’s accuracy is calculated as$${P}_{j}=frac{{p}_{{jj}}}{p{.}_{j}}$$
    (4)
    where pjj is the mapped proportion of area of class j that belongs to reference class j and p.j is total proportion of area of reference class j.Variance of overall accuracy (V (Ô)) (Eq. 5) is computed as$$Vleft(hat{O}right)={sum }_{i=1}^{q}{W}_{i}^{2}hat{{U}_{i}}left(1-hat{{U}_{i}}right)/left({n}_{i}-1right)$$
    (5)
    where q is the number of map classes, Wi is the proportion of area mapped to class i, Ûi is the class wise user’s accuracy and ni is the number of sample units in stratum i.Variance of user’s accuracy (V(Ûi)) is estimated according to Eq. 6:$$Vleft({hat{U}}_{i}right)={hat{U}}_{i}left(1-{hat{U}}_{i}right)/left({n}_{i}-1right)$$
    (6)
    and variance of producer’s accuracy of reference class j = k is estimated according to Eq. 7:$$Vleft({hat{P}}_{J}right)=frac{1}{{hat{N}}_{.j}^{2}}left[frac{{N}_{j.}^{2}{left(1-{hat{P}}_{j}right)}^{2}* {hat{U}}_{j}left(1-{hat{U}}_{j}right)}{{n}_{j.}-1}+{hat{P}}_{j}^{2}{sum }_{ine j}^{q}{N}_{i.}^{2}frac{{n}_{{ij}}}{{n}_{i.}}left(1-frac{{n}_{{ij}}}{{n}_{i.}}right)/left({n}_{i.}-1right)right]$$
    (7)
    Where ({hat{N}}_{.j}) is equal to ({sum }_{i=1}^{q}frac{{N}_{i.}}{{n}_{i}.}{n}_{{ij}}) the estimate of marginal total number of pixels of reference class j; Nj., the marginal total of map class j, nj. is the total number of sample units in class j and ni. is the marginal total number of pixels in mapped class i. Standard errors for variance estimates can be obtained simply by taking the square root of the variances.Finally, when a confusion matrix expressed in terms of area proportions is used, the proportion of area ({hat{p}}_{.k}) (Eq. 8) of mapped class k is expressed as:$${hat{p}}_{.k}={sum }_{i=1}^{q}{W}_{i}frac{{n}_{{ik}}}{{n}_{i}}$$
    (8)
    The standard error (Sleft({hat{P}}_{.k}right)) (Eq. 9) of the estimated proportion of mapped class k is:$$Sleft({hat{p}}_{.k}right)=sqrt{{sum }_{i}frac{{W}_{i}{hat{p}}_{{ik}}-{hat{p}}_{{ik}}^{2}}{{n}_{i.}-1}}$$
    (9)
    The associated area and standard error of area of class k, ({hat{A}}_{k}) and (Sleft({hat{A}}_{k}right)) are computed as Eq. 10 and Eq. 11:$${hat{A}}_{k}={hat{p}}_{.k}A$$
    (10)
    $$Sleft({hat{A}}_{k}right)=Sleft({hat{p}}_{.k}right)A$$
    (11)
    where A is the total pixel counted area of the map. A 95% confidence interval can be obtained by multiplying (Sleft({hat{A}}_{k}right)) by 1.96.We delivered final planted area estimates for winter wheat, other winter cereals, and rapeseed by mid-August 2022, mid-September 2023, and mid-June 2024 and mid-July 2025, respectively, to SSSU and FAO partners. The winter wheat planted area was derived as 85.7% of the total winter cereal areas. This proportion was computed from the report by the Ministry of Agricultural Policy and Food of Ukraine on winter crop planted areas for the 2022 season, released in December 2021 (SSSU, 2024). Standard error for winter wheat was adjusted in the same manner. From 2025 onward, we started using the previous season winter wheat to other winter cereals ratio, published in final SSSU statistics (State Statistics Service of Ukraine, 2024).Remote Sensing imagery interpretationThe assignation of a sample unit, in our case pixels, or a cluster to a class is driven by a deductive approach with varying complexity based on the number of strata and their definitions. In a five classes setup, such as during the 2023 growing season, the labeler must first address the question Is this pixel cropland? The recognition of non-cropland classes is usually straightforward and will mostly be grassland, water, forested and built-up areas.Once it is established that a sample unit belongs to cropland, the next question will be Does this sample unit belong to winter crops? Winter crops are typically planted in the autumn of the previous year and should exhibit a pre-winter green-up signal, appearing red in false color composites (as shown in the top row of Figs. 6, 7). Following an often cloudy and/or snowy winter period (illustrated in the second row of Figs. 6, 7), winter crops emerge from dormancy and resume growth. As demonstrated in the fourth row of Figs. 6, 7, a winter crop should be clearly photosynthetically active in April and reach peak Normalized Difference Vegetation Index (NDVI) values in May in Ukraine. We recommend using false color NIR-R-G composites for early season annotation as the NIR channel is more sensitive to the vegetation’s chlorophyll. If the observed sample unit meets the criteria for winter crops, the annotator should then carefully inspect the imagery in true color (R-G-B) mode, looking for the characteristic rapeseed flowering signal from mid-April to the end of May. Onset and peak flowering are visible in 2024-04-29 and 2024-05-13 composites in Fig. 6.Fig. 6: Rapeseed Planet image time-series.The black star represents the sample unit to be annotated, corresponding NDVI values and composite end dates are displayed in the top left of each image. Composites up to mid-April are visualized in false color mode (NIR-R-G), subsequent ones are shown in true colors (R-G-B) to detect rapeseed flowering visible in 2024-04-29 and 2024-05-13 composites.Full size imageFig. 7: Winter cereal Planet image time-series.The black star represents the sample unit to be annotated, corresponding NDVI values and composite end dates are displayed in the top left of each image. Composites up to mid-April are visualized in false color mode (NIR-R-G), subsequent ones are shown in true colors (R-G-B), in order to distinguish between rapeseed and winter cereals during the flowering period.Full size imageIf no flowering is observed in this period, the annotator can safely conclude the sample unit is a winter cereal. Additionally, very high photosynthetic activity in the pre-winter period, is a non-conclusive hint that a winter crop might be rapeseed. Usually sown first, rapeseed has more time to establish than other winter crops, holds more chlorophyll and has therefore often higher NDVI values in autumn (see top row in Figs. 6, 7).The last rows of Figs. 6, 7, cover the senescence to harvest phase of winter crops, usually starting in June. During this period, the annotator should expect a decrease in NDVI values as the crop dries, ultimately resulting in a clear harvest signal around end of July (visible in 2024-07-22 composite in Fig. 7, South from the sample unit).Spring cereals can often be mistaken for winter cereals, particularly by less experienced annotators who may overlook the early season signals. Spring cereals exhibit a growth pattern similar to that of winter crops, peaking in May-June and maturing for harvest in July. However, unlike winter cereals, spring cereals are not planted in autumn and thus experience a delayed green-up, often revealing bare soils until early April, followed by a rapid green-up, as illustrated in Fig. 8.Fig. 8: Spring crop Planet image time-series.The black star represents the sample unit to be annotated, corresponding NDVI values and composite end dates are displayed in the top left of each image. Composites up to mid-April are visualized in false color mode (NIR-R-G), subsequent ones are shown in true colors (R-G-B).Full size imageIf a crop is neither classified as non-crop, winter, nor spring crop, the labeler should then consider the question, Is this sample unit a summer crop or is it non-cultivated? In Ukraine, most summer crops display a bare soil signal until the end of May (rows 1 to 4 in Fig. 9), followed by rapid green-up in June and July (bottom row in Fig. 9). The annotator can further categorize non-cultivated cropland into two types: barren plots, which are often managed through tillage, typically do not exhibit significant vegetation signals throughout the growing season, as illustrated in Fig. 10.Fig. 9: Summer crop Planet image time-series.The black star represents the sample unit to be annotated, corresponding NDVI values and composite end dates are displayed in the top left of each image. Composites up to mid-April are visualized in false color mode (NIR-R-G), subsequent ones are shown in true colors (R-G-B).Full size imageFig. 10: Barren plot Planet image time-series.The black star represents the sample unit to be annotated, corresponding NDVI values and composite end dates are displayed in the top left of each image. Composites up to mid-April are visualized in false color mode (NIR-R-G), subsequent ones are shown in true colors (R-G-B).Full size imageSecondly, we define fallow as cropland covered in naturally grown vegetation. This vegetation greens up alongside winter crops but senesces in autumn with summer crops. When annotating grass-covered fallows, particularly those found along the frontline due to cropland abandonment, the annotator should look for “always green” plots, as shown in Fig. 11. Confusions can arise between fallow and cropped plots, especially during early June annotation. To mitigate this risk, we recommend “context-aware annotation,” as grassy fallows are often spatially co-located along the frontline, disrupting the typical winter crop/summer crop mosaic that characterizes Ukraine’s cropland. Fallow may be terminologically wrong, considering most weeded-in fields are, in reality, abandoned.Fig. 11: Fallow plot covered in grasses Planet image time-series.The black star represents the sample unit to be annotated, corresponding NDVI values and composite end dates are displayed in the top left of each image. Composites up to mid-April are visualized in false color mode (NIR-R-G), subsequent ones are shown in true colors (R-G-B).Full size imageWhile labeling, the annotator may encounter sample units located at the edges between two classes. In such cases, we advise conducting a peer review to reach a consensual decision on whether the sample unit primarily represents one class or the other. Additionally, we recommend utilizing very high-resolution imagery, such as Google Satellite background maps, to aid in this decision-making process.

    Data availability

    Planet two-week composites were shared with NASA Harvest through a Planet-NASA Harvest agreement. Planet daily data was accessed through the NASA CSDA program. Sentinel-1 Synthetic Aperture Radar Ground Range Detected data is openly available in Google Earth Engine [h]. Sentinel-2 Multi Spectral Instrument optical imagery is openly available in Google Earth Engine [here]. Oblast boundaries extracted from the FAO GAUL Level-1 dataset are openly available in Google Earth Engine [h]. The ESA World Cereals active cropland layer 2021 is openly available in Google Earth Engine [h]. Institute for the Study of War and AEI’s Critical Threats Project occupation status vector data is available upon request [h]. Shapefiles of sample unit locations, corresponding stratum and annotated class values are openly available for seasons 2022 to 2025 on GitHub [h]. Google Earth Engine java script API code to build a simple Sentinel-2 based annotation tool is openly available on GitHub [h].
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    Download referencesAcknowledgementsThe authors thank Oleg Prokopenko from the State Statistics Service of Ukraine for his valuable comments and insights on the work realized. We also acknowledge the Kyiv Polytechnique Institute (KPI), Kernel, and IMC for their collaboration and willingness to share ground data, although these datasets were not used in the present analysis. We also thank Planet and the Institute for the Study of War and AEI’s Critical Threats Project for providing satellite imagery and conflict boundaries for this research project. Planet two-week composites were shared with NASA Harvest through a Planet-NASA Harvest agreement. Planet daily data was accessed through the NASA CSDA program. KPI data collection was funded by the European Commission through the joint World Bank/EU project ‘Supporting Transparent Land Governance in Ukraine’ [grant numbers ENI/2017/387–093 and ENI/2020/418–654]. This project was supported by funding from NASA Harvest (NASA cooperative agreement: 80NSSC23M0032 P00002), USAID (grant number: 80NSSC18K1571) and FAO (grant number: 720BHA22IO00188).Author informationAuthors and AffiliationsICube, University of Strasbourg, Illkirch-Graffenstaden, Strasbourg, FranceJosef Wagner, Shabarinath S. Nair, Manav Gupta, Fangjie Li, Mae Chevassu, Jean Rehbinder, Françoise Nerry & Inbal Becker-ReshefDepartment of Geographical Sciences, University of Maryland, College Park, MD, USASergii Skakun, Sheila Baber, Oleksandra Oliinyk, Abhishek Kotcharlakota, Nataliia Kussul, Mary Mitkish & Inbal Becker-ReshefCollege of Information, University of Maryland, College Park, MD, USASergii SkakunSchool of Earth, Atmosphere and Environment, Monash University, Clayton, VIC, AustraliaYuval SadehKernel, Kyiv, UkraineDanylo PoliakovIMC, Kyiv, UkraineBohdan Vaskivskyi & Oleksii MisiuraNational Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”, Kyiv, UkraineNataliia KussulSpace Research Institute NASU-SSAU, Kyiv, UkraineNataliia KussulUnited Nations Food and Agriculture Organization, Rome, ItalyDmytro Prykhodko, Oleksandr Sikachyna & Andry RajaoberisonAuthorsJosef WagnerView author publicationsSearch author on:PubMed Google ScholarSergii SkakunView author publicationsSearch author on:PubMed Google ScholarShabarinath S. NairView author publicationsSearch author on:PubMed Google ScholarYuval SadehView author publicationsSearch author on:PubMed Google ScholarSheila BaberView author publicationsSearch author on:PubMed Google ScholarOleksandra OliinykView author publicationsSearch author on:PubMed Google ScholarAbhishek KotcharlakotaView author publicationsSearch author on:PubMed Google ScholarManav GuptaView author publicationsSearch author on:PubMed Google ScholarDanylo PoliakovView author publicationsSearch author on:PubMed Google ScholarBohdan VaskivskyiView author publicationsSearch author on:PubMed Google ScholarOleksii MisiuraView author publicationsSearch author on:PubMed Google ScholarNataliia KussulView author publicationsSearch author on:PubMed Google ScholarDmytro PrykhodkoView author publicationsSearch author on:PubMed Google ScholarOleksandr SikachynaView author publicationsSearch author on:PubMed Google ScholarAndry RajaoberisonView author publicationsSearch author on:PubMed Google ScholarFangjie LiView author publicationsSearch author on:PubMed Google ScholarMae ChevassuView author publicationsSearch author on:PubMed Google ScholarJean RehbinderView author publicationsSearch author on:PubMed Google ScholarFrançoise NerryView author publicationsSearch author on:PubMed Google ScholarMary MitkishView author publicationsSearch author on:PubMed Google ScholarInbal Becker-ReshefView author publicationsSearch author on:PubMed Google ScholarContributionsJosef Wagner: Conceptualization, Methodology, Software, Validation, Formal Analysis, Investigation, Writing – original draft, Visualization. Sergii Skakun: Methodology, Validation, Investigation, Writing – Review & Editing. Shabarinath S. Nair: Conceptualization, Methodology, Validation, Formal Analysis, Investigation, Writing – Review & Editing. Sheila Baber: Methodology, Validation, Writing – Review & Editing. Yuval Sadeh: Methodology, Validation, Writing – Review & Editing. Oleksandra Oliinyk: Data Curation, Investigation, Resources. Abhishek Kotcharlakota: Methodology, Validation. Manav Gupta: Validation, Data curation. Danilo Poliakov: Resources. Bohdan Vaskivskyi: Resources. Oleksii Misiura: Resources. Nataliaa Kussul: Resources, Writing – Review & Editing. Dmytro Prykhodko: Conceptualization, Writing – Review & Editing. Oleksandr Sikachyna: Conceptualization, Writing – Review & Editing. Andry Rajaoberison: Conceptualization, Writing – Review & Editing. Fangjie Li: Validation. Ma(bar{{rm{e}}}) Chevassu: Visualization. Jean Rehbinder: Supervision, Writing – Review & Editing. Françoise Nerry: Supervision. Mary Mitkish: Writing – Review & Editing. Inbal Becker-Reshef: Conceptualization, Methodology, Writing – Review & Editing, Supervision, Funding acquisition.Corresponding authorCorrespondence to
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    Housing modifications for heat adaptation, thermal comfort and malaria vector control in rural African settlements

    AbstractThe rapid increase in global temperatures coupled with persistent malaria transmission presents substantial health burdens in sub-Saharan Africa. Here this randomized pilot field study assessed the feasibility of sustainable housing modifications via passive cooling approaches and vector proofing. Forty houses were randomly allocated to four arms: cool-roof, cross-ventilation, mat-ceiling or control. Doors, windows and eaves of the intervention houses (not control) were screened for malaria mosquito vectors. Indoor temperature and humidity were monitored continuously to assess Heat Index (HI), predicted mean value and psychrometric charts. The HI in cool-roof houses was the lowest (daytime −3.3 °C, P < 0.001; nighttime −2.4 °C, P < 0.01). Mat-ceiling houses lowered daytime HI but increased nighttime HI compared to control. No differences in HI were observed for cross-ventilation houses. Screening reduced the number of female Anopheles funestus mosquitoes by 77% and the number of Culex mosquitoes by 58% compared to control houses. Eighty-five percent of the households expressed willingness to use their resources for housing intervention. Cool-roofs combined with vector proofing is an effective, practical and sustainable housing modification for heat adaptation and for reducing indoor mosquito numbers in rural African households.

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    MainClimate change and associated extreme weather events are adversely affecting the health of the human population globally1. Sub-Saharan Africa (SSA) is projected to experience some of the highest increases in temperatures2,3, which are expected to result in severe consequences due to the region’s limited adaptive capacity and preexisting health burdens4.Although outdoor exposures to extreme heat are widely studied, household indoor environments, where people spend most of their time, are often overlooked5. In rural SSA, houses are often constructed with materials and designs that are poorly suited to dissipate heat, such as metal roofing, limited shading and inadequate ventilation6. Furthermore, indoor heat gain is largely driven by raised outdoor temperatures and solar radiation7 as well as internal heat generated by building occupants, installed equipment and activities such as cooking. Elevated indoor temperatures not only lead to heat stress and reduced productivity but also increase reliance on active cooling systems such as electric fans, thereby increasing energy demands and the use of fossil fuels8.In recent decades, several validated indices have been developed to assess heat-related health risk; one of these indices is the HI, which combines temperature and humidity9,10. In addition, to mitigate heat stress resulting from environmental heat load, it is important to ensure that the ambient temperature in an indoor living space is maintained within the thermal comfort zone (TCZ), which is the range within which most of the occupants are predicted to feel thermally comfortable. The TCZ is based on temperature, humidity, radiant temperature and airflow, according to different complex models11.Passive cooling strategies are critical, particularly where active systems are unavailable and/or unaffordable8. A common architectural approach to indoor cooling is the inclusion of openings in a building´s exterior surface—for example, doors, windows and vents8. These openings facilitate indoor and outdoor heat transfer through ventilation—that is, both cross-ventilation and stack-ventilation. The size, number and orientation of these openings relative to the building layout substantially influence their cooling effectiveness. In practice, however, many households in SSA have openings that are often undersized, poorly oriented or absent due to cost constraints, cultural preferences or structural concerns, limiting their capacity to mitigate indoor heat accumulation.Although essential for ventilation, these openings also serve as entry points for mosquitoes, particularly Anopheles species that transmit malaria12,13, further risking the health of occupants. The world observed an increase of 11% in malaria burden in 2023, above the 2022 levels14. The SSA region bears the highest burden, accounting for 94% of malaria cases and 95% of estimated mortality globally14. Although the exact causes of this rise are multifactorial, climate change is a key driver, altering temperature, humidity and rainfall patterns in ways that expand mosquito habitats, enhance vector survival and increase malaria transmission risk15. Additionally, climate-induced temperature increases further reduce long-lasting insecticidal net (LLIN) use during hot nights16, compounding malaria risk.Malaria-transmitting mosquitoes are highly adapted to feeding indoors at night17,18. Field studies from western Kenya have shown that human−vector interactions that lead to malaria transmission still occur mostly indoors, throughout the night and morning, despite widespread use of LLINs19,20. Therefore, house designs that simultaneously prevent mosquito entry and maintain thermal comfort is a priority in SSA21.House modifications involving the screening of doors, windows and eaves with insect mesh were previously demonstrated to reduce malaria transmission22,23. Consequently, the World Health Organization has given an interim recommendation for the use of untreated insect screens as a supplementary control measure against malaria24. However, addition of screens to these openings could potentially reduce airflow and increase indoor temperatures, further jeopardizing the comfort of the occupants. An experimental study in The Gambia assessing how house design affects malaria mosquito density, temperature and humidity observed that screening of eaves of both thatched and metal-roofed houses increased indoor temperatures, whereas increasing ventilation in metal-roofed houses made them as cool as thatched houses with open eaves21.Critical considerations on house design and its impact on indoor thermal comfort and mosquito control are necessary in every building design. This pilot, mixed-methods study, conducted in Siaya County, western Kenya, tested the feasibility and acceptability of low-cost, sustainable house modifications aimed at improving passive cooling and reducing mosquito entry. The study also evaluated implementation costs, community engagement strategies and participant recruitment processes. Findings from this pilot study will inform a forthcoming larger cluster randomized trial (Wellcome Trust grant number 226750/Z/22/Z), involving approximately 300 households, which will assess the epidemiological and environmental impacts of scalable housing interventions on malaria transmission and indoor thermal comfort.ResultsHousing modificationsRural houses were typically rectangular in shape, with walls made of mud and sticks and roofs of corrugated iron sheets. Unmodified (control) houses had open eaves, unscreened or absent windows, unscreened doors, no ceilings and unpainted roofs (Fig. 1a). Mat-ceiling houses were fitted with horizontally installed papyrus reed mats across the roof space, above the eaves, as ceiling for cooling (Fig. 1b). Cool-roof houses received white reflective paint on the roofs to reduce heat gain (Fig. 1c). Cross-ventilation houses received new, screened windows to enhance indoor airflow (Fig. 1g,f). All intervention houses with cool-roof, mat-ceiling and cross-ventilation received screened doors (Fig. 1d) and eave screening (Fig. 1e) for mosquito control, whereas control houses did not receive any vector screening. For cool-roof and mat-ceiling houses, no additional windows were added; existing ones were screened for mosquito control.Fig. 1: Pictures of different housing modification approaches.Unmodified house (a), mat-ceiling (b), external view of house with cool-roof (c), screened door (d), screened eave (e), internal view of screened window (f) and external view of screened window for cross-ventilation (g).Source dataFull size imageBaseline assessment of house characteristicsTable 1 presents a description of the study structures before modification. All structures had corrugated iron roofs, with most (97.5%, 39) having mud walls. Most of the houses (67.5%, 27) did not have windows, 10.0% (4) had one window, 15.0% (6) had two windows and 7.5% (3) had three windows. Most of the houses (97.5%, 39) had open eaves on all sides, and 2.5% (1) had open eaves on two sides of the wall, typical for gable roof design. Most of the houses had double rooms (62.5%, 25), followed by single rooms (27.5%, 11) and three rooms (10.0%, 4). The houses were evenly distributed across the study arms, with no significant differences in wall type, roof type, number of windows, presence of eaves and number of rooms.Table 1 Baseline characteristics of houses across the study armsFull size tableIndoor thermal environmentWe measured the indoor HI, temperature and relative humidity a few days before the start of the modification (Extended Data Table 1). These data indicate a high degree of homogeneity of the indoor environmental conditions, regardless of whether data from a whole day, during daytime hours or at night were considered.Figure 2 shows daily mean values (±95% confidence interval), temperature and relative humidity for the different study arms, after the housing modifications. Data shown correspond to the representative post-intervention period—May—1 month after implementation.Fig. 2: Daily mean values (± 95% confidence interval) of HI, temperature and relative humidity after the intervention in May.The left panel displays daytime data (7:00−19:00) for heat index (a), temperature (c) and relative humidity (e), and the right panel shows nighttime data (19:00–7:00) for heat index (b), temperature (d) and relative humidity (f), across all study arms.Source dataFull size imageHI and temperature were higher during the day (7:00−19:00; Fig. 2a,c) than at night (Fig. 2b,d). By contrast, humidity was observed to be higher at night than during the day (Fig. 2e,f). The HI was highest during the day in houses with cross-ventilation (mCV) and in control houses (mCL), whereas, at night, mat-ceiling houses (mMC) consistently recorded the highest HI values across all groups (Fig. 2a,b). Cool-roof houses (mCR) showed the lowest HI and the lowest indoor temperature but also the highest relative humidity, regardless of time of day (Supplementary Tables 1 and 2). Table 2 reports descriptive statistics for HI across study arms.Table 2 Indoor HI (°C) after intervention in MayFull size tableThe findings suggest that cool-roof houses reduced indoor heat out of all interventions. In addition, houses with cross-ventilation and mat-ceiling had the highest indoor heat during the day and night, respectively.Thermal comfortNext, we measured thermal comfort of the housing designs using psychrometric charts. The psychrometric charts were based on the indoor environment data, collected on each day (24 hours) throughout the month of May, across all the study arms (Fig. 3). Extended Data Fig. 1 shows the same data separately for daytime (7:00–19:00) and nighttime (19:00–7:00) measurements. Each point in the charts represents a paired measurement of dry-bulb temperature and humidity ratio, and the red polygons indicate the TCZ. Across the 24-hour measurement periods over multiple days, most houses with cool-roof and mat-ceiling were within the TCZ, whereas control and cross-ventilation houses were mostly above the TCZ. Similarly, during daytime measurements, cool-roof and mat-ceiling houses were largely within the TCZ (Extended Data Fig. 1a). At night, mat-ceiling houses fitted best with TCZ (Extended Data Fig. 1b), maintaining the warmest indoor conditions.Fig. 3: Psychrometric charts of 24-hour indoor temperature and humidity conditions, in the month of May, across study arms.Each panel represents one arm; each point represents a paired measurement of dry-bulb temperature and humidity ratio; and the red polygon marks the TCZ.Source dataFull size imageExtended Data Table 2 summarizes the predicted mean vote (PMV) values across study arms for the same time window. PMV is a thermal comfort index that predicts the mean thermal sensation of a group of people on a seven-point scale from −3 (too cold) to +3 (too hot), with 0 being neutral. The daily PMV values were higher than +3 for control, mat-ceiling and cross-ventilation houses and lower for cool-roof houses. Additionally, Extended Data Fig. 2 provides a graphical description of the PMV with respect to daytime and nighttime data. During the day, houses with cool-roofs had PMV values closest to zero (0 represents neutral or comfortable thermal sensation). At night, houses with mat-ceiling had PMV values closest to the comfortable thermal sensation zone. This observation, therefore, coincides with the psychrometric charts.The results suggest that houses with cool-roofs or mat-ceilings provided the greatest thermal comfort, in contrast to control and cross-ventilation houses, which did not. From a health perspective, these findings suggest that houses with cool-roof or mat-ceiling designs could be better interventions to prevent heat-related illness.Mosquito dataWe assessed the entry of both Anopheles and Culex mosquitoes, before and after housing interventions. Anopheles gambiae s.s, A. funestus and Anopheles arabiensis of the genus Anopheles are the primary species that transmit malaria in the region. Both A. gambiae s.s. and A. arabiensis are part of the A. gambiae s.l. (complex). No further analysis was performed to distinguish between the two species and are reported as A. gambiae s.l. Culex is the genus of mosquitoes of several species that transmit a number of arbovirus infections. A total of 8,297 Anopheles and 2,840 Culex mosquitoes were collected indoors by Centers for Diseases Control and Prevention (CDC) light traps. More A. funestus were collected prior to any house modification (n = 6,958; 84.2%) than after the intervention (n = 1,303; 15.8%). On the contrary, fewer Culex species were collected before modification (n = 974; 34.3%) than afterwards (n = 1,866; 65.7%); however, the numbers were higher in unmodified houses compared to modified ones. Only two A. gambiae s.l. were collected in the pre-modification period, and 34 were collected in the post-modification period. Therefore, the species was not subjected to further analysis because the numbers were too low for any meaningful statistical analysis and interpretation.The baseline assessment of mosquito numbers showed no difference in indoor vector densities among the different study arms. After any modification, screening significantly reduced the number of female A. funestus mosquitoes and lowered the counts of Culex mosquitoes. In modified houses, the mean nightly density of female A. funestus was 1.3 mosquitoes per house compared to 5.6 in control houses, representing a 77% reduction (P < 0.001). Similarly, the density of Culex spp. decreased from an average of 4.8 (before modification) to 2.0 (after modification) mosquitoes per house, a 58% reduction (P = 0.004), as reported in Fig. 4 and Extended Data Table 3.Fig. 4: Mosquito densities after intervention.The bar plot represents comparison of mean densities of female A. funestus and Culex species across the different study arms (n = 120). The points represent mosquito species-specific mean density per structure. In all bars, error bar is plotted as mean ± s.d. Data are representative of three technical replicates (three collections per structure per arm).Source dataFull size imageCost of modificationExtended Data Table 4 presents the costs incurred in housing modification for vector proofing (screening) and passive cooling. The cost of modifying a single house for passive cooling and vector proofing was estimated at US $189.12. Modification for passive cooling using cool-roof was the most expensive at approximately US $145.14 per structure, followed by mat-ceiling at US $79.00. The cost of a single window for cross-ventilation was estimated at US $37.36, and the cost per structure varied by the size of the house and the number of windows required. The exchange rate for US Dollars to Kenya Shillings was US $1 = KES 133.66 as of April 2023 when the modifications were conducted.Community knowledge, attitude and perception on house modificationKnowledge, attitude and perception surveys were conducted on 28 and 26 households before and after modification, respectively. The participants interviewed were mostly women: 75.0% (21/27) before modification and 76.9% (20/26) after modification. The mean age of the participants was 52 years in the pre-modification period and 54 years in the post-modification period, with the median age being 51 years and 58 years, respectively. The highest level of education attained by the study participants was primary education. The main source of income for the study population was small-scale farming (53.6% (15/28)) and small-scale businesses (25.0% (7/28)). Other sources of income included skilled labor at 7.1% (2/28), donations at 7.1% (2/28) and charcoal burning at 7.1% (2/28). The assessment of community knowledge, attitude and perception of housing modification for mosquito control and thermal comfort was targeted at the 26 intervention houses. At the beginning of the survey, 42.9% (12/28) of the households reported having windows. This increased to 61.5% (16/26) in the post-modification period as some houses received windows for cross-ventilation.All 26 respondents surveyed in the post-modification period reported that house modification reduced indoor temperatures and mosquito numbers. Most of the respondents (76.9%, 20/26) perceived a substantial reduction in indoor temperatures after the modifications. An additional 15.4% (4/26) reported moderate reduction, and 7.7% (2/26) reported only a slight reduction. Most respondents in both the pre-modification (64.3%, 18/28) and post-modification (65.4%, 17/26) surveys mentioned the importance of building designs that reflect their cultural values, especially when building the initial ‘starter’ home. This first structure is typically built within a day, guided by specific cultural practices, and is considered temporary, with the intention of replacing it afterwards with a more permanent dwelling. Before modification, 14.3% (4/28) of the respondents, and 19.2% (5/26) of the respondents after modification, felt that the reflection of cultural values in building design was somewhat important. In addition, 21.4% (6/28) and 15.4% (4/26) of respondents in the pre-modification and post-modification surveys, respectively, reported that they did not prioritize cultural considerations in housing design.The majority (71.4% (20/28) and 96.2% (25/26)) of the respondents in the pre-modification and post-modification periods, respectively, expressed willingness to adopt new house designs that are different from their cultural preferences if they help to improve thermal comfort and mosquito control. The proportion of respondents willing to use their family resources increased from 78.5% (22/28) in the pre-modification survey to 84.6% (22/26) in the post-modification survey.The main importance of windows, identified by participants, was to allow fresh air and light into the house. Additional benefits mentioned included access to the house when the door is locked, visibility of the outdoors and the ability to use windows as an outlet for business purposes if part of the house served as a shop. The main reason given for not having windows installed was inadequate finances. Other reasons provided included: ‘The current house is a temporary structure before building the desired house’; ‘I did not think about it then’; ‘it is a cottage, so it has no window’; ‘I just built it without windows’; ‘I left to fix later’; and ‘fear of house collapsing if the windows are installed after construction’. The disadvantages of having windows stated during the pre-modification survey were insecurity, mosquito entry, allowing dust into the house and superstitious beliefs, such as being used by witches to harm people in the house. However, after building new windows in the intervention phase, most participants noted that there were no real disadvantages to having windows. Among the houses that had windows, 66.7% (8/12) of respondents at baseline reported opening them daily compared to 92.9% (13/14) after the modification.DiscussionThis study demonstrates that targeted housing modifications can effectively address the dual challenges of extreme heat and malaria transmission in rural African settings. The integration of passive, sustainable, cooling housing modifications with vector proofing resulted in significant reductions in indoor HI and mosquito densities and improved thermal comfort levels compared to controls. These findings inform climate change adaptation and malaria control in resource-limited settings. Climate models project that equatorial SSA will experience temperature increases of at least 1−2 °C in the coming decades25. Although this temperature rise may seem small, it has important implications for human health and functioning. This is because the human body must maintain its core temperature within a narrow range of around 37 °C ± 0.5 °C. Even slight deviations of ±1.5 °C from this core temperature can substantially affect physical and mental performance, and persistent larger deviations exceeding 3 °C can be life-threatening26,27. The body can tolerate additional heat stress for only brief periods. Consequently, even small but sustained increases in environmental temperature, especially when protracted for a long time, directly impact human health, causing a range of conditions from dehydration and cardiovascular stress to physical and mental impairment, exhaustion and heat stroke. In rural African communities, where active cooling options are limited, passive cooling approaches that reduce indoor heat exposure represent a critical adaptation strategy for protecting health.Among the passive cooling options evaluated, cool-roofs effectively reduce HI during the day, but they are unlikely to improve comfort at night because the thin mud walls have low thermal mass and do not store much heat. Mat-ceilings appear to better insulate houses and, therefore, maintain consistent temperatures during the day and night. However, they are likely to be ineffective in warmer months as the ceiling prevents heat from radiating out at night. Mat-ceilings might, therefore, induce heat stress and perform the worst, especially due to heat trapping and reduced indoor space for air circulation. The use of cross-ventilation for cooling in this setting would benefit from co-creation on window design and community sensitization to promote appropriate use of windows for indoor cooling.The application of white reflective coating on iron sheet roofs, which were the hottest part of the house during the day, effectively reflected solar radiation, reducing indoor mean temperatures by 2.8 ± 0.2 °C compared to control houses. This finding aligns with studies from other tropical regions: in North Australia, light-colored roofs showed 30% lower total heat gain than dark-colored ones, and, in The Gambia, white-roofed houses were consistently cooler and more comfortable than those with bare metal roofs28,29. Consequently, passive cool-roofs are observed as a means of reducing energy cooling loads to satisfy human comfort requirements in hot climates30. The measure of how much solar energy a building’s exterior surface reflects away has been applied on walls as well to reduce daytime heat gain31. The effectiveness of cool-roofs is particularly relevant in SSA, where corrugated metal roofing is widespread in both rural and urban buildings. These roofs typically allow substantial solar energy flux into living spaces32. With global warming intensifying this heat burden, cool-roofs offer a practical, passive solution for thermal comfort in resource-poor communities. Consistent with other studies, the data presented here demonstrate the benefit of reflective roofs in lowering indoor HI in resource-poor, rural African communities.Although cool-roofs had the lowest indoor temperatures, they had over 10% higher relative humidity levels compared to control. Additionally, relative humidity—the ratio of the current absolute humidity to the maximum possible absolute humidity at a given air temperature—was observed to increase when air temperature levels decreased. It is generally recommended to keep relative humidity indoors between 40% and 60% for comfort and health33; studies have shown that higher relative humidity levels of between 50% and 70% are fairly tolerated at lower temperatures of between 26 °C and 30 °C34. But when temperatures are elevated above 37 °C at 70% relative humidity, heat stress sets in with increased heart rate, respiratory rate and mean skin temperature34. Although high relative humidity at lower temperatures is tolerable, it also promotes dampness and the growth of fungi and mites and creates moisture problems in indoor building materials with poor air quality, which altogether increases health risks. It is, therefore, essential to balance both temperature and humidity levels to achieve thermal comfort indoors. A combination of cool-roofs and natural ventilation has been recommended to increase indoor thermal comfort30 by balancing both temperature levels and humidity. In this study, six of the 10 houses that received cool-roofs did not have windows, hence contributing to the high humidity levels observed in such houses. Sufficient ventilation in combination with other cooling options is, therefore, critical in achieving thermal comfort indoors.Mat-ceilings reduced daytime HI by 1.3 °C but increased nighttime HI by 2.0 °C compared to control houses. Although the mats provided daytime insulation from roof heat, they likely trapped heat released by walls at night. Despite reduced headroom and air circulation, houses with mat-ceilings maintained the most stable daily temperature (23.2−29.9 °C) and humidity (56.5−72.7%) ranges, closest to human comfort levels (22−27 °C and 40−60%). Performance could be improved through roof space venting, better mat alignment and enhanced cross-ventilation.Cross-ventilation was not achieved due to behavioral and structural barriers. Residents frequently closed windows for security reasons, particularly at night, and 67.5% of houses lacked windows altogether, at baseline. Additionally, the study was conducted during colder months of the year, and the residents are more likely to maximize the use of cross-ventilation for cooling in the hotter months of the year. These factors potentially prevented meaningful cooling, with cross-ventilated houses showing higher nighttime HI than controls, likely due to eave screening that reduced natural airflow21. The limited effectiveness highlights challenges in real-world housing interventions where security concerns and cultural practices influence resident behavior. Future designs should incorporate permanently open, secure ventilation systems or combine cross-ventilation with other passive cooling strategies35.House modifications significantly reduced indoor mosquito densities, with female A. funestus numbers 77% lower in screened versus unscreened houses. This effectiveness aligns with findings from multiple studies across SSA demonstrating that screening eaves, windows and doors successfully reduce mosquito entry36,37. Although mosquito control in the Global North primarily focuses on outdoors due to widespread indoor screening38, house modifications are particularly crucial in regions like western Kenya where mosquito biting remains high indoors despite bed net use19,20. The importance of house screening is highlighted by recent findings showing that 87% of A. funestus and 88% of A. gambiae bites still occur indoors even when bed nets are used19. Other studies have demonstrated that mosquito activity peaks in the early morning as people leave their bed nets and extends into other indoor spaces such as schools19,39. Because open eaves are the primary route for mosquito entry when doors and windows are closed at night12,18, comprehensive screening of all entry points is essential for effective vector control.However, screening can potentially reduce airflow and increase indoor temperatures, as demonstrated in The Gambia where screened houses were warmer than unscreened ones40. Our study addresses this challenge by combining vector proofing with sustainable passive cooling strategies; the aim is to create living spaces that are both mosquito free and thermally comfortable. With the limitations of being an in-field pilot study (for example, small sample size, heterogeneity of houses and absence of airflow measures), future studies would need to demonstrate whether such an integrated approach could yield broader co-benefits beyond our measured outcomes, including sleep quality, economic productivity, heat stress, malaria incidence and other health outcomes.The adoption of such interventions would depend not only on technical effectiveness but also on community uptake. In our study, acceptance of the modifications was high, with 85% of households expressing a willingness to invest their resources on similar improvements. This represents an increase from the 79% observed during pre-intervention, suggesting that experiencing the benefits firsthand strengthened community buy-in. Furthermore, 96% of the respondents expressed willingness to adopt new house designs that differ from traditional preferences if they improved thermal comfort and mosquito control. This openness to innovation, while maintaining respect for cultural values, suggests promising potential for scaling up such interventions. Implementation costs and structural challenges present important considerations for scaling. The total cost of screening per house (US $189.12) protects approximately four individuals per house throughout the day and night and lasts longer, making it relatively more cost-effective compared to repeated distributions of bed nets every 3 years at approximately US $5−7 per net for every two people with only partial protection. However, challenges encountered during installation, particularly with door modifications and existing structural limitations, highlight the need for standardized approaches and skilled implementation. Future interventions might benefit from incorporating these modifications into initial construction rather than retrofitting existing structures.This study has several limitations. Although airflow is critical for thermal comfort and a major functionality of cross-ventilation, indoor airflow measurements were not performed in this pilot study, due to study constraints. We will consider this aspect in the upcoming larger randomized controlled trial. Direct airflow measurement in this setting presented technical challenges due to behavioral factors—residents frequently closed windows for security and space needs, which would have confounded continuous airflow monitoring. Future studies should incorporate standardized airflow measurement protocols that account for these behavioral patterns. The use of preexisting structures that vary in size and shape may have resulted in indoor climate variability among the houses. This may limit the generalizability of the findings from this evaluation. However, retrofitting existing houses is less costly and most suitable for local context. Furthermore, the approach promotes community participation and ownership and preserves the community’s cultural values and practices in housing construction.Another limitation is that the study was conducted during a cooler period (April−June) rather than during the hottest months (January−March), which may have underestimated the full thermal stress experienced by residents during peak heat periods. The seasonal timing was chosen for logistical reasons, which might limit the generalizability of findings to year-round thermal comfort assessment. Due to cultural predisposition to leave doors open during the day, some households were observed to prop their door screens open, which potentially reduced the effectiveness of the screened doors in limiting mosquito entry into the house.Additionally, to provide a more comprehensive understanding of thermal comfort, we provided psychrometric charts and PMV based on ASHRAE Standards 55. However, we could not determine if such standards, developed in high-income countries where residential buildings are different (for example, larger and made of concrete/cement), might not be fully relevant to rural SSA homes (for example, smaller and mostly made of mud), due to relevant structural differences. The short evaluation period also limited our ability to assess long-term durability and performance across different seasons. Although we observed significant reductions in vector populations, the study duration precluded assessment of epidemiological impacts on malaria transmission. Collection and counting of mosquitoes were performed by the same staff; hence, blinding was not possible. However, potential bias in mosquito data was minimized by standardized measurement protocol and instrument, consistent timing of collections and data validation procedures. Due to the small sample size and the short duration of this pilot study, a cost-effectiveness evaluation and detailed analysis of confounding factors on mosquito catch sizes were not included. The limitations observed in this feasibility pilot evaluation will be addressed in a cluster randomized trial, with the most effective modification being applied in approximately 300 houses.In conclusion, housing modifications that combine passive cooling with vector proofing represent a sustainable approach to improving health outcomes in rural communities. However, successful scaling will require careful consideration of humidity control, structural challenges and implementation costs. For future scale-up and implementation, the housing modifications would need to be optimized for different climatic zones, evaluating long-term durability and cost-effectiveness and the epidemiological impacts considered, in order to facilitate broader adoption of these interventions. The dual benefits of reduced heat stress and mosquito control, coupled with strong community acceptance, suggest the potential for climate change adaptation and malaria control in SSA.MethodsStudy site description and population characteristicsThis study was conducted in Kadenge Ratuoro village (0.0242° N, 34.1749° E) with an altitude of 1,140 m, in Alego Usonga sub-county, Siaya County. The study area is within the Kenya Medical Research Institute (KEMRI)/CDC Health and Demographic Surveillance System (HDSS)41. The residents are of the Luo ethnic group, subsist on farming, fishing and trade and live in small houses, clustered into family social units of relatives called compounds. Supplementary Fig. 1 is a pictorial and technical representation of an unmodified house in the study area. Houses are typically rectangular and constructed of stick frames (wattle), compacted soil or cement foundation and dirt or cement floor37,42. The walls are either mud or cemented with an average thickness of approximately 0.20 m. The roofs are mostly of corrugated galvanized iron sheets. A few houses have either thatched or clay tile roofs. Most of the houses have two rooms, with an average internal floor area of 19.88 m2 and a headroom of 2.24 m. Doors are approximately 0.85 × 2.02 m (1.71 m2) and are either unframed or framed with wood to create a jam and sash. Windows vary greatly in size, with an average size of 1.37 m2 each; however, most of the houses do not have windows. For context, 67.5% of the houses in this study did not have windows at the start of the study. The eaves are usually open and are approximately 3.33 m2 (0.22 m height × 19.40 m perimeter). The open eaves, in addition to doors and windows, provide ventilation into the houses, allowing entry of light and fresh air. Unfortunately, eaves are also the main route for unlimited entry of mosquitoes into the houses.Malaria transmission in Siaya County is stable throughout the year, with a prevalence of 37% in children between 6 months and 14 years of age16, with the Alego Usonga sub-county having a prevalence of 50%. A. gambiae, A. funestus and A. arabiensis are the main malaria vector species in the region. Both A. gambiae and A. funestus are considered the primary malaria vectors in SSA because they feed more frequently indoors and on humans43,44. A. funestus is capable of sustaining malaria transmission even in dry seasons due to its preference for permanent breeding habitats45. A. arabiensis, on the other hand, is a more opportunistic43,44 feeder with lower vectoral capacity compared to the first two. The vector is, however, capable of sustaining residual malaria transmission where both A. gambiae and A. funestus have been controlled46. The region has a bimodal rainfall pattern, with long rains between March and May and short rains between October and December. The region has typical SSA tropical climatic conditions. A temperature suitability index for malaria transmission shows that the western Kenya region has ambient temperature and adequate rainfall suitable for endemic malaria transmission16,47. The average wet-bulb temperature range within the HDSS has been estimated at between 17 °C and 35 °C.Study designWe conducted a pilot, randomized field study assessing the impact of housing modification on indoor thermal comfort and mosquito numbers. The 40 houses were randomly allocated to mCV, mCR, mMC or mCL, n = 10 houses per arm. Quantitative and qualitative data collections were conducted before and after house modification.Mobilization and recruitment of study householdsExtended Data Fig. 3 illustrates the selection and randomization of the study houses. Home visits were conducted to enumerate and characterize houses within a section of the study village. A total of 47 compounds with 84 houses where people slept were enumerated. For every active structure in each compound, structural features, including wall type, roof type, presence or absence of ceiling, eave type and the number of windows, doors and rooms, were recorded. From the 47 compounds with a pool of 84 houses, to standardize data collection, n = 40 houses, all with identical characteristics—mud walls, open eaves, iron roofs and not more than three rooms—were selected for the study. A single house that met the selection criteria was identified per compound after a discussion with the compound head.RandomizationRepresentatives of the 40 selected structures were invited to a meeting where a random, transparent and fair allocation of houses to the different study arms was conducted. Forty raffle tickets, labeled with the different study arms—mCR, mCV, mMC or mCL (10 tickets for each study arm)—were provided in a transparent container. The tickets were folded to conceal labels and were mixed up in the container. The house representatives were invited to draw a single ticket each. The houses were allocated to different treatment arms based on the selection of the house representative.Structural modificationStructural modification of houses was conducted by a building professional identified based on previous experience with similar modifications. After the randomization of houses into the various study arms, the specific house characteristics, including floor area, numbers and sizes of doors and windows and the presence and sizes of open eaves, were collected to guide modification. The building expert established a workshop within the study area where all materials, including doors, windows and pieces of timber for eaves and ceiling modifications, were fabricated before installation in the various houses. Modifications were conducted based on the randomization for the three passive cooling options as follows (Fig. 1). For all houses that received new windows for cross-ventilation, a standard-size window was made based on the average size of rooms in the study houses. The modified houses were assessed by the structural engineer on the project to ascertain the quality of the changes. At the end of the data collection, all the control houses were modified with screened doors, windows and eaves and a cool-roof for indoor cooling.Cross-validationCross-validation (mCV) was achieved by installing screened windows on the opposite walls of each room. This involved a complete overhaul of the existing windows and/or the creation of new ones if no windows existed in a house. The windows were made of cypress battens to create two batten window leaves, each measuring 360 × 600 mm with an area of 0.46 m2. Each window leaf hung on two 50-mm ordinary hinges and was secured at the center using a barrel bolt latch. A fiberglass insect mesh (Streme Limited 12) laid between two sheets of coffee tray mesh (ALS Limited, Trading Division) was attached to the window frame for insect screening. The insect screen was installed outwards, whereas the window panels opened inwards. To install the windows, a section of the wall was cut to create space for the window if none existed before or adjusted if the original window was smaller. After the installation of the modified window, the remaining gaps in the wall were filled with mud to achieve the same finish as the original wall (Supplementary Fig. 2).Cool-roof system and insect-proof housingIron-roofed houses were painted with a reflective white coat to reduce the amount of heat conducted into the house, hence lowering internal temperatures. Two coats of paint were applied to the roofs. Crown Roofmaster (Crown Paint Industries), an extremely durable, weather-resistant, self-priming acrylic resin-based paint with a waterborne topcoat and matte finish, was used.Mat-ceiling and insect-proof housingLocally made papyrus mats were installed horizontally, covering the roof space just above the eaves. Locally sourced round poles were used as structural bearers and cross-binders in the ceiling substructure (brandering) to support the mat finish. Timber battens were nailed below the mats to fasten them securely to the round poles forming the brandering (Supplementary Fig. 3).Mosquito proofingAll houses that received passive cooling options were screened for mosquito control. The doors were modified by introducing wooden frames and panels in addition to the originally existing door. The existing doors opened inwards, whereas the newly introduced screened doors opened outwards. The screened door panels were made of wooden frames and fiberglass insect mesh laid between two sheets of coffee tray mesh (CTM). The doors hung on two self-closing hinges to keep them always closed (Supplementary Fig. 4). The windows were also screened as already described above. The eaves were screened by introducing a piece of timber at the edge of the wall just before the eave space and another piece of timber on the roof directly above the wall. Fiberglass insect mesh was then attached to the two pieces of timber, hence covering the eave space. An overlap of the insect mesh was tacked into the grooves between the timber and the corrugated iron sheet to block gaps between the corrugated iron sheet and the timber.Indoor thermal environmentDaily temperature and humidity were collected every 15 minutes in both modified and control houses from March to July 2023 using Onset HOBO UX100-003 data loggers. The data loggers were placed indoors, hanging at approximately 1.5 m from the floor, in the sleeping zone, away from walkways and out of reach of children to avoid interference with the daily activities of household members. Data download was conducted twice during the study period. We used temperature and humidity to calculate the HI10,48 based on an algorithm from the US National Weather Service, which is implemented in the R package weathermetrics49. HI is also known as the apparent temperature. It is the heat felt by the human body when relative humidity is combined with the air temperature. More than a dozen indices have been developed in the recent past to describe the complex interaction among ambient air temperature, relative humidity, wind speed and radiation and their impact on human health and performance9. The Wet-Bulb Globe Temperature (WBGT) is a measure of heat stress that combines air temperature, humidity, wind speed, sun angle and cloud cover to indicate how hot it feels in direct sunlight50. It is the most widely used index to assess heat stress in humans and to recommend rest/work cycles at different physical work intensities, especially under hot and humid conditions51. However, the WBGT is difficult and costly to measure directly, and most observations are sparse and based on estimations. In addition, the WBGT is more informative for workplaces. For these reasons, we opted for the HI, which combines temperature and humidity, the most crucial factors in this specific indoor environment, especially at night, as ventilation can be dramatically reduced by the absence of openings or windows closed due to safety reasons. To verify our approach, we conducted a small side test to compare HI measured with a HOBO device and a portable hygrometer designed to measure WBGT (model PCE-WB 20SD; PCE Instruments) directly. The results showed complete agreement; in addition, we made a direct comparison between the HI from HOBO and the WBGT index directly measured from such a hygrometer and observed the same pattern (see Extended Data Figs. 4 and 5 for comparison description).To provide a more comprehensive overview of the impact of the house modification on thermal comfort, we provided psychrometric charts and predicted mean vote (PMV), metrics used to assess thermal comfort in building design and other applications. Both are established models for standards such as ASHRAE 55 and ISO. Input variables for the psychrometric chart were temperature and relative humidity from the data logger (tPost data) and humidity ratio calculated using the R package PsychroLib52. For these charts, we also added a TCZ based on the estimated effects of clothing, metabolism and air speed. Data points falling outside this zone suggest that occupants may be experiencing thermal discomfort. Boundaries of the TCZ were determined using the CBE Thermal Comfort tool, a free online tool that implements thermal comfort calculations from standards53. We used the so-called ‘psychrometric (air temperature)’ (https://comfort.cbe.berkeley.edu) method and entered the following into the calculation mask: a mean radiant temperature of 24 °C (because ASHRAE Standard 55-2017 recommends temperature between 19.4 °C and 27.8 °C, mean 23.6 °C), a metabolic rate of 1 MET (because 0.8 MET corresponds to sleep and 1.2 MET corresponds to standing relaxed), an air speed of 0.1 m s−1 and clothing varying between 0.5 clo and 0.6 clo (because these corresponds to typical summer indoor clothing). Based on this input, the limits of the TCZ at 0% relative humidity are 28 °C and 34.8 °C and, at 100% relative humidity, 23.4 °C and 28.0 °C53.The PMV is a point thermal sensation scale to quantify the degree of thermal comfort. It generally ranges from −3 to +3 (that is, −3 cold, −2 cool, −1 slightly cool, +1 slightly warm, +2 warm and +3 hot), with 0 representing neutral comfort. The farther the values are from zero, the greater the perceived thermal discomfort. The PMV was calculated using the method by Fanger implemented in the R package comf49. The input consisted of six parameters: air temperature from the indoor data logger, radiant temperature (=air temperature), relative humidity from the indoor data logger, air speed of 0.1 m s−1, clothing level of 0.5 clo and metabolic rate of 1 MET. Here, fixed values for air speed, clothing level and metabolic rate were the same as used for TCZ in the psychrometric chart (see above). It is important to note that, in the frame of this study, we focused specifically on day and nighttime measurements separately, because residents spend the longest time at home especially during the night, whereas, during daytime, they might be mostly outdoors, due to the different occupational activities.Mosquito collectionMosquito collection was performed indoors using the CDC Miniature Light Trap (model 512; John W. Hock Company). Collections were conducted twice before modification (baseline) and three times after modification in each house. The light traps were set in the sleeping area, next to an occupied bed net, at approximately 1.5 m from the floor. The traps were run from 18:00 to 7:00 the next morning. During the mosquito collection period, the collector administered a brief questionnaire to collect information on household characteristics, including roof type, wall type, presence of eaves, presence and use of malaria control products such as bed nets, presence of cattle and number of people who slept in the house the previous night. The location of each house was recorded using the Global Positioning System. The collected mosquitoes were identified to genus levels as either Anopheles or Culex and in each genus as either male or female based on morphological features. The anophelines were further identified morphologically54,55 to species level as either A. gambiae s.l. or A. funestus s.l. All the collected mosquitoes were counted for each day, trap and house, and the counts were recorded based on genus, species and sex. All the mosquitoes that were morphologically identified as A. funestus s.l. were further identified as species by polymerase chain reaction (PCR)56, and all were confirmed to be A. funestus s.s. The mosquitoes identified as A. gambiae s.l. were not subjected to further analysis because the numbers were so low. Data on household characteristics and mosquito information were collected on a CommCare (Dimagi, Inc.) application run on an Android tablet and transmitted to a project cloud server.Collection of data on community knowledge, attitude and perception of house modificationA structured questionnaire assessing community perception, knowledge and attitude toward house modification for vector control and temperature reduction was administered to intervention households before and after house modification. Data were collected on the community’s building practices, including reasons for the inclusion or exclusion of certain building elements such as windows, eaves spaces, ceiling, wall and roof types. The questionnaire further assessed the community’s understanding of the relationship between the various building elements and the entry of mosquitoes into houses and indoor heat levels. Additional questions were administered to gauge the community’s perception of changes made to their houses, their potential contribution to house cooling and reduction of mosquito entry as well as their willingness to use their resources to modify their houses. In the post-modification survey, the perceived benefits and risks of house modification and the community’s willingness to continue using the modifications beyond the lifetime of the study were assessed.Thermal imagingThermal images of the houses were taken using a FLIR T450sc camera (Teledyne). The images were taken by the same operator with the same camera on different days but within the same time slots. In addition, a camera built-in laser pointer was used to ensure a precise distance and pointing for subsequent images of different houses. In this way, confounders were minimized, ensuring a standardization of measurements in the photographed houses. Images were taken early in the morning before sunrise, at midday and in the evening after sunset to assess the source of heating in the houses at different times of the day. Images were taken centered on a specific reference point, identified by using a built-in laser pointer, to ensure reproducibility and to compare different times of the day. A set of n = 4 houses was selected, as easily accessible for photographs at all times of the day and representing the different structural modifications—mCR, mCV and mMC—as well as control (Extended Data Fig. 6).Economic costThe costs for housing modification were based on material and labor costs in cases where the raw materials were used directly in the modification process—for instance, in the application of cool-roof paint and installation of mat-ceiling and eave screens. Screened windows and doors were procured as complete units including the costs of installation. Material, labor and item costs for windows and doors were provided within the market rates.Data management and analysisField data were collected using CommCare software run on Android tablets. Every participating house was identified by a unique code, and a collection code was generated by the tablet for every mosquito sampling effort. These codes were used to track data generated from the different study components for ease of management. Individual mosquitoes from each collection were placed in microcentrifuge tubes labeled with pre-printed barcodes and linked to the field data using a house code and a collection code. Results of species identification by PCR were linked to individual mosquitoes by the unique barcode label.StatisticsSample size considerationBecause this was an external pilot study, a sample size calculation was not performed. The objectives of the pilot study were to assess the feasibility of combining housing modification for passive cooling with malaria vector control, community acceptance and engagement, identification of implementation costs and challenges and development of data collection tools for the main trial. A sample of 10 structures per treatment arm was considered sufficient based on recommendations by Whitehead et al.57 for an external pilot with a continuous outcome variable. However, the outcome of the pilot was not used to inform the sample size for the main trial.Indoor environmental data and thermal comfortTo assess the indoor environment of houses before and after their modification (mCL, mCR, mCV and mMC), each temperature and relative humidity recording was divided into tPre and tPost. The tPre set includes data from 4 days at the end of March, and the tPost set data comprises data from the entire month of May. Each recording was then grouped into three different subsets: 24hTime, data covering a full day; dayTime, data covering periods from 7:00 to 19:00; and nightTime, data covering periods from 19:00 to 7:00. To ensure data quality, each was checked for completeness. Only datasets with a completeness of at least 95% were considered for further analysis. In addition to indoor temperature and relative humidity, the HI was calculated using the R package weathermetrics10. Means and 95% confidence intervals were calculated for temperature, relative humidity and HI at different timepoints (tPre: 24−27 March; tPost1: 1−7 May; tPost2: 8−15 May; tPost3: 16−23 May; tPost4: 24−31 May). Variability in indoor environmental data between the different groups of houses was assessed separately for tPre and tPost using linear mixed-effect models. The tPre model consists of time (days 1−4), modification (mCL, mCR, mCV and mMC) and their interactions as fixed effects and house as a random effect with random intercepts for house. The tPost model consists of time (tPost1, tPost2, tPost3 and tPost4) and modification (mCL, mCR, mCV and mMC) and their interactions as fixed effects and house as a random effect with random intercepts for house. Linear mixed-effect models were run using the R package afex. Analyses and graphical illustrations were performed using R version 4.3.3 (ref. 58). Linear mixed-effect models were run using the R package afex. Estimated marginal means and contrasts were calculated using the R package emmeans. Figures were created using the R packages ggplot2 and cowplot.Mosquito dataVector abundance was assessed using descriptive statistics (means, proportions and 95% confidence intervals). For inferential analysis, we used rate ratios as a measure of intervention effect59. Generalized linear mixed models (GLMMs) using Template Model Builder (glmmTMB) were fitted using negative binomial distribution for analysis of mosquito counts among different study arms. Study arm (treatment allocation), presence of deterrence (use of mosquito coil indoors, fire burning indoors and/or use of insecticide spray can) and presence of domestic animals indoors with their interaction were considered as fixed effects. Models were adjusted for repeated measures using the structure ID as a random effect. The rate ratios were obtained by exponentiating the model coefficient on the log rate ratio scale.All data analyses were performed using R statistical software version 4.4.1, and the significance level was set at α = 0.05.ConfoundingThe following confounders were accounted for in the analysis of mosquito data: use of mosquito coils, use of insecticide spray cans, open cook fires and the presence of cattle indoors. No confounders were considered in the heat stress analysis.Ethics approvalThe study received ethical review and approval from the KEMRI Scientific Ethics Review Unit (SERU 4796). Before being included in the study, written informed consent was obtained from every household for modification of houses and mosquito collection.Inclusion and ethics statementThis project was designed through a partnership among KEMRI, Habitat for Humanity International and Charité – Universitätsmedizin Berlin. A section of Kadenge Ratuoro village in Siaya County was selected as a study site by KEMRI in consultation with the village elder. It is within the KEMRI/CDC HDSS and has high mosquito numbers and malaria burden given its proximity to Yala swamp. Allocation of interventions to the study structures was conducted randomly by lottery with the participation of structure representatives.In study implementation, the KEMRI team led the intervention design, community engagement and data collection, whereas Habitat for Humanity International led the sourcing of funds and provided technical oversight for housing modifications. All team members collaborated in data ownership, intellectual property and authorship of the publication relating to the work.As housing modification involved major alteration in the house design, measures were put in place to guard the safety and privacy of study participants. The housing modification workers were trained on safeguarding and good clinical practice. At the end of the data collection, the control houses were modified, and the study results were deiminated to all participants.Previous work on housing modification in the region guided the design of this study. Additionally, findings from similar investigations in the regions were taken into account in the citations for this paper.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

    Data availability

    All data are available on GitHub (https://github.com/abongoben/Housing-modifications-data-and-codes). Source data are provided with this paper.
    Code availability

    All programming codes are available on GitHub (https://github.com/abongoben/Housing-modifications-data-and-codes).
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    Download referencesAcknowledgementsWe would like to acknowledge the contribution of the staff at KEMRI-Centre for Global Health Research (CGHR) who assisted with data collection and analysis of mosquito samples, the local contractor and artisans for housing modifications work and the residents of Kedenge Ratuoro village who volunteered their houses for modification. The work was supported by funds from SeaFreight Labs through Habitat for Humanity International (J.S. and J.O.) and Wellcome Trust grant number 226750/Z/22/Z (B.A., E.O. and D.K.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.Author informationAuthor notesThese authors contributed equally: Eric Ochomo, Martina Anna Maggioni.Authors and AffiliationsKenya Medical Research Institute – Centre for Global Health Research, (KEMRI-CGHR), Kisumu, KenyaBernard Abong’o, Daniel Kwaro, Teresa Bange, Vincent Moshi & Eric OchomoCharité – Universitätsmedizin Berlin, Institute of Physiology, Center for Space Medicine and Extreme Environments Berlin, Berlin, GermanyDaniel Kwaro & Stefan MendtHabitat for Humanity International, Nairobi, KenyaJacob Simwero & Jane OtimaVector Group, Liverpool School of Tropical Medicine, Liverpool, UKEric OchomoCharité – Universitätsmedizin Berlin, Charité Center for Global Health (CCGH), Center for Space Medicine and Environments Berlin, Berlin, GermanyMartina Anna MaggioniDepartment of Biomedical Sciences for Health, Università degli Studi di Milano, Milano, ItalyMartina Anna MaggioniHeidelberg Institute of Global Health (HIGH), Faculty of Medicine and University Hospital, Heidelberg University, Heidelberg, GermanyMartina Anna MaggioniAuthorsBernard Abong’oView author publicationsSearch author on:PubMed Google ScholarDaniel KwaroView author publicationsSearch author on:PubMed Google ScholarTeresa BangeView author publicationsSearch author on:PubMed Google ScholarVincent MoshiView author publicationsSearch author on:PubMed Google ScholarJacob SimweroView author publicationsSearch author on:PubMed Google ScholarJane OtimaView author publicationsSearch author on:PubMed Google ScholarStefan MendtView author publicationsSearch author on:PubMed Google ScholarEric OchomoView author publicationsSearch author on:PubMed Google ScholarMartina Anna MaggioniView author publicationsSearch author on:PubMed Google ScholarContributionsB.A., J.S., J.O. and E.O. conceived the idea and wrote the protocol. J.S. and J.O. secured the funding. B.A., T.B., J.S. and E.O. implemented the study. B.A., V.M. and S.M. performed the data analysis and prepared figures. M.A.M. and D.K. provided technical support, expertise and supervision for the manuscript. B.A. wrote the initial draft of the manuscript. All authors reviewed and approved the final manuscript.Corresponding authorCorrespondence to
    Bernard Abong’o.Ethics declarations

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    Nature Medicine thanks Matthew Chersich, Lorenz von Seidlein and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Ming Yang, in collaboration with the Nature Medicine team.

    Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Psychrometric charts of indoor conditions during daytime and nighttime in May.Panel a shows conditions from 7:00am to 7:00 pm (daytime), and panel b from 7:00 pm to 7:00am (nighttime), across all study arms. Each point represents a paired measurement of dry-bulb temperature and humidity ratio. Red polygons indicate the Thermal Comfort Zone (TCZ).Source dataExtended Data Fig. 2 Predicted Mean Vote (PMV) during daytime and nighttime in May.Boxplots show the distribution of PMV values for daytime (7:00am–7:00 pm) and nighttime (7:00pm–7:00am) across study arms (n = 31 days). The box plot shows the median (black horizontal line), the 25th and 75th percentiles (lower and upper edges of the coloured box), and whiskers extending up to 1.5 times the interquartile range from the box boundaries. Data points that lie outside these whisker limits are displayed individually as outliers (black dots). Positive values indicate warm thermal sensation, with 0 representing neutral comfort or comfortable thermal sensation. Differences reflect the effect of each intervention on perceived thermal comfort.Source dataExtended Data Fig. 3 Recruitment process overview.CONSORT diagram showing the selection, enrolment, and randomization of houses into the four study arms.Extended Data Fig. 4 Comparison of measurements from April to July 2023 in two houses with cross ventilation modifications.Temperature and relative humidity were recorded using HOBO thermohygrometers (Onset Computer Corporation, USA), while the Wet Bulb Globe Temperature (WBGT) index was measured using the PCE-WB 20 SD WBGT meter (PCE Instruments, Germany). Panel (a) show the indoor Wet Bulb Globe Temperature (WBGT) index measured directly with the PCE device in each household. Panel (b) presents a household-level comparison of Heat Index (HI) values measured using both the HOBO and PCE devices. Panel (c) compares indoor temperature readings from the two devices, while panel d shows the corresponding comparison for relative humidity.Source dataExtended Data Fig. 5 Comparison of Wet Bulb Globe Temperature (WBGT) and Heat Index (HI) measurements from April to July 2023.WBGT was directly measured using the PCE-WB 20 SD device, while HI was calculated from temperature and relative humidity recorded with HOBO thermohygrometers. Panel (a) shows WBGT and HI values per household across the study period. Panel (b) displays the difference between HI and WBGT, which consistently ranges from about 2.5°C to 4.5°C, with WBGT values being lower. Although WBGT is better suited for environments involving physical exertion and solar radiation, and HI is more appropriate for indoor resting conditions, both indices followed similar temporal patterns.Source dataExtended Data Fig. 6 Thermal performance of housing modifications across time of day.Thermal images of houses with cross ventilation, cool roof, and mat ceiling interventions taken in the morning (6:00–7:00am), afternoon (12:00–2:00 pm), and evening (7:00–8:00 pm), showing differences in surface temperature and heat retention.Source dataExtended Data Table 1 Descriptive statistics of indoor environment before house modificationFull size tableExtended Data Table 2 Indoor PMV values after intervention, for the different cooling solutions in MayFull size tableExtended Data Table 3 Comparison of mean mosquito densities of female A. funestus and Culex species among control (mCL), cool-roof (mCR), cross-ventilation (mCV) and mat-ceiling (mMC) houses, before and after modificationFull size tableExtended Data Table 4 Costs of housing modificationsFull size tableSupplementary informationSupplementary Figs. 1−4 and Supplementary Tables 1 and 2.Reporting SummarySource dataSource Data Fig. 1Images of unmodified and different house modification features.Source Data Fig. 2Daily temperature, humidity and HI of houses in different study arms for the month of May.Source Data Fig. 3Daily (24 hours) temperature and humidity ratio by study arm.Source Data Fig. 4A. funestus and Culex species densities by study arm.Source Data Extended Data Fig. 1Daytime and nighttime temperature and relative humidity by study arm.Source Data Extended Data Fig. 2Daytime and nighttime PMV values by study arm.Source Data Extended Data Fig. 4Relative humidity, temperature, HI and WBGT data comparison between two houses.Source Data Extended Data Fig. 5WBGT and HI from two structures.Source Data Extended Data Fig. 6Thermal images of structures with different modifications at different times of the day.Source Data Extended Data Table 1Daily (24 hours) daytime and nighttime temperature, relative humidity and HI.Source Data Extended Data Table 2PMV values by study arm.Source Data Extended Data Table 3Counts of A. funestus and Culex species before and after modification by study arm.Rights and permissions
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    Reprints and permissionsAbout this articleCite this articleAbong’o, B., Kwaro, D., Bange, T. et al. Housing modifications for heat adaptation, thermal comfort and malaria vector control in rural African settlements.
    Nat Med (2026). https://doi.org/10.1038/s41591-025-04104-9Download citationReceived: 06 August 2024Accepted: 05 November 2025Published: 05 January 2026Version of record: 05 January 2026DOI: https://doi.org/10.1038/s41591-025-04104-9Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    The sexual reproductive strategy in clonal Scopolia carniolica is based on obligatory cross-pollination and self-incompatibility

    Abstract

    Understanding the reproductive biology and breeding system of rare species is crucial for effective conservation. We examined floral biology, assessed the true pollinators, and investigated the reproductive effect of six pollination treatments (spontaneous and induced autogamy, geitonogamy, spontaneous and supplemental cross-pollination, control – flowers exposed to natural pollinators) on the fruit and seed set and their quality (size, viability) in two populations (artificial and natural) of clonal Scopolia carniolica (Solanaceae), a rare species distributed across Central and South-Eastern Europe. Hermaphroditic flowers of S. carniolica represent movement herkogamy and incomplete protogyny. Pollinators are necessary for transferring pollen onto stigmas (there was no fruit and seed set after a test for spontaneous self-pollination), and the species shows the system of obligatorily xenogamy (only cross-pollination guarantees the production of seeds with fertile embryos ensuring viable offspring) and self-incompatibility (no seeds are produced after within-flower self-pollination). Although fruits can be developed through geitonogamous self-pollination, this is associated with late-acting self-incompatibility because the seeds produced are non-viable (contain no embryos and endosperm). Bumble bees are the most effective pollinators; honeybees also contribute to pollen deposition. Conserving pollinator diversity is essential for rare clonal S. carniolica, sustaining reproduction, preventing inbreeding depression, and supporting long-term population viability.

    IntroductionStudies of reproductive biology and pollination ecology are essential for predicting plant population survival1. The results can help to identify factors disturbing the reproduction of individuals and may be crucial for the implementation of conservation programs aimed at maintaining or restoring rare, narrowly distributed, or endangered populations2,3.Flowering plants exhibit a wide array of floral adaptations and reproductive strategies that have an impact on their fitness, genetic makeup, and population dynamics4,5. Most angiosperm species (ca. 78% in the temperate-zone and 94% in the tropical zone) rely on insect vectors for pollen transfer to mediate the pollination process6. However, their mating systems can vary widely – from autonomous selfing (self-pollination without external assistance) via mixed mating systems (combining sexual and asexual reproduction or involving selfing and outcrossing) to obligate outcrossing (relying entirely on cross-pollination)7,8,9. In general, cross-pollination is considered more advantageous than self-pollination due to a higher proportion of heterozygous individuals and increased genetic diversity of offspring8,10. Nevertheless, reproductive assurance is an important selective force that can strengthen self-fertilization in order to secure offspring production in environmental conditions non-conducive for cross-fertilization, e.g. a lack of compatible mates or pollinator limitation9,10.A large proportion of angiosperm flowers are hermaphroditic, with the potential for self-pollination11. Self-pollination may take place within a flower (autogamy – autonomous or vector-mediated) or between flowers (geitonogamy) of the same individual12 and references therein). Despite the possibility for self-pollination, a large number of species have evolved an extraordinary variety of adaptive mechanisms, e.g. spatial and temporal separation of male and female function or separation of male and female sex organs onto different individuals in order to force transfer of cross-pollen and ensure cross-fertilization13,14,15,16.The only strategy that guarantees full protection against self-fertilization is the self-incompatibility (SI) system17,18. The self-incompatibility phenomenon is regarded as an effective mechanism for preventing inbreeding depression9. There are two main types of self-incompatibility, i.e. gametophytic (GSI) and sporophytic (SSI)19,20. In the Solanaceae family, the self-incompatibility mechanism is primary gametophytic (GSI), which is based on the interaction between proteins present in the pollen and in the pistil21,22,23. Less commonly, the late-acting self-incompatibility phenomenon occurs in other Solanaceae plants, in which the incompatibility reaction is delayed and arises after fertilization24,25. The degree of self-incompatibility can vary not only between species but also within a single species (among populations) or even individual plants12,26. This variability includes species being polymorphic for SI, with some populations being self-incompatible (SI) and others self-compatible (SC), and individual plants potentially transitioning from SI to SC over their lifespan27,28.Presently, there is evidence that a majority of small angiosperm populations occurring in an altered environment (e.g. ecological conditions and pollinator assemblages have been modified by anthropogenic disturbance or climate change) are subjected to pollen limitation resulting from a lack of pollinators29,30. Therefore, well-organized conservation strategies require information on plant-pollinator interactions, which are fundamental for evaluation of the reproductive capacity of individuals and the capability of a population for maintenance and regeneration in both natural and human-changed ecosystems6,31.The genus Scopolia Jacq. includes three species; two species are distributed across Central and South-Eastern Europe and the Caucasus region and one occurs in Japan and the Korean Peninsula32. Among them, Scopolia carniolica is a rare species and has been entered in the IUCN Red List of Threatened Species and classified as LC (Least Concern)33. In Poland, after the latest regulation, S. carniolica is under partial protection34.Our previous research evidenced that S. carniolica flowers exhibit several traits that support entomophily: a large-size corolla, a visual guide that can serve as a signal pattern of floral rewards, abundant nectar and pollen attractants, the nectar composition (sucrose-dominant nectar), and a papillate wet stigma35.Despite the importance of conservation of the rare S. carniolica species, its reproductive biology is unknown. Therefore, the aim of the present study was to examine the aspects of floral morphology and biology as well as the breeding system of S. carniolica in two populations. Specifically, we (i) studied the period of stigma receptivity and timing of pollen release during the course of anthesis and determined pollen quantity and quality, (ii) compared the fruit and seed set in diverse pollination experiments, (iii) examined the pollination treatment effects on seed viability, (iv) tried to assess whether the species is pollen-limited, i.e. if its reproduction is restricted by insufficient pollination service, and (v) attempted to identify the effective pollinator(s) of the species by means of the effectiveness of pollen transfer and deposition onto the stigma.Materials and methodsStudy species Scopolia carniolica Jacq. (Solanaceae Juss.) is a perennial plant that develops vertical green above-ground stems (ca. 40–60 cm high) and underground rhizomes33,36. It prefers moist, shaded habitats in beech forests, especially on slopes and in valleys. Flowering occurs in early spring (from early April to early May). Its hermaphroditic flowers are set in leaf axils on relatively long pedicels36. The flowers are large (size = 20–25 mm in length; 7–10 mm in diameter) with campanulate brownish-violet corolla and the presence of nectar and pollen attractants37,38. Our previous study revealed that the flower life-span is ca. 3.3 days; sucrose-dominant nectar is secreted throughout the flower life-span, with a peak coinciding with the beginning of pollen release from the anthers. One flower produces ca. 1.67 ± 0.47 mg of nectar, 0.54 ± 0.22 mg of sugars, and 1.95 ± 0.56 mg of pollen35. The species was described as protogynous and insect-pollinated39. Bumble bee queens are reported as the main floral visitors35. Fertilization occurs one day after pollination40. The fruit is a capsule that matures at the end of May and beginning of June (unpublished own observations).Study locations and populationsThe study was carried out in two S. carniolica populations (artificial and natural) located at a distance of ca. 130 km away from each other (Fig. 1S a – b). The research in the artificial population was conducted in 2018 and 2019. The artificial population was located in the city of Lublin (L-population), SE Poland (51° 16’ N, 22° 30’ E, 200 m a.s.l.), within an area belonging to the Botanical Garden of Maria Curie-Skłodowska University. This population was established in 1975 and originated from natural sites in the Bieszczady Mountains (SE Poland): inventory number 877 from Zasan (49o23’N 22o25’E) and 2166 from Widełki (49o07’N 22o41’E). Each population covered an area of ca. 20 m2. The plants were grown on loess-origin soil. The surrounding vegetation consisted of species associated with the communities of the Fagetalia sylvaticae order (e.g. Fagus sylvatica L., Ranunculus cassubicus L., Symphytum cordatum L., Scilla bifolia L., Corydalis solida (L.) Clairv., Corydalis cava (L.) Schweigg. and Körte, Allium ursinum L., Galium odoratum (L.) Scop.).A similar experiment was performed in 2023 in a natural population located in Potoki Forestry Division (P-population), Tomaszow Lubelski Forest District, SE Poland (50°18’ N 23°30’ E). This population covered an area of ca. 60 m2 and occurred in the ground flora of typical Fagetum carpaticum forest (= Carpathian beech forest) composed predominantly of Pulmonaria obscura L., Anemone nemorosa L., Galium odoratum (L.) Scop., Ranunculus ficaria L., and Lathraea squamaria L. The soil was fertile brown typical for beech wood41.According to the Köppen classification42, both populations occur in the temperate climate zone (Dfb – warm-summer humid continental climate).Reproductive biology observations and pollen transfer effectivenessStigma receptivityThe receptivity of the stigma was assessed in stigmas dissected from buds and 1-, 2-, 3, and 4-day-old flowers. Entire gynoecia were excised from randomly selected flowers, placed on a microscope slide, and covered with a drop of 3% hydrogen peroxide (H₂O₂) to detect peroxidase activity. Stigmas that produced bubbles within 2–4 min were considered receptive43. The proportion of ‘bubbling stigmas’ in relation to all that were checked was determined (n = 20 stigmas for every point of flower life-span per year, i.e. 300 stigmas in total were checked, with 8–10 plants sampled from each population per year). These observations were conducted under a binocular stereomicroscope (NIKON SMZ-2B).Pollen quantity and qualityFlowers were selected randomly from at least six individuals and collected before pollen presentation at the bud stage (n = 20 flowers per population). All anthers from individual flowers were placed separately in Eppendorf tubes (2.0 ml) and, after pollen grains were released, 2 ml of distilled water was added. Then, anther tissues were removed thoroughly, and the pollen grains were counted in 20-µl samples using a hemocytometer (Bürker chamber) under a light microscope at 40× magnification. The results were expressed as the average number of pollen grains per 2 ml of suspension using the formula described by44, giving the number of pollen grains per flower.Pollen grain viability was determined in pollen collected during pollen presentation, i.e. in 2-, 3-, and 4-day-old flowers. At each time point, pollen was collected from n = 5 flowers originating from 2 to 3 plants. Pollen was stained with 2% acetocarmine and fixed in glycerogelatin). Pollen grains which stained red were considered viable, while deformed or unstained grains were regarded as non-viable. For each time point, 4 repetitions x 100 pollen grains were examined across different fields of view45.Number of ovulesA total of 40 flowers (n = 20 flowers per population) randomly selected from different individuals were examined for the number of ovules formed per ovary. The ovaries were cut lengthwise manually, and the ovules were counted under a stereoscopic microscope (Nikon SMZ-2B). The ovule number and pollen quantity data were used to assess the P/O – the pollen-to-ovule ratio was estimated according to Cruden7.Reproductive and breeding system studyPollination treatmentsThe flowers were marked at the bud stage (ca. 2–3 days prior to anthesis). Five flowers were labeled on three different days, i.e. in total 20 flowers were examined for each pollination treatment in each population. The flowers were selected randomly from at least six different plants.The following treatments were performed: (1) spontaneous self-pollination (= flowers enclosed in a fine mesh bag to prevent access by insect visitors) to evaluate the possibility of autogamy, (2) induced self-pollination (= obligate autogamy; flowers isolated and hand-pollinated within the same flower with self-pollen), (3) geitonogamy (flowers isolated and hand-pollinated with pollen collected from a different flower in the same individual plant); (4) open out-crossed pollination (non-manipulated flowers available to insects (= control) to demonstrate insect importance in pollination, (5) induced cross-pollination (xenogamy; buds were emasculated before supplemental manual pollination with fresh cross-pollen collected from different individual plants and then isolated from further insect visits), (6) supplemental cross-pollination (flowers hand-pollinated with pollen collected from different individual plants, uncovered for open-pollination) to check pollen limitation. The flowers were also tested for apomixis, (flowers were emasculated and then bagged with isolators; n = 10 flowers per year per population). Since S. carniolica is capable of vegetative propagation via rhizomes, pollen for cross-pollination originated from individual plants occurring > 15 m from pollen receptor individuals to ensure that it was derived from different genets.Fruit and seed collection and analysis of phenotypic featuresFruits obtained from the pollination treatments were harvested in the beginning of July prior to opening and the number of fruits in relation to pollinated flowers was established. In the laboratory, we determined (i) the fruit size based on their length measured with an electronic caliper (accuracy of 0.1 mm) – the measurements were made along the longitudinal axis from the capsule base to the top point, (ii) the number of seeds per each capsule, and (iii) the mass of 1,000 seeds.Seed viability was checked using a TTC test (2,3,5-triphenyl tetrazolium chloride test)46,47, and performed on seeds that were at the same stage of development. The main indicator of seed suitability for testing was a change in the capsule color from green to greenish-light brown and the presence of dark brown, hard, and dry seeds inside the capsule. The seeds (n = 40 per pollination treatment per year) were soaked in distilled water in a thermal chamber at a temperature of 20–25 °C for 24 h. Then, they were filtered and scarified, i.e. cut in such a way that the two halves (cotyledons) remained connected. Next, the seeds were flooded with a 0.1% solution of 2,3,5-triphenyltetrazolium chloride with distilled water and placed in a thermal chamber at 35 °C for 24 h. The seeds were filtered and then cut longitudinally. They were classified as either (i) viable – with the embryo evenly stained red or (ii) non-viable – with the embryo completely unstained or light pink or with the absence of an embryo and endosperm. Seeds were viewed under a stereoscopic microscope.Pollen transfer and deposition effectivenessPollen transfer and pollen deposition experiments were conducted in 2018 in the L-population and in 2023 in the P-population. To determine the ability of insect pollinators to transfer S. carniolica pollen, the method described in detail by Zych48 was applied. Briefly, we captured flower-visiting insects using an entomological net and carefully removed the pollen from their bodies and transferred it onto a microscope slide (stained with a 2% acetocarmine, fixed in glycerogelatin). The number of pollen grains of S. carniolica and ‘other’ species was counted. The visiting insects were assigned to the following groups: Bombus spp., Apis mellifera, and solitary bees (n = 3–12 individuals were examined for each group of insects).To establish the effectiveness of a pollinator in depositing pollen onto the stigma, we determined the quantity and purity of the pollen load deposited during a single visit. First, we randomly selected flowers at the bud stage (n = 24 in the L-population and n = 9 in P-population) from different plants. The flowers were emasculated and enclosed in tulle isolators to exclude stigma contamination. Then, after the flower opened, the isolator was carefully removed and the insect was allowed to visit the flower once. After this single visit, the flower was re-isolated and labeled, indicating the type of the visiting insect. On the next day, the stigmas were removed from the experimental flowers with clean forceps and microscopic slides were made (stained with 2% acetocarmine, fixed in glycerogelatin)49. The number of S. carniolica pollen grains and pollen of ‘other’ species was counted in each sample. Control flowers were subjected to identical experimental conditions but were excluded from insect visitation (n = 10 per population). These observations were made using a light microscope (Nikon Eclipse E200; Tokyo, Japan).Statistical analysesAnalysis of variance (ANOVA) was used to test the significance of differences between mean values of the analyzed floral features (pollen viability, stigma receptivity between floral phases) and pollen production between the populations; prior to performing any tests, the data were checked for normality (Shapiro-Wilk test). If the data were not normally distributed, the log transformation (ln) was applied.A generalized linear model (GLM) was used to assess the effects of population and pollination treatment on fruit set and number of seeds produced per capsule. For percentage of set fruits, a beta regression model was used to analyze continuous response variables bounded within the unit interval (0,1). The model was fitted using the betareg package in R, employing a logit link function to map the percentage data onto the real line. For the seed number (response variable), a negative binomial (NB) regression model was used to account for overdispersion in the count data. The explanatory variables included year, population, and treatment. A log link function was applied to relate the expected seed count to the linear predictor. The model was fitted using the glm.nb() function from the MASS package in R (v4.3.150).A generalized linear model (GLM) with a Gaussian distribution and a log link function was applied to assess the effects of explanatory variables (pollination treatment and population) on fruit length and seed weight. Seed viability (proportion of viable seeds per fruit) was analyzed using a beta regression with a logit link (betareg package, R), which is appropriate for modeling proportional data bounded between 0 and 1.The generalized linear models were built using the R programming language (R Core Team, 2023) in R Studio, version 1.3.1093 (PBC).The Kruskal–Wallis test was used to analyze the number of pollen grains transported on insect bodies and deposited on the stigma. Statistica 13.3 software (Statsoft, Kraków, Poland) was applied for these calculations.ResultsReproductive biology: flower featuresFlowering of Scopolia carniolica occurs in early spring, with the timing varying between years. Depending on the year, flowering begins either in early April and extends to the first days of May, or from mid-April to the beginning of May (Fig. 2S). The species has hermaphroditic flowers. Initially, in the buds and 1-day-old flowers, the pistil is positioned above the anthers, but later, the anther-stigma distance shortened during anthesis and the stigma was placed almost at the level of the pollen-bearing anthers or slightly above the anthers (Fig. 1a-b). Stigma receptivity differed significantly over the flower life-span (F4,25 = 576.85, P < 0.001) (Fig. 1c-g). It started in the floral bud stage (8.5 ± 2.0% of receptive stigmas) and gradually increased, reaching a maximum on the 3rd day of anthesis (97.0 ± 2.0%), and lasted until the end of the flower life-span. Anther dehiscence began on the 2nd day of anthesis. The average pollen viability was high (79.9 ± 5.0%) and differed significantly across the pollen presentation time (F 2,21 = 181.48, P < 0.001); the highest viability was recorded on the 3rd day of anthesis (91.9 ± 3.0%), whereas the lowest value was obtained in the 4th day (72.8 ± 3.0%). From the second day until the end of the flower life-span, an overlapping period of both female and male functions was observed (Fig. 2).Fig. 1Macrophotographs of S. carniolica showing: (a) flower with stamens before pollen presentation and anthers clearly separated in a 1-day-old flower; (b) stigma placed slightly above the pollen presenting anthers in a 3-day-old flower, (c-g) stigma receptivity evaluated using the peroxidase test at various flower stages: (c) bud; (d) 1- day-old flower; (e) 2-day-old flower; (f) 3-day-old flower; (g) 4-day-old flower.Full size imageFig. 2Changes in stigma receptivity and pollen viability in S. carniolica. Values are means calculated across populations and study years. Whiskers represent standard errors. Different letters indicate statistically significant differences between flower ages (a-d for stigma receptivity; A-C for pollen viability) at p < 0.05, based on HSD Tukey’s test.Full size imageThe pollen productivity varied between the populations (F1,38 = 16.04, p = 0.0002). It amounted to 46,149.0 ± 16,334.0 pollen grains per flower in the L-population and was ca. 55% higher in the P-population (mean = 72,423.9 ± 23,468.2 pollen grains per flower). The number of ovules per ovary was similar in both populations (p > 0.05; 43.2 ± 13.4 in the L-population and 45.5 ± 12.6 in the P-population). The average calculated P/O ratio was 1102.1 ± 303.1 in the L-population and 1692.5 ± 677.8 in the P-population.Fruit and seed setApomixis was not identified in S. carniolica. Pollination treatment had a strong effect on the reproductive performance of the species (beta regression, logit link; Fig. 3a). Neither population nor year significantly influenced fruit set. No fruits developed when insect vectors were excluded (spontaneous self-pollination, treatment 1), indicating the absence of autonomous within-flower self-pollination (Table 1). The highest fruits set were recorded in the natural and induced cross-pollination treatments (treatments 4, 5; estimate = −0.1845, odds ratio [OR] = 0.83, p > 0.05). Relative to open cross-pollination (GLM reference treatment 4), induced self-pollination (treatment 2) resulted in a 90% reduction (OR = 0.10, p < 0.001) while geitonogamous pollination (treatment 3) showed a 64% reduction (OR = 0.363, p < 0.001) in fruit set. Supplemental cross-pollination (treatment 6) reduced fruit set by approximately 61% (OR = 0.39, p < 0.001) (Tabel 1 S).Table 1 Effects of population and pollination treatment on fruit set in S. carniolica analyzed using a beta regression GLM with a logit link function. Estimates are expressed relative to the cross- pollination (treatment 4), which served as the reference category.Full size tableNo seeds were produced after within-flower self-pollination and induced self-pollination, indicating self-incompatibility (Fig. 3b). Seed production was similar between open and induced cross-pollination, (multiplicative effect = 0.84–0.90, p > 0.05; Table 2). Geitonogamy (treatment 3) resulted in a 37–46% reduction in seed number compared with open cross-pollination (multiplicative effect = 0.54–0.63, p < 0.01). However, the seeds formed were non-functional. Supplemental cross-pollination (treatment 6) reduced strongly seed number by 39–49% (multiplicative effect = 0.51–0.61, p < 0.001) compared to open cross-pollination. Population origin also influenced seed set, with plants from P-population producing approximately 31% fewer seeds than those from L-population (multiplicative effect = 0.69, p < 0.001). Year had no detectable effect on seed production in L-population (Table S2).Fig. 3Average fruit set (a) and seed number per fruit (b) in S. carniolica subjected to different pollination treatments: 1 – spontaneous self-pollination, 2-induced self-pollination, 3 – geitonogamous pollination, 4 – open out-crossed pollination, 5 – induced cross-pollination (xenogamy), 6 – supplemental cross-pollination. Significance codes: *** p < 0.001, ** p < 0.01, ns = not significant, based on generalized linear models (GLMs). Note: no fruit or seeds were produced in spontaneous self-pollination (treatment 1) or induced self- pollination (treatment 2); therefore, values for these treatments are not available.Full size imageTable 2 Effects of population and pollination treatment on seed number in S. carniolica analyzed using a negative binomial generalized linear model (GLM). The model used a log link function. Significant negative coefficients indicate reduced seed set to the cross-pollination (treatment 4), which served as the reference category.Full size tableFruit and seed qualityPopulation had no significant effect on any of the measured fruit and seed quality traits (fruit length: p = 0.086; seed weight: p = 0.612; seed viability: p = 0.518; full model outputs are provided in Supplementary materials; Tables S3-S5). Therefore, subsequent summaries focused on the effects of pollination treatments, which strongly influenced fruit and seed traits (Table 3). Overall, open cross-pollination maintained the highest fruit and seed quality. The largest fruits were produced in the natural and induced cross-pollination groups (treatments 4, 5, Fig. 4). In contrast, induced self-pollination reduced fruit length by 38.3% (multiplicative effect = 0.62, p < 0.001), geitonogamous pollination by 18.5% (0.82, p < 0.001), and supplemental cross-pollination by 15.0% (0.85, p < 0.01).Table 3 Effects of different pollination treatments on fruit length, 1000-seed weight, and seed viability in S. carniolica, relative to open cross-pollination. Estimates from generalized linear models (Gaussian family; log link) and beta regression models (logit link) are shown. Multiplicative effects (exp[Estimate]) and approximate percent changes relative to open pollination are provided for fruit length and seed weight. Seed viability estimates are presented on the logit scale of the beta regression model.Full size tableFor seed weight, geitonogamous fruits produced seeds about 62% lighter than those from open cross-pollination (estimate = 0.38, p < 0.001), whereas induced and supplemental cross-pollination treatments did not differ significantly from open cross-pollination (p > 0.05).The seeds developed in the cross-pollination treatment (treatments 4–6) were fully formed, i.e. they had well-developed embryos and endosperm (Fig. 4). Beta regression analysis revealed that the seed viability did not differ significantly between induced and open cross-pollination (p < 0.05), whereas supplemental cross-pollination significantly reduced seed viability, compared to cross-pollination (p < 0.001; Tables 3 and 5 S). The seeds produced in the geitonogamous pollination treatment were non-viable, as they had no embryo and no endosperm.Fig. 4Appearance of fruits and seeds developed after different pollination treatments in S. carniolica: 1 – spontaneous self-pollination, 2 – induced self-pollination, 3 – geitonogamous pollination, 4 – open out- crossed pollination, 5 – induced cross-pollination (xenogamy), 6 supplemental cross-pollination. Seed viability was assessed using the TTC test. The symbol X indicates absence of fruit or seed set.Full size imagePollen transfer and deposition effectivenessAll investigated insect visitors carried the pollen of S. carniolica and ‘other’ plant species. We found differences in the number of pollen grains of both Scopolia (Kruskal–Wallis test: H (2, N = 33) = 7.28, P = 0.026) and ‘other’ species (H (2, N = 33) = 8.02, P = 0.018) carried by the various insect groups. The number of conspecific pollen grains carried by Bombus species (mean = 551.06 ± 379.79, range 8.0–904.0, n = 15) was statistically similar to that carried by Apis mellifera (mean = 323.8 ± 367.7, range 3.0–1405.0, n = 11; Fig. 5a). Solitary bees transferred the smallest amount of S. carniolica pollen grains on their bodies (mean = 82.8 ± 70.9, range 4.0–194.0, n = 7). The pollen loads carried by bumble bees were the most diverse with a high share of pollen of ‘other‘ plants. No ‘other’ pollen grains were noted on the S. carniolica stigmas.The number of pollen grains deposited on the stigma per single visit also differed between the insect groups (H (2, N = 33) = 20.09, P = 0.0001). The highest amount of cognate pollen on the stigma during a single visit (mean = 413.9 ± 354.4, range 7.0–886.0, n = 15; Fig. 5b) was deposited by bumble bees, revealing their greatest effectiveness. Significantly less pollen was left by Apis mellifera (mean = 60.9 ± 73.9, range 2.0–215.0, n = 11), whereas only 2.1 ± 1.4 pollen grains (range 1.0–5.0, n = 7) were found to be loaded by solitary bees.Fig. 5The number of S. carniolica pollen grains carried by three insect groups (Bombus spp., Apis mellifera, and solitary bees): (a) on insects’ body, (b) deposited on stigma. The mark within each box indicates the median; box boundaries represent the 25th and 75th percentiles; whiskers indicate the range of non-outlier values; asterisks (*) mark extreme values. Values represent means across observations. Different letters indicate significant differences between groups according to the Kruskal-Wallis test (p < 0.05).Full size imageDiscussionOur study provides the first description of the floral traits related to reproductive biology and the breeding system in Scopolia carniolica. The floral morphology and reproductive biology of S. carniolica indicate that the species has evolved mechanisms promoting cross-pollination, while maintaining the potential for self-pollination. The species is strongly self-incompatible and strictly dependent on insect-mediated cross-pollination, as self-pollination and pollinator exclusion produced no fruits or viable seeds. Natural and induced cross-pollination yielded the highest fruit and seed set, whereas within-flower self-pollination and geitonogamous pollination produced few or non-functional seeds, highlighting the critical role of pollinators in reproductive success. Population-level differences in seed production, suggesting that reproductive success across populations may be subject to limitations (local pollinator activity, compatible pollen availability, pollen quality, timing of pollination). However, for this rare species, conserving pollinator communities (mainly bumblebees), maintaining population connectivity, and preventing habitat fragmentation are critical to ensure effective cross-pollination and long-term survival.Floral traits: support of both cross- and self-pollination Scopolia carniolica flowers have evolved morphological and biological characteristics that encourage cross-pollination but still carry the risk of self-pollination. We observed the changes in the position of the stigma in relation to the anthers, i.e. a phenomenon described by Boucher et al.51 as movement herkogamy. This phenomenon was reported in Solanaceae species, e.g. in Solanum lycopersicum52. Herkogamy has long been regarded as an important floral trait that plays an advantageous role in increasing outcrossing rates and reducing self-pollination53,54. In S. carniolica, the shortening of the anther-pistil distance was observed during anthesis – the stigma reached the level of pollen-presenting anthers or was located slightly above the anthers in the 2-day-old flowers, which might increase the likelihood of automatic self-pollination.In particular, we documented incomplete protogyny at the intrafloral level based on the criteria of Lloyd and Webb54. Knuth39 described S. carniolica flowers as protogynous, but the study did not consider the receptivity of stigmas. We found that the stigma became receptive before anther dehiscence and remained functional throughout pollen presentation up to the end of anthesis. In general, such an overlapping of sexual functions might allow within-flower self-pollination8,54,55. However, even if self-pollen was deposited on the stigma (due to the anther-stigma position and overlapping stigma receptivity with pollen presentation), no seed production was recorded after a treatment which allowed for spontaneous within-flower self-pollination (treatment 1), indicating that S. carniolica relies on outcrossing for reproduction and the presence of self-incompatibility phenomena that prevent self-fertilization.At the population level, the protogyny in S. carniolica is ‘asynchronous’ – flowers in the female and male sexual phases are present at the same time in multiple ramets per individual genets, which allows geitonogamous pollination (e.g56,57,58,59). The promotion of geitonogamy by ‘asynchronous protogyny’ was demonstrated e.g. in species from the genus Scrophularia, Scrophulariaceae60,61. Indeed, in S. carniolica, the seeds developed after geitonogamy were non-viable as they formed no embryos and endosperm, indicating the presence of late-acting self-incompatibility.Breeding system: obligatory out-crossing with self-incompatibilityWe analyzed various traits in order to gain broader knowledge of the plant mating system, i.e. in addition to the fruit set and size, we evaluated the degree of the seed set and estimated the ability of seeds to express vital function by measuring the metabolic activity of the embryo (63,64). However, the seed viability results assessed with the TTC test (2,3,5-triphenyl tetrazolium chloride test) have to be considered as a ‘potential’ for seedling establishment and do not indicate the seed and seedling performance in non-optimal environmental conditions for the species or its role in population stability62.The results of the pollination experiment evidenced that S. carniolica is unable to self-pollinate effectively but relies on pollinators to ensure fertilization and successful seed and fruit development, as no fruits and seeds were set when insects were excluded. The floral attributes (e.g. showy corolla, presence of primary attractants, predominance of sucrose in nectar, changes in nectar sugar composition) described in our previous complementary article35 are in concordance with the idea of entomophily in this species39,40. Another feature of our study species that is crucial for successful sexual reproduction is cross-pollination. Specifically, this assumption is supported by the highest fruit and seed set noticed in the cross-pollinated flowers (both natural and induced). Likewise, only seeds produced by the cross-pollinated flowers had fully formed embryos, indicating the outcrossed nature of species and the benefits of cross-pollination for viable offspring. As shown by literature data, an obligatory cross-pollination mating system is a reproductive strategy where cross-pollination is essential for fertilization and seed production, which indicates that the species cannot reproduce effectively through self-pollination63,64,65,66. In fact, self-pollination in S. carniolica (whether autonomous or induced within-flower self-pollination) consistently fails to produce seeds, indicating strong outcrossing and self-incompatibility20,67,68,69,70. Self-incompatibility is further proved by the results of induced geitonogamous self-pollination, where fruits may develop but their seeds are non-viable and have no embryos and endosperm, consequently leading to infertile offspring. The production of non-viable embryos following geitonogamous pollination may indicate a system of late-acting self-incompatibility19,28,71. Late-acting self-incompatibility is widely described in angiosperms (e.g24). and has also been reported within the Solanaceae family, e.g. in Lycium cestroides72. It is a phenomenon where self-pollen tubes are allowed to reach the ovary and successfully penetrate the ovules, but the developing seeds are non-viable, leading to self-sterility10,73. Further research should include detailed histological analyses of post-pollination events on stigmas, pistils, and ovaries with self- and cross-pollen in order to identify where the self-incompatibility barrier actually occurs in S. carniolica and try to indicate a possible mechanism for controlling self-incompatibility at the molecular and genetic levels.Facultative xenogamy was indicated by the P/O ratio, as its value (1102.1 ± 303.1–1692.5 ± 677.8) falls within the range of 1,000–10,0007. However, the P/O ratio is based only on the ‘pollination success’ and does not analyze the offspring quality.Surprisingly, supplemental cross-pollination did not improve fruit set or seed production; instead, it considerably reduced them compared to naturally and experimentally cross-pollinated flowers in both studied populations. There are several possible causes of these unexpected results. It is possible that the amount of pollen applied during supplementation was too large (the stigmata received both insect-applied pollen, including self-pollen and hand-applied pollen) and the stigmatic surface was clogged, hence pollen germination was blocked and therefore many ovules were not fertilized. In fact, the presence of movement herkogamy (discussed above) in the flowers of S. carniolica may also potentially contribute to blocking the stigma with self-pollen. Research conducted by Opedal74 demonstrated that the reduced anther-pistil distance during movement herkogamy can be disadvantageous – it restricts stigma’s access to cross-pollen and increases self-pollen deposition, leading to stigma clogging and increased competition between self- and cross-pollen, possibly hindering cross-pollination. In consequence, it can negatively impact the seed set, particularly in self-incompatible species75. Other studies has also shown that excessive pollen coverage on the stigma hinders the fertilization process in Collinsia heterophylla (Plantaginaceae)76 and Silene alba (Caryophyllaceae)75,77, supporting the idea that over-pollination can negatively affect reproductive success. In S. carniolica, these mechanisms likely explain why supplemental cross-pollination reduced rather than improved fruit and seed set, highlighting the importance of pollen quality, timing, and stigma receptivity in this strongly outcrossed, self-incompatible species. These findings also raise the question of whether reproductive success in S. carniolica is truly limited by pollen quantity, or rather by the quality, compatibility, and timing of pollen transfer – a question addressed in the following section.Possibility of pollen limitationDespite the dependence on cross-pollination, our data indicate that natural pollination provides near-optimal reproductive outcomes. However, based on our data it is not possible to clearly state whether the species is pollen limited due to insufficient pollen amount on the stigmas. In strongly self-incompatible S. carniolica the disparity in seed number between open cross-pollination and supplemental cross-pollination may likely reflect pollen interference, where the added pollen, potentially incompatible, disrupted fertilization by naturally deposited pollen. These results indicate that S. carniolica reproduction may be dependent on the quality, source, and timing of pollen transfer, rather than the total amount of pollen received. While the species is entirely cross-pollen-dependent, natural pollination appears to provide an optimal balance for successful reproduction. Given that pollen viability fluctuates throughout the floral lifespan, reproductive output is likely constrained by both pollen compatibility and temporal variation in pollen viability. Literature data indicate that differences in the timing of pollination, whether occurring at different times of day, flower maturity stages, or across the flowering season, may influence reproductive outcomes by altering pollen transfer efficiency, pollen tube growth, or fertilization success28,78,79,80. In the Solanaceae, the timing of pollination is a key factor determining reproductive success. Both the time of day and the age of the flower may influence pollen viability, stigma receptivity, and the rate of pollen tube growth. In Solanum lycopersicum, pollination performed in the morning hours (07:00–09:00) resulted in higher fruit set, by approximately 18–25%, compared to pollination conducted at midday81,82. The disparity has been attributed to lower morning temperatures, which maintain higher pollen viability and accelerate pollen tube growth, thereby increasing the probability of successful fertilization. The age of the flower may also influence reproductive outcomes. In S. lycopersicum and Capsicum annuum, pollination on the day of anthesis resulted in high fruit-set rates (70–90%), whereas a 24-hour delay reduced fruit set to 30–60%83,84,85. An even more stranger effect has been observed in Nicotiana tabacum, where a one-day delay resulted in more than a 60% reduction in seed number78. These results indicate the existence of a narrow ‘time window’ during which stigma receptivity and pollen viability are optimal. Reproductive success may also be influenced by flowering phenology. For example, in Solanum sisymbriifolium, fruit set reaches ~ 70% at the start of the flowering season but falls below 35% toward the end, accompanied by fewer seeds per fruit80. This decline has been attributed to reduced pollinator activity and decreasing pollen quality as the season progresses. It seems that the observed differences in seed number among S. carniolica populations reflect not just pollen availability, but mainly variation in pollen quality, availability of compatible pollen, and timing of transfer. However, pollination success and subsequent fruit/seed development are not solely determined by pollinator behavior or abundance and pollen traits. Various pre- and post-pollination factors, including availability of resources, can also significantly impact the pollination/fertilization outcomes86. Furthermore, the outcrossing sexual reproduction may be limited by e.g. the characteristics of the population (when the population is too small and/or too uniform genetically) or intense competition for pollinator service among individual plants87,88.PollinatorsWe used two indicators (the number of pollen grains transferred on insect bodies and the number of pollen grains deposited on stigmas during a single visit) as a proxy for pollinator effectiveness. Bumble bees and solitary bees (mainly Andrena fulva) were indicated as pollinators of S. carniolica by Knuth39. We found that solitary bees deposited almost no pollen, demonstrating that they are not the true pollinators of our study species. Presumably, morphological and chemical adaptations in S. carniolica (e.g. numerous non-glandular trichomes on the inner surface of the corolla and the presence of various chemical compounds in corolla cells reported by Tymoszuk et al.35 restricted the pollen deposition by short-tongued solitary bees. Indeed, morphological and chemical adaptations, alongside pollinator behavior, play a crucial role in controlling access to floral rewards and sexual organs, ultimately influencing pollinator effectiveness89.Our research evidenced that bumble bees deposited substantially more pollen to S. carniolica flowers than is needed for fertilization of all ovules (ca. 9-fold, on average), while honey bees loaded slightly higher amounts (ca. 1.5-fold, on average) than required. Therefore, it seems that both pollinator types can provide sufficient pollination, which highlights their importance in the reproductive success of S. carniolica, even if honey bees might be less efficient. However, adequate pollination of individual flowers does not guarantee overall pollination success at the population level. As reported by many authors, even if the average pollen load is large enough to fertilize all ovules, some flowers may still experience insufficient pollination and fail to set al.l possible seeds (e.g16,90). Given the predicted decline of bumblebee populations in Europe87,88, the self-incompatibility of S. carniolica may potentially accelerating population declines and leading to a contraction of its geographic range.We are aware of several limitations of this study. First, experiments were conducted in only two populations (artificial vs. natural), and observations were temporally separated, occurring in different years for each population. This was partly due to challenges in locating sufficiently large natural populations. Second, pollination treatments in the natural population were restricted to a single year because of permit limitations, which may not fully capture interannual variability in sexual reproduction, such as fluctuations in seed production. Third, seed quality was assessed solely using the tetrazolium test. While this test provides a useful measure of embryo viability, it does not fully reflect germination potential under natural conditions. Future studies should address these limitations by examining the biological and genetic basis of self-incompatibility in S. carniolica and validating pollination results across multiple populations to better assess the role of pollen limitation in reproductive success. Additionally, multi-year observations and complementary seed quality assessments, such as germination trials under variable environmental conditions, would provide a more comprehensive understanding of the factors regulating reproduction in this species.In summary, reproductive success in the clonal, self-incompatible, and strongly outcrossed S. carniolica depends on pollen quality, compatibility, and timing rather than quantity. Natural pollination provides near-optimal outcomes, while excessive or incompatible pollen can hinder fertilization. Bumble bees and honey bees are the main effective pollinators, highlighting their key role in reproduction. In this context, the conservation of S. carniolica should focus on maintaining abundant and diverse pollinator communities ensuring genetic diversity and spatial mixing of clones. These factors are crucial to sustain viable populations and promote long-term reproductive success in this species.

    Data availability

    The raw datasets used during the current study are available from the corresponding authors on reasonable request.
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    Download referencesAcknowledgementsWe thank Grazyna Szymczak, PhD, the Director of the Botanical Garden in Lublin for providing access to the S. carniolica population. Our thanks also go to Mykhaylo Chernetskyy, PhD, Jacek Jachuła, PhD, Monika Strzałkowska-Abramek PhD, and Hubert Rydzewski, MSc for their invaluable support during the field observations. We are thankful to the three anonymous reviewers for their valuable comments, which significantly improved the paper.FundingThis research received no external funding.Author informationAuthors and AffiliationsDepartment of Botany, Mycology and Ecology, Institute of Biological Sciences, Maria Curie- Sklodowska University, 19 Akademicka St, 20-033, Lublin, PolandKarolina Tymoszuk-RydzewskaDepartment of Botany and Plant Physiology, University of Life Sciences in Lublin, 15 Akademicka St, 20-950, Lublin, PolandBożena Denisow & Ernest StawiarzDepartment of Applied Mathematics and Computer Sciences, University of Life Sciences in Lublin, 28 Głęboka St, 20-612, Lublin, PolandJacek MielniczukAuthorsKarolina Tymoszuk-RydzewskaView author publicationsSearch author on:PubMed Google ScholarBożena DenisowView author publicationsSearch author on:PubMed Google ScholarJacek MielniczukView author publicationsSearch author on:PubMed Google ScholarErnest StawiarzView author publicationsSearch author on:PubMed Google ScholarContributionsK.T., B.D. – conceptualized the study; K.T. – conducted the field observations; K.T., B.D. – performed the laboratory procedures; E.S. – performed pollen analyses; K.T., J.M., B.D. – performed the statistical analyses; K.T. – took the photographs; K.T. – designed the tables and figures; K.T., B.D. – wrote the main manuscript text; B.D. – supervised the study. All authors reviewed the manuscript.Corresponding authorsCorrespondence to
    Karolina Tymoszuk-Rydzewska or Bożena Denisow.Ethics declarations

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    Reprints and permissionsAbout this articleCite this articleTymoszuk-Rydzewska, K., Denisow, B., Mielniczuk, J. et al. The sexual reproductive strategy in clonal Scopolia carniolica is based on obligatory cross-pollination and self-incompatibility.
    Sci Rep 16, 302 (2026). https://doi.org/10.1038/s41598-025-29571-5Download citationReceived: 27 August 2025Accepted: 18 November 2025Published: 05 January 2026Version of record: 05 January 2026DOI: https://doi.org/10.1038/s41598-025-29571-5Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    KeywordsIncomplete protogynyGeitonogamyFruit setSeed setSeed viabilityBumble beesP/O ratio More

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    Quantitative assessment of ecological security and its influencing factors in the Danjiangkou Reservoir based on a health–risk–service framework

    AbstractThe Danjiangkou Reservoir (DR) serves as the primary water source for the Middle Route of China’s South-to-North Water Diversion Project (SNWDP), and its ecological security (ES) is critical to water supply safety in North China and to broader regional sustainability. However, systematic assessments of ES in the DR region remain limited. In this study, a health–risk–service framework was developed to evaluate the evolution of the ecological security in DR across three benchmark years (2003, 2013, and 2023). Furthermore, the XGBoost–SHAP model was employed to uncover the dominant natural, anthropogenic, and landscape influential factors behind ES variation. The results indicate that: (1) The proposed framework effectively captures the ES status of DR, with a strong correlation between ecological security index (ESI) and remote sensing ecological index (RSEI) (R² > 0.8, P < 0.001); (2) ESI exhibited a fluctuating upward trend over time, with over 95% of the area classified as Medium or above in terms of ecological security. The ESI hotspots were primarily distributed in the northern and southern regions, which are dominated by forest cover, whereas the cold spots were mainly concentrated in the central region, characterized by cropland and built-up land; (3) Results from the XGBoost–SHAP model revealed that ESI is influenced by multiple factors in a nonlinear fashion. NDVI and LPI were the primary positive contributors, whereas HDI and urbanization had negative impacts, with all these relationships exhibiting nonlinear threshold effects. Notably, threshold effects were identified within specific ranges of these variables. This framework provides a practical approach for evaluating ESI in reservoir regions and offers a scientific foundation for ecological protection and source-area ecological security management in cross-basin water diversion projects such as the DR.

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    Download referencesFundingThis study was supported by the National Key Research and Development Program of China (Grant No. 2022YFF0711601); the Opening Fund of Key Laboratory of Geological Survey and Evaluation of Ministry of Education (Grant No. GLAB2022ZR01) and the Fundamental Research Funds for the Central Universities; the Programs national natural science foundation of China (42471475).Author informationAuthors and AffiliationsSchool of Geography and Information Engineering, China University of Geosciences, Wuhan, 430074, ChinaYinghui Chang & Liang WuSchool of Computer Science, China University of Geosciences, Wuhan, 430074, ChinaLiang Wu, Zhanlong Chen & Chuncheng YangAuthorsYinghui ChangView author publicationsSearch author on:PubMed Google ScholarLiang WuView author publicationsSearch author on:PubMed Google ScholarZhanlong ChenView author publicationsSearch author on:PubMed Google ScholarChuncheng YangView author publicationsSearch author on:PubMed Google ScholarContributionsY.C. (Yinghui Chang) contributed to the study design and wrote the manuscript; L.W. (Liang Wu) discussed the original idea, revised the manuscript; Z.C. (Zhanlong Chen) and C.Y. (Chuncheng Yang) were involved in drafting and checking of the manuscript. All authors contributed to the article and approved the submitted version.Corresponding authorCorrespondence to
    Liang Wu.Ethics declarations

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    Reprints and permissionsAbout this articleCite this articleChang, Y., Wu, L., Chen, Z. et al. Quantitative assessment of ecological security and its influencing factors in the Danjiangkou Reservoir based on a health–risk–service framework.
    Sci Rep (2026). https://doi.org/10.1038/s41598-025-34039-7Download citationReceived: 19 October 2025Accepted: 24 December 2025Published: 05 January 2026DOI: https://doi.org/10.1038/s41598-025-34039-7Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    KeywordsEcosystem securityAssessment systemDriving factorsXGBoost-SHAPDanjiangkou Reservoir More

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    Phage-mediated resistome dynamics in global aquifers

    AbstractWhile mobile genetic elements (MGEs) critically influence antibiotic resistance gene (ARG) dissemination, the regulatory role of bacteriophages as unique MGEs remains enigmatic in natural ecosystems. Through a global-scale phage-resistome interrogation spanning 840 groundwater metagenomes, we established a large aquifer resistome repository and uncovered three paradigm-shifting discoveries. First, phages harboured markedly fewer ARGs compared to plasmids and integrative elements, but their bacterial hosts paradoxically maintained the highest anti-phage defence gene inventories, showing an evolutionary equilibrium where investment in phage defence constrains ARG acquisition. Second, lytic phages demonstrated dual functionality characterized with directly suppressing ARG transmission through host lysis while indirectly enriching defence genes that inhibit horizontal gene transfer. Third, vertical inheritance sustained ARGs in 11.2% of MGE-free groundwater microbes. We further extended linkages between ARG profiles, phage defences and biogeochemical genes, revealing phage-mediated co-occurrence of ARGs and denitrification genes in shared hosts. These findings pioneer a phage-centric framework for resistome evolution, guiding phage-based ARG mitigation in groundwater ecosystems.

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    Fig. 1: A global atlas of groundwater resistomes reveals expansive ARG landscapes.Fig. 2: MGE-mediated ARG mobilization patterns in global groundwater.Fig. 3: Interplay between phage infection, host defence systems and ARG dissemination.Fig. 4: Vertical gene transfer sustains ARG persistence in aquifer microbiomes.Fig. 5: Phage–host dynamics link microbial resistance, defence and aquifer ecosystem functions.

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    Data availability

    Domestic groundwater data generated for this study have been deposited in the NCBI Sequence Read Archive under accession code PRJNA858913. Publicly available groundwater metagenomes are listed with their BioProject accession numbers in Supplementary Table 2. Source data are provided with the paper.
    Code availability

    The R scripts used are publicly available via Zenodo at https://doi.org/10.5281/zenodo.17540538 (ref. 76).
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    Cao, H. Phage-mediated resistome dynamics in global aquifers. Zenodo https://doi.org/10.5281/zenodo.17540538 (2025).Download referencesAcknowledgementsThis work was supported by the National Natural Science Foundation of China (grant numbers U2240205 and 51721006 to J.R.N.).Author informationAuthor notesJinren NiPresent address: College of Environmental Sciences and Engineering, Peking University, Beijing, P. R. ChinaAuthors and AffiliationsEco-environment and Resource Efficiency Research Laboratory, School of Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen, People’s Republic of ChinaHuaiyu Cao, Songfeng Liu, Jiawen Wang & Jinren NiEnvironmental Microbiome and Innovative Genomics Laboratory, College of Environmental Sciences and Engineering, Peking University, Beijing, People’s Republic of ChinaHuaiyu Cao, Pinggui Cai & Pengwei LiEnvironmental Protection Key Laboratory of All Material Fluxes in River Ecosystems, Ministry of Ecology and Environment, Beijing, People’s Republic of ChinaPengwei Li & Jinren NiAuthorsHuaiyu CaoView author publicationsSearch author on:PubMed Google ScholarSongfeng LiuView author publicationsSearch author on:PubMed Google ScholarPinggui CaiView author publicationsSearch author on:PubMed Google ScholarPengwei LiView author publicationsSearch author on:PubMed Google ScholarJiawen WangView author publicationsSearch author on:PubMed Google ScholarJinren NiView author publicationsSearch author on:PubMed Google ScholarContributionsJ.R.N. designed the research. H.Y.C., S.F.L. and P.G.C. conducted the statistical analysis with help of P.W.L. and J.W.W. H.Y.C. and J.R.N. wrote the paper. All the authors read and approved the final paper.Corresponding authorCorrespondence to
    Jinren Ni.Ethics declarations

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    The authors declare no competing interests.

    Peer review

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    Nature Water thanks Liping Ma, Yanni Sun and Pingfeng Yu for their contribution to the peer review of this work.

    Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Composition and geographic distribution of antibiotic resistance genes (ARGs) across global aquifer metagenomes.a. Total abundance and prevalence of ARGs detected in all metagenomes. The majority of ARGs (6,935 of 9,681) are sparsely distributed, appearing in < 10% of samples (peripheral ARGs); whereas a core set of 392 ARGs occurs in > 75% of samples (core ARGs). b. Relative abundance of ARG types based on read-level profiling; MLS: macrolide-lincosamide-streptogramin. c. Upset plot showing ARG subtypes uniquely detected in specific continental combinations. d. Composition of ARGs by resistance mechanisms based on MAG-level analysis, reflecting host-associated ARG preferences.Source dataExtended Data Fig. 2 Characterization of mobile genetic elements (MGEs) associated with transferable ARGs in groundwater.a. Functional composition of annotated MGEs linked to ARGs in groundwater metagenomes. b. Distribution of transferable ARGs across different MGE types. Transferable ARGs were categorized as MGE-single if they were associated with only one type of MGE, and as MGE-multi if detected in association with more than one type of MGE. c. MGE repertoire associated with the most pervasive transferable ARGs (detected in > 100 MGE sequences). For each ARG, both the number and diversity of linked MGEs are shown.Source dataExtended Data Fig. 3 Distribution and characteristics of bacterial defence systems in groundwater microbiomes.a. Composition of defence gene families identified across all groundwater MAGs. b. Distribution of defence-encoding genomes across the ten most represented bacterial phyla. Bars indicate the proportion of genomes carrying defence systems, with the total number of unique DGs per phylum shown alongside. c. Defence gene (DG) counts in MAGs predicted to be phage-susceptible (P-phage, n = 1,458) versus those without phage-linked contigs (NP-phage, n = 1,452). Each point represents the number of DGs in an individual host genome. Statistical significance was evaluated using a two-sided Wilcoxon rank-sum test (p < 2 × 10−16). d. DG counts in L-phage (n = 908) versus NL-phage (n = 376) hosts. Each point represents an individual host genome. Box plots indicate the median (center line and red point), interquartile range (box), and 1.5 × the interquartile range (whiskers). Statistical significance was determined using a two-sided Wilcoxon rank-sum test. e. Number of ARG types associated with NP-phage, NL-phage, and L-phage hosts.Source dataExtended Data Fig. 4 Defence system distribution between P-phage and NP-phage MAGs across aquatic ecosystems (freshwater, marine, wastewater).P-phage MAGs consistently encoded more defence systems (DSs) than NP-phage MAGs, with significant differences detected across all ecosystems (two-sided Wilcoxon rank-sum test; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). The magnitude of phage–host antagonism decreased along the gradient from freshwater to marine and wastewater environments. Each point represents the number of DSs in an individual host genome. Box plots indicate the median (center line and red point), interquartile range (box), and 1.5 × the interquartile range (whiskers). Sample sizes and exact p values were as follows: Freshwater: NP-phage (n = 432) and P-phage (n = 249), p = 3.12 × 10⁻11; Marine: NP-phage (n = 252) and P-phage (n = 379), p = 6.82 × 10⁻4; Wastewater: NP-phage (n = 203) and P-phage (n = 210), p = 8.26 × 10⁻3.Source dataExtended Data Fig. 5 Vertical gene transfer sustains ARG persistence in aquifer microbiomes.Comparison between the MAG-based phylogenetic tree and the phylogeny of the rsmA resistance gene within two bacterial genera: 202FULL6113 (a) and JADFDG01 (b). Topological similarity between the two phylogenies was assessed using Robinson–Foulds (RF) distance.Source dataExtended Data Fig. 6 Assessment of vertical and horizontal contributions to ARG dissemination in groundwater microbial communities.Procrustes analysis at the phylum (a) and genus (b) levels compares microbial community composition (read-based) with ARG subtype profiles. Higher Procrustes m2 values indicate greater deviation from vertical inheritance, suggesting stronger influence of HGT on ARG distribution. Statistical significance was evaluated using a two-sided Procrustes permutation test (999 permutations; p < 0.001).Source dataExtended Data Fig. 7 Phage–host interactions shape microbial nitrogen cycling potential in aquifer ecosystems.a. Module completeness of functional gene markers associated with eight nitrogen cycling processes in P-phage (n = 1,458) and NP-phage (n = 1,452) genomes. Each bar represents the mean metabolic completeness for the corresponding nitrogen transformation process within each group, and error bars indicate the standard error of the mean (SEM). Statistical differences between groups were evaluated using a two-sided Wilcoxon rank-sum test; ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Exact p-values for A–H are: A, p = 1.36 × 10−2; B, p = 0.19; C, p = 8.37 × 10−7; D, p = 5.24 × 10−3; E, p = 2.39 × 10−3; F, p = 0.30; G, p = 2.73 × 10−12; H, p = 1.97 × 10−6. b. Contribution of lytic phages to nitrogen cycling in all phage-infected microbial hosts, illustrated by a metabolic pathway map highlighting their role in aquifer nitrogen transformations.Source dataSupplementary informationReporting SummarySupplementary TablesSupplementary Tables 1–14.Source dataSource Data Fig. 1Statistical source data.Source Data Fig. 2Statistical source data.Source Data Fig. 3Statistical source data.Source Data Fig. 4Statistical source dataSource Data Fig. 5Statistical source data.Source Data Extended Data Fig. 1Statistical source data.Source Data Extended Data Fig. 2Statistical source data.Source Data Extended Data Fig. 3Statistical source data.Source Data Extended Data Fig. 4Statistical source data.Source Data Extended Data Fig. 5Statistical source data.Source Data Extended Data Fig. 6Statistical source data.Source Data Extended Data Fig. 7Statistical source data.Rights and permissionsSpringer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Reprints and permissionsAbout this articleCite this articleCao, H., Liu, S., Cai, P. et al. Phage-mediated resistome dynamics in global aquifers.
    Nat Water (2026). https://doi.org/10.1038/s44221-025-00558-wDownload citationReceived: 01 July 2025Accepted: 11 November 2025Published: 05 January 2026Version of record: 05 January 2026DOI: https://doi.org/10.1038/s44221-025-00558-wShare this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    Integrating transformer-based learning and Sentinel-2 bare soil composites for soil organic carbon mapping in the black soil region of Northeast China

    AbstractAccurate assessment of soil organic carbon (SOC) is essential for sustainable cropland management and carbon sequestration monitoring. However, high-resolution SOC mapping remains challenging due to two persistent limitations: (1) the difficulty of extracting true bare-soil reflectance—especially when single-date imagery is used and spectral signals remain influenced by vegetation, residue, and soil moisture; and (2) reliance on models that require large training datasets and may underperform in typical small-sample soil survey settings. To address these challenges, we developed an approach that integrates multi-temporal Sentinel-2 bare-soil composites with a transformer-based foundation model—Tabular Prior-data Fitted Network (TabPFN)—for SOC prediction in the black soil region of Northeast China. Bare soil pixels were extracted using a Normalized Difference Vegetation Index threshold (0.1–0.4), and two compositing strategies—the 50th percentile (P50) and 90th percentile (P90)—were compared. We systematically evaluated three advanced algorithms: TabPFN, convolutional neural network (CNN), and Extreme Gradient Boosting (XGBoost). Results demonstrated that the TabPFN model coupled with P50 composites achieved the highest prediction accuracy (R2 = 0.78, RMSE = 1.90 g kg⁻1), outperforming CNN and XGBoost by 4–6%. TabPFN’s distinct advantage lies in its design as a prior-data fitted transformer, which enables robust generalization from limited samples (N = 174) without extensive hyperparameter tuning, effectively addressing the “small data” challenge pervasive in digital soil mapping. SHapley Additive exPlanations analysis indicated that shortwave infrared band (B12) and precipitation have the greatest effect on model output, indicating joint role of soil spectral response and climate variability. This is one of the first studies to apply the TabPFN architecture to SOC estimation, offering a novel, interpretable, and scalable workflow that bridges the gap between data scarcity and model complexity. The proposed framework provides a reliable tool for high-resolution SOC mapping in heterogeneous croplands, supporting precision agriculture and long-term carbon accounting initiatives.

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    The datasets analyzed during the current study are not publicly available due to existing agreements and data-use restrictions but are available from the corresponding author on reasonable request.
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    Download referencesFundingThis research was funded by the “Study on the Retrogressive Erosion Mechanism of Gully Heads with Different Parent Materials in Black Soil Regions of Low Mountains and Hills” project of Natural Science Foundation of Jilin Province, China (20250102200JC).Author informationAuthors and AffiliationsCollege of Economics and Management, Jilin Agricultural University, Changchun, 130118, ChinaNa Chen, Zhikang Wei, Ling Zhao & Song WuCollege of Earth Sciences, Jilin University, Changchun, 130061, ChinaXuancheng JinModern Industry College, Jilin Jianzhu University, Changchun, 130118, ChinaNan LinCollege of Resources and Environment, Jilin Agricultural University, Changchun, 130118, ChinaFan YangAuthorsNa ChenView author publicationsSearch author on:PubMed Google ScholarZhikang WeiView author publicationsSearch author on:PubMed Google ScholarXuancheng JinView author publicationsSearch author on:PubMed Google ScholarNan LinView author publicationsSearch author on:PubMed Google ScholarFan YangView author publicationsSearch author on:PubMed Google ScholarLing ZhaoView author publicationsSearch author on:PubMed Google ScholarSong WuView author publicationsSearch author on:PubMed Google ScholarContributionsConceptualization, Song Wu, Na Chen, and Zhikang Wei; methodology, Na Chen and Xuancheng Jin; software, Nan Lin; validation, Nan Lin; formal analysis, Zhikang Wei; resources, Ling Zhao and Na Chen; data curation, Xuancheng Jin; writing—original draft preparation, Song Wu, Zhikang Wei and Na Chen; writing—review and editing, Xuancheng Jin, Na Chen and Song Wu; visualization, Song Wu and Xuancheng Jin; supervision, Nan Lin; project administration, Song Wu; funding acquisition, Fan Yang. All authors have read and agreed to the published version of the manuscript.Corresponding authorCorrespondence to
    Song Wu.Ethics declarations

    Competing interests
    The authors declare no competing interests.

    Additional informationPublisher’s noteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary InformationSupplementary Information.Rights and permissions
    Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
    Reprints and permissionsAbout this articleCite this articleChen, N., Wei, Z., Jin, X. et al. Integrating transformer-based learning and Sentinel-2 bare soil composites for soil organic carbon mapping in the black soil region of Northeast China.
    Sci Rep (2026). https://doi.org/10.1038/s41598-025-33682-4Download citationReceived: 14 November 2025Accepted: 22 December 2025Published: 05 January 2026DOI: https://doi.org/10.1038/s41598-025-33682-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    KeywordsSoil organic carbonSentinel-2Digital soil mappingBare soil compositeTabPFNSHAP More

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    Assessing mining impacts in the deep sea

    New studies that document the effect of polymetallic nodule mining vehicles on deep-sea biodiversity suggest that keeping up with technological innovations will be key to more realistic impact assessments of deep-sea mining.

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    Fig. 1: Comparison of tracks from abyssal polymetallic nodule mining experiments and their respective disturbers or prototype collectors.

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    Competing interests
    The authors are involved in scientific research projects that are funded in part by prospective deep-sea mining companies, which has provided valuable insights into technical and operational aspects of test mining operations. These companies did not fund this publication, nor did they contribute to or have any influence on the findings or opinions expressed by the authors.

    Rights and permissionsReprints and permissionsAbout this articleCite this articleIngels, J., Leduc, D., Ullmann, A. et al. Assessing mining impacts in the deep sea.
    Nat Ecol Evol (2026). https://doi.org/10.1038/s41559-025-02965-4Download citationPublished: 05 January 2026Version of record: 05 January 2026DOI: https://doi.org/10.1038/s41559-025-02965-4Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    Multi-omics comparison of two emerging storage pests (Necrobia rufipes and Tribolium castaneum) of dried black soldier fly larvae product

    AbstractThe black soldier fly (BSF) larvae is a rich and promising source of alternative protein that continues to increasingly gain global traction as a functional ingredient for sustainable livestock and fish production. The key setback to postharvest processing of stored BSF larvae (BSFL) products is the significant damage caused by two notable storage pests (Tribolium castaneum and Necrobia rufipes). Here, we present a comparative analysis of the complete mitochondrial genomes and gut microbiome profiles of T. castaneum and N. rufipes. The study mitogenomes were similar in size and structure to other coleopteran mitogenomes. The gut microbiome profiles of the two pests showed a high abundance of bacteria in the Proteobacteria and Firmicutes phyla. However, T. castaneum had 78% more phyla represented within its microbiome than N. rufipes. The most abundant genera in T. castaneum were Staphylococcus and Streptococcus, while in N. rufipes, the dominant genera were Klebsiella and Synechococcus. We also identified the presence of potentially clinically harmful microbial genera (Stenotrophomonas maltophilia) in the gut of T. castaneum and N. rufipes in relatively high abundance. These results provide insight into potential harmful associations in the gut of the storage pest, picked from contaminated, poorly processed BSFL products.

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    Data availability

    All sequences generated in this study were deposited in the GenBank database ( [www.ncbi.nlm.nih.gov/genbank](http:/www.ncbi.nlm.nih.gov/genbank) ) under the BioProject number: PRJNA995429 and accession number: OR450807.1.
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    R Development Core Team. R: A language and environment for statistical computing. In R Foundation for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0. http://www.R-project.org (2008).Download referencesAcknowledgementsThe authors gratefully acknowledge the financial support for this research by the following organizations and agencies: Australian Centre for International Agricultural Research (ACIAR) (ProteinAfrica: LS/2020/154), Rockefeller Foundation (WAVE-IN: 2021 FOD 030); IKEA Foundation (G-2204-02144); European Commission (NESTLER Project: 101060762 and INNOECOFOOD project: 101136739), the Curt Bergfors Foundation Food Planet Prize Award; the Swedish International Development Cooperation Agency (Sida); the Swiss Agency for Development and Cooperation (SDC); the Australian Centre for International Agricultural Research (ACIAR); the Government of Norway; the German Federal Ministry for Economic Cooperation and Development (BMZ); and the Government of the Republic of Kenya. The views expressed herein do not necessarily reflect the official opinion of the donors. We thank Mr Fidelis Levi Ombura, Ms Maureen Adhiambo, and Mr Eric Rachami for their technical assistance.FundingThis research was funded by the following organizations and agencies: Australian Centre for International Agricultural Research (ACIAR) (ProteinAfrica: LS/2020/154), Rockefeller Foundation (WAVE-IN: 2021 FOD 030); IKEA Foundation (G-2204-02144); European Commission (NESTLER Project: 101060762 and INNOECOFOOD project: 101136739), the Curt Bergfors Foundation Food Planet Prize Award.Author informationAuthors and AffiliationsInternational Centre of Insect Physiology and Ecology, Nairobi, KenyaInusa Jacob Ajene, Chrysantus M. Tanga, Komivi S. Akutse, Samantha W. Karanu & Fathiya M. KhamisCollege of Tropical Agriculture and Human Resilience, Komohana Research and Extension Center, University of Hawaii at Manoa, Hilo, Hawai’i, 96720, USAInusa Jacob AjeneUnit for Environmental Sciences and Management, North-West University, Potchefstroom, 2520, South AfricaKomivi S. AkutseDepartment of Zoology and Entomology, University of Pretoria, Hatfield, South AfricaFathiya M. KhamisAuthorsInusa Jacob AjeneView author publicationsSearch author on:PubMed Google ScholarChrysantus M. TangaView author publicationsSearch author on:PubMed Google ScholarKomivi S. AkutseView author publicationsSearch author on:PubMed Google ScholarSamantha W. KaranuView author publicationsSearch author on:PubMed Google ScholarFathiya M. KhamisView author publicationsSearch author on:PubMed Google ScholarContributionsIJA: Conceptualization; data curation; investigation; formal analysis; methodology; validation; visualization; writing—original draft; writing— review and editing. CMT: Conceptualization; funding acquisition; resources; project administration; supervision; writing—original draft; writing—review and editing. KSA: Data curation; resources; validation; writing—review and editing. SWK: Data curation; investigation; formal analysis; methodology; writing—review and editing. FMK: Conceptualization; methodology; resources; supervision; validation; writing—review and editing. All authors have read and approved the manuscript.Corresponding authorsCorrespondence to
    Inusa Jacob Ajene or Fathiya M. Khamis.Ethics declarations

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    Reprints and permissionsAbout this articleCite this articleAjene, I.J., Tanga, C.M., Akutse, K.S. et al. Multi-omics comparison of two emerging storage pests (Necrobia rufipes and Tribolium castaneum) of dried black soldier fly larvae product.
    Sci Rep (2026). https://doi.org/10.1038/s41598-025-34902-7Download citationReceived: 25 September 2025Accepted: 31 December 2025Published: 05 January 2026DOI: https://doi.org/10.1038/s41598-025-34902-7Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy shareable link to clipboard
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    KeywordsGut microbiomeMitogenomePostharvest storage pestRed flour beetleRed-legged ham beetle More