Ecology

Archaeoglobales as systematic (electro)lithoautotrophs of the communityWe have evidenced the development of microbial electrotrophic communities and metabolic activity supported by current consumption (Fig. 1), product production (Fig. 2), and qPCRs (Fig. 3). These data suggest that growth did occur from energy supplied by the cathode. Our study is the first to show the possibility of growth of biofilm from environments harboring natural electric current in the total absence of soluble electron donors. To further discuss the putative mechanism, it is necessary to have a look at our conditions unfavorable for water electrolysis (see Supplementary Fig. S2). The equilibrium potential for water reduction into hydrogen at 80 °C, pH 7, and 1 atm was calculated at − 0.490 V vs SHE in pure water. The operational reduction potential is expected to be lower than the theoretical value due to internal resistances (from electrical connections, electrolytes, ionic membranes, etc.)16 and overpotentials (electrode material). This was confirmed with the on-set potential of H2 evolution measured at − 0.830 mV vs SHE in both experimental condition and abiotic control, indicating the absence of catalytic effect of putative hydrogenases secreted by the biofilm or metals from inoculum. Also, during preliminar potentials screening, the increase in current consumption and H2 production was observed only below − 0.7 V vs SHE (Supplementary Figs. S1 and S2). In addition, the presence of catalytic waves observed by CV with midpoint potentials between − 0.217 V to − 0.639 V indicate the implication of enzymes directly connected to the surface of the electrode (see Supplementary Fig. S2). Finally, the fixation of 267–1596 Coulombs day−1 into organics (Fig. 1) exceeds the maximum theoretical abiotic generation of hydrogen from abiotic current (~ 3 C day−1) 90- to 530-fold17.Therefore, under our experimental conditions, the biofilm growth should be largely ensured by a significant part of a direct transfer of electrons from the cathode, thereby demonstrating the presence of electrolithoautotroph microorganisms. This is supported by obtaining a similar biodiversity on sulfate with the cathode poised at − 300 mV [compared to − 590 mV vs SHE (Fig. 3)], whose potential is 190 mV more positive than the Equilibrium potential of H2 evolution (− 490 mV vs SHE), with then no electrochemical possibility of H2 production, even at molecular level.Taxonomic analysis of the enriched microbial communities at the end of the experiments showed the systematic presence of Archaeoglobales on cathodes. Moreover, the qPCR and MiSeq data (Fig. 3) highlighted a strong correlation between current consumption and density of Archaeoglobales in the biofilm (Supplementary Fig. S4, R2 = 0.945).The OTUs were related to some Archaeoglobales strains with 95–98% identities. Thus, we assume that under our experimental conditions new specific electrotrophic metabolisms or new electrolithoautotrophic Archaeoglobaceae species were enriched on the cathode. They were retrieved in all conditions and belonged to the only order in our communities exhibiting autotrophic metabolism. Autotrophic growth in the Archaeoglobales order is ensured mainly through using H2 as energy source and requires both branches of the reductive acetyl-CoA/Wood-Ljungdahl pathway for CO2 fixation18. Terminal electron acceptors used by this order include sulfate, nitrate, poorly crystalline Fe (III) oxide, and sulfur oxyanions19. Moreover, Archaeoglobus fulgidus has been recently shown to grow on iron by directly snatching electrons under carbon starvation during the corrosion process20. Furthermore, Ferroglobus and Geoglobus species were shown to be exoelectrogens in pure culture in a microbial electrosynthesis cell12 and have been enriched within a microbial electrolysis cell11,13. Given these elements, the identified Archaeoglobales species could be, under our electrolithoautotrophic conditions, the first colonizers of the electrode during the first days of growth. This hypothesis was confirmed into a more detailed study focusing on the enrichment on nitrate21.The growth of Archaeoglobales species in presence of oxygen is a surprising finding. Archaeoglobales have a strictly anaerobic metabolism, and the reductive acetyl-CoA pathway is very sensitive to the presence of oxygen22. This can be firstly explained by the low solubility of oxygen at 80 °C. Secondly, carbon cloth mesh reduces oxygen in the environment, allowing for anaerobic development of microorganisms into a protective biofilm23. This observation was supported by the near absence of Archaeoglobales in the liquid medium (Fig. 3). One of the hypotheses concerns direct interspecies electron transfer (DIET)24,25, with Archaeoglobales transferring electrons to another microorganism as an electron acceptor. Research into DIET is in its early stages, and further investigations are required to better understand the diversity of microorganisms and the mechanism of carbon and electron flows in anaerobic environments25 such as hydrothermal ecosystems.Electrosynthesis of organic compoundsAccumulation of pyruvate, glycerol and acetate was measured, while another set of compounds that appeared transiently were essentially detectable in the first few days of biofilm growth (Supplementary Table S1). They included amino acids (threonine, alanine) and volatile fatty acids (formate, succinate, lactate, acetoacetate, 3-hydroxyisovalerate) whose concentrations did not exceed 0.1 mM. Despite their thermostability, this transient production suggests they were used by microbial communities developing on the electrode in interaction with the primary producers during enrichment.On the other hand, in presence of nitrate, sulfate and oxygen as electron acceptors, the liquid media accumulated mainly acetate, glycerol, and pyruvate (Fig. 1). Coulombic efficiency calculations (Fig. 2) showed that electron content of the carbon products represented 60–90% of electrons consumed, the rest being potentially used directly for biomass or transferred to an electron acceptor. This concurs with the energy yield from the Wood-Ljungdahl pathway of Archaeoglobales, with only 5% of carbon flux directed to the production of biomass and the other 95% diverted to the production of small organic end-products excreted from the cell26.Pyruvate is a central intermediate of CO2 uptake by the reducing pathway of the acetyl-CoA/WL pathway27. It can be used to drive the anabolic reactions needed for biosynthesis of cellular constituents. Theoretically, the only explanation for improved production and accumulation of pyruvate (up to 5 mM in the liquid media of sulfate experiment) would be that pyruvate-consuming enzymes were inhibited or that pyruvate influx exceeded its conversion rate. Here we could suggest that in-cell electron over-feeding at the cathode leads to significant production of pyruvate when the electron acceptor runs out.In an ecophysiological context, similar pyruvate and glycerol production could occur on hydrothermal chimney walls into which electric current propagates28. The electrotroph biofilms would continually receive electrons, leading to an excess of intracellular reducing power which would be counterbalanced by overproduction of glycerol and pyruvate29,30. Furthermore, these products can serve as carbon and energy sources for heterotrophic microorganisms or for fermentation. In our experiments, pyruvate and glycerol concentrations varied over time, suggesting they were being consumed by heterotrophic microorganisms. Acetate production would thus result from the fermentation of pyruvate or other compounds produced by electrotrophic Archaeoglobales.Enrichment of rich heterotrophic biodiversity from electrotrophic Archaeoglobales communityDuring our enrichment experiments, the development of effective and specific biodiversity was dependent on the electron acceptors used (Fig. 3). Heatmap analyses (Supplementary Fig. S3) showed four distinct communities for the three electron acceptors and the initial inoculum. Thus, at the lower taxonomic level of the biodiversity analysis, most OTUs are not common to multiple enrichments, except for one OTU of Thermococcales that was found in both the nitrate and sulfate experiments. This suggests a real specificity of the communities and a specific evolution or adaptation of the members of the shared phyla to the different electron acceptors available in the environment. However, the various enrichments also showed the presence of Thermococcales regardless of the electron acceptors used, thus demonstrating a strong interaction between Thermococcales, assumed to be heterotrophs, and Archaeoglobales, the only demonstrated autotrophs. Moreover, members of these two groups have frequently been found together in various hydrothermal sites4,5,31,32, where they are considered potential primary colonizers33,34,35,36,37. After Thermococcales, the rest of the heterotrophic biodiversity was specific to each electron acceptor.On nitrate, two additional phylogenetic groups were retrieved: Desulfurococcales and Thermales. OTUs of Desulfurococcales are mainly affiliated to Thermodiscus or Aeropyrum species, which are hyperthermophilic and heterotrophic Crenarchaeota growing by fermentation of complex organic compounds or sulfur/oxygen reduction (Huber and Stetter, 2015). Concerning Thermales, a new taxon was enriched on cathode and only affiliated to Vulcanithermus mediatlanticus with similarity of 90%. This new taxon of Thermales (OTU 15, Supplementary Fig. S3) was also enriched up to 2% on the cathode of sulfate enrichment. Thermales are thermophilic (30–80 °C) and heterotrophic bacteria whose only four genera (Marinithermus, Oceanithermus, Rhabdothermus, and Vulcanithermus) are all retrieved in marine hydrothermal systems. They can grow under aerobic, microaerophilic and some anaerobic conditions with several inorganic electron acceptors such as nitrate, nitrite, Fe (III) and elemental sulfur38. All of the Thermales species can utilize the pyruvate as carbon and energy source with the sulfate or nitrate as electron acceptors.Pseudomonadales and Bacillales were found in the oxygen experiment. Most Pseudomonas are known to be aerobic and mesophilic bacteria, with a few thermophilic species (up to 65 °C)39,40. There have already been some reports of mesophilic Pseudomonas species growing in thermophilic conditions in composting environments41. Moreover, some Pseudomonas sp. are known to be electroactive in microbial fuel cells through long-distance extracellular electron transport42,43,44, and were dominant on the cathodes of a benthic microbial fuel cell on a deep-ocean cold seep45. In Bacillales, the Geobacillus spp. and some Bacillus sp. are known to be mainly (hyper)thermophilic aerobic and heterotrophic Firmicutes46.Hydrothermal electric current: a new energy source for the development of primary producersThe presence of so many heterotrophs in an initially autotrophic condition points to the hypothesis of a trophic relationship inside the electrotrophic community (Fig. 5). This suggests that the only autotrophs retrieved in all communities, the Archaeoglobales, might be the first colonizer of the electrode, using CO2 as carbon source and the cathode as energy source. Models using the REACT module of the Geochemist’s Workbench (GWB) and based on electron donor acceptor availability predicted low abundances of Archaeoglobales ( More

Pesticide residues in organic productsNon-use of synthetic pesticides is a major characteristic of organic farming, with the objectives of protecting (a) the environment, (b) consumer health, and (c) farm worker health. In consumer studies, “no chemical pesticides” is usually mentioned as one of the most important criteria for buying organic food1,2. These consumer expectations are mostly met in what is referred to in objective (b). Both European and U.S. sources consistently found the percentage of samples with residues of pesticides above the limit of quantification ( > LOQ) to be much lower in organic than in conventional food (Fig. 1a, see also Supplementary Fig. 1). This is especially true when it comes to fresh fruits and vegetables (Fig. 1c), which are known to be the most critical food groups in terms of pesticide residues3. It is elucidating, however, to not only look at the number of samples with an (unknown) level of residues  > LOQ, but to quantify the residues found per sample. In many cases, more than one substance is found in a sample, therefore one meaningful indicator is the mean cumulative pesticide load per sample (MCPL, see Supplementary Table 1). This is represented in Fig. 1b for three out of the four datasets. The food authority CVUA (Chemisches- und Veterinäruntersuchungsamt) in Baden-Württemberg, Germany, has been comparing pesticide residues between organic and conventional food since 2002. In 2019, on average the residues in organic produce were more than 150 times lower than in the corresponding conventional products4 (Ratio Org./Conv., bottom of Fig. 1d). The USDA (U.S. Department of Agriculture) numbers tend to be higher than the European ones, both in percentages (Fig. 1a,c) and in MCPL (Fig. 1b). One reason for this is probably USDA’s risk-oriented sampling approach, in which some highly contaminated commodities are over-represented, as compared to their importance in most people’s diet (Supplementary Table 2, column C). If we correct this possible bias by assuming that every commodity would have been sampled with the same frequency, the MCPL across all commodities is cut by 40% (Supplementary Table 2, last row). Different LOQs and numbers of analytes covered by USDA on one hand, and different European laboratories on the other hand, also make comparison difficult.Figure 1Pesticide residues in conventional and organic food in tests conducted by four organisations: EFSA (European Food Safety Authority) collects official data from all EU member states3, CVUA from one federal state in Germany4, USDA from government laboratories across the U.S.5, while Eurofins is a commercial laboratory in Germany. Figures in brackets represent the number of samples. The legend is valid for (a), (b) and (c). In order to increase the number of samples (represented in brackets) and thus their representativeness, figures from several years were grouped together, as available from each organisation. Black bars symbolise standard errors across years. (a) Shows the percentage of samples with residues above the limit of quantification (LOQ), for all types of food ( available from two organisations only). (b) Represents the mean cumulative pesticide load (MCPL) for fruits and vegetables (available from three organisations). (c) Similar to (a), but for fresh fruits and vegetables only (CVUA uses “above 0.01 mg/kg” instead of LOQ, but this is identical for most substances). The same datasets were used for (b) and (c). (d) Multi-layer sieving model for residue testing at different points of the organic supply chain. The data above the white arrows are from the commercial laboratory Eurofins, and mostly represent the situation before products are released to the market, while the figures below the white arrows are from CVUA, representing the situation on the market (both wholesale and retail). Ratios from “before market” to “on market” are shown in the white arrows. In this process, the MCPL remains in the same range for conventional products (blue rectangle to the right), while it is reduced massively for organic products (green trapezium in the centre). As a result of this sieving mechanism, residues in samples from the market are 150 and more times lower in organic than in conventional produce (trapezium at the bottom). This shows that the process represented by the blue arrows works fairly well—which is not always the case for the investigation of the origin of such residues, symbolised by the yellow arrows.Full size imageOrganic businesses’ testing strategiesUnfortunately, the generally good news for consumers with respect to objective (b) does not always mean that objectives (a) and (c) are also met. With the steady growth of the organic market and globalisation of supply chains, integrity of the system is often at stake. Organic products mostly fetch higher prices, and therefore also attract fraud6,7. Since pesticide residues are easily detectable parameters, often indicating non-compliance with organic production rules, many organic businesses test each batch for such residues, before placing it on the market. Positive results should then lead to an investigation of the origin of the found residues: Did an organic farmer spray? Do the residues come from drift, from ubiquitous contamination, or from (avoidable or unavoidable) contamination during processing, transport, storage? Were organic and conventional products mixed at some point of the supply chain—or is somebody simply labelling conventional products as “organic”? The idea behind this is depicted in Fig. 1d. The filter process as such, and the exclusion of contaminated batches from the organic market, as represented by the blue and red arrows, often work well. Thus, there are remarkably lower average amounts of residues after undergoing this filtering process. Residues in organic produce reported from the market were reduced by 22 and 89 times in fruits and vegetables, respectively, compared to the levels reported by the commercial laboratory, which represent mostly pre-market samples, while the values for conventional samples remained in the same range. This shows that market actors often remove problematic batches by declaring them conventional. In Supplementary Tables 3 and 4 we provide further explanations why the datasets “before release to the market” and “on the market” in Fig. 1d are comparable.We do not have test results from a commercial laboratory in the U.S. that could be compared to Eurofins data. But, as opposed to the other sources of information, the USDA database identifies the country of origin of each sample. Anybody working in international organic certification would expect residues in imported food to be higher than in domestic products, because fraud is more widespread when the distance is bigger between producers on the one hand, and consumers and the competent authorities on the other. The U.S. data, however, suggest the opposite trend: Not only at the aggregate level, but also for most individual commodities, the MCPL is lower in imported than in domestic products (Supplementary Table 2, columns J and K). The reason is probably that samples are tested before signing purchase contracts, and products rejected or bought as conventional, if they do not comply with the expectations.This is good quality control practice—the problem is that the information about the “downgrading” of organic products to conventional is not always reaching the certification bodies (CBs), thus impeding the investigation of the origin of residues and the exclusion of fraudulent actors from the market (yellow arrows in Fig. 1d). It is in the nature of things that these processes are not publicly known and therefore cannot be quantified, but in Supplementary Fig. 2 we present anecdotic evidence, which also suggests that for some market actors the definition of “organic” is limited to “free of pesticide residues”.Certifiers’ testing strategiesThe two most important markets for organic food are the EU, where the “organic” label is legally governed by an EU Regulation, and the USA, where the corresponding rule is the National Organic Program (NOP). Although they have different approaches on how to deal with spray-drift and with residues (Supplementary Table 5), both regulations require CBs to take samples from at least 5% of their clients every year. A large amount of data is being generated through this mechanism, but the sampling procedures and interpretation of results often do not allow deriving clear results. A recent unpublished BSc thesis at the University of Kassel revealed that 80% of the samples by CBs in ten EU member countries are taken of final products, but only 20% from the field or during the production process. This suggests that not only for market actors, but also for many CBs, the purpose of sampling and testing is limited to ensuring that food sold on the market with an organic claim, is free of pesticide residues, without digging deeper to find the origin of contamination.The differentiation between active use and non-intentional contamination is difficult, if only final products are tested. Plant (mainly leaf) samples from the field have several advantages in this regard: (a) Often, there is a long time span between pesticide application and harvest. Because of dissipation of the residues, nothing or only traces may be found in the final product (Supplementary Table 6). Field samples can be taken during or shortly after a suspected pesticide application, so that the dissipation effect is reduced and residues are found even for substances with a short half-life. (b) Leaves have a surface/weight ratio between 10 and 118 cm2/g8, whereas for fruits this ratio is between 0.6 and 2.29, and for seeds between 2 and 10 cm2/g only10,11,12. Residues in leaves are therefore normally higher than in seeds, fruits or roots, which makes interpretation of test results easier. (c) Field sampling allows taking separate samples from centre and margin of the field, as explained below in more detail.Unfortunately, if CBs take field samples at all, they often take them only from field margins13,14 (“let’s see if there is a drift problem”). Positive results are then attributed to spray-drift, and farmers are required to establish buffers—without even considering the possibility of residues originating from an application by the organic farmer. Such procedures open the door for fraudulent use of pesticides by organic farmers.Other CBs have established so-called “action levels”, below which they consider the presence of residues in organic products to be the result of ubiquitous environmental contamination, with no need to investigate their origin13. While such thresholds may be necessary for specific cases (see below concerning the banana industry), using this approach as a general procedure disregards not only the spatial distribution, but also temporal dynamics of pesticides in plant tissue. As opposed to soil, half-lives in plant tissue exposed to UV radiation and weather, are relatively short for most modern pesticides15. A residue level of 0.02 mg/kg, used by some CBs as “action level”, is typically reached one to two months after the application of a pesticide, in some cases even after only five days (Supplementary Table 6).The time that has elapsed since an application, however, is unknown in most cases. Spraying records kept by conventional neighbours are normally not part of the inspection. In case of suspicious test results in samples from the organic farm, such records may sometimes be accessed as part of a follow-up investigation, but at that point the organic farmer may have asked the neighbour to manipulate the records. And if the organic farmer has sprayed, he or she obviously tries to hide this fact. This situation makes interpretation of low levels of residues found in samples from organic fields even more challenging, and increases the importance of being able to differentiate application from drift through other methods.Two forms of spray-driftOver the past decades, a distinction has been made between short distance primary spray-drift during the application, and long distance secondary spray-drift occurring after the application16. The latter was attributed to evaporation and considered to play a role only for pesticides with high vapour pressure17. On the one hand, recent studies have shown that evaporation and long-distance transport can already play a role during, not only after application18. On the other hand, long-distance transport has been found to be linked not only to evaporation. Pesticides adherent to dust from wind erosion can contaminate large areas19. In the present context, we use the terms short-range and long-range drift, instead of primary and secondary drift (Fig. 2).Figure 2Simplified model of short-range vs. long-range drift originating from air-blast spraying in a fruit orchard. The specific values for pesticide concentrations (mg/kg) expected for different downwind distances from the orchard can vary by a factor 10 or more, depending on the applied substance, dose, weather conditions, vegetation, etc., but the graph provides an approximate estimate of the ratios that can be expected. In the case presented here, pesticide concentration in fruit leaves immediately after the application is 15 mg/kg. In the area of short-range direct drift, deposit decreases exponentially, so that at 100 m distance, we can expect to find only 0.01 mg/kg. At further distances, deposits are often below this level.Full size imageLong-range driftLong-range drift is so far poorly understood, can lead to (normally very low) residues at distances as far as thousands of km19, and happens in the form of vapour or molecules adhering to dust. The main factors influencing long-range drift are vapour pressure of the pesticide, capacity of adherence to dust, incidence of wind erosion, and temperature inversion in the atmosphere17. Long-range pesticide drift has recently received more attention21,22,23,24,25. Examples have been used in the context of organic certification for supporting the argument of ubiquity of pesticides, linked to the assumption that low- or even medium-level residues in organic products are often derived from their omnipresence in the environment26,27.Cases from Brazil (endosulfan in soybeans), Montana (USA) and Saskatchewan (Canada) (glyphosate in khorasan wheat) and Germany (pendimethalin and prosulfocarb in different crops) have been quoted to demonstrate the ubiquity of pesticides27. None of these case studies, however, provides solid evidence for the assumption that long-distance transport of pesticides leads to residues in organic food above the level of, say, 0.01 to 0.03 mg/kg. The problem of the herbicides pendimethalin and prosulfocarb being subject to long-distance drift because of their high vapour pressure, has been known for a long time28, but this phenomenon cannot be extrapolated to other substances. Even for these herbicides, there is no evidence that residues at larger distances could be above the indicated levels. Across 15 vegetation samples from nature reserves in Germany, on average, 0.009 mg/kg pendimethalin and 0.004 mg/kg prosulfocarb were found29. Exceptions may exist, e.g., when pesticide applications are followed by heavy wind erosion, as seems to be the case in some of the North American wheat growing areas, where glyphosate is used for cereal desiccation shortly before harvest.In a survey in Switzerland30, neonicotinoid residues were found in 93% of plant samples from organic farms (as compared to 100% of samples from conventional farms), thus supporting the ubiquity suspicion. But there were substantial quantitative differences between organic and conventional farms (Fig. 3). The average sum of neonicotinoid residues in plant and soil samples from organic farms was lower by a factor of 11 than that of plant samples from conventional farms. For soil samples, this factor was as high as 71. Even the highest value for one single substance (imidacloprid) found in organic plants (2.13 µg/kg = 0.00213 mg/kg) would be below the limit of quantification (LOQ) used for this substance in most screenings (0.01 mg/kg).Figure 3Maximum and average residues of neonicotinoid insecticides in soil and plant samples from organic farms, integrated crop production (“IP Suisse”: this program involves reduced pesticide application) and conventional farms in Switzerland. The figures represent the sums of acetamiprid, chlothianidin, imidacloprid, thiacloprid and thiamethoxan. Figures in brackets represent standard errors.(Data from Humann-Guilleminot et al.30).Full size imageIn a study in Germany29, the MCPL in natural vegetation in five reference areas (average distance from arable fields  >3 km) was 0.003 mg/kg, and in 15 nature conservation areas (average distance from arable fields 143 m) it was 0.006 mg/kg, but in three buffer zones (average distance 54 m) it was 5.4 mg/kg. To make figures comparable with other data in this article, we have subtracted the concentration of non-agricultural pesticides from the total amounts, and divided the numbers by a factor five, because the residues in this study refer to dry matter, while all the others use fresh matter. Although 5.4 mg/kg at 54 m distance is a disturbingly high value, the survey confirms that concentrations at larger distances do not exceed the “traces” level. The intention of this article is not to put in doubt the environmental damage caused by such traces. What we try to show is that the “ubiquity” argument may sometimes be hiding cases of fraudulent pesticide use by organic farmers.Short-range driftAs opposed to long-range drift, short-range drift is well understood, has its impact mainly in a range from 1 m up to a maximum of 1,000 m (for aerial spraying), happens in the form of droplets, and is not substance specific. The main factors influencing this form of drift are droplet size, windspeed, and height of the boom (nozzles) above soil17,19,31,32,33. The fact that long-range drift is poorly understood and leads to low concentrations of certain substances over wide areas, should not stop certification bodies (CBs) from using the available knowledge about short-range drift as a tool for assessing farmers’ compliance with organic production rules. The dynamics of short-range spray-drift have been widely studied in the context of preventing liability problems due to herbicide damage, contamination of water bodies and natural habitats, and direct risks for human settlements19,31,32,33,34,35,36. Pesticide deposit decreases exponentially with increasing distance from the field on which the substance is applied. With a tractor boom sprayer, deposit at 25 m distance is expected to be only 1% of that in the target field. While distances are greater for air-blast or aerial spraying, the basic principle of exponential decrease is the same (Fig. 2 and Supplementary Fig. 3).ObjectivesThe objectives of our study are: (I) to demonstrate that appropriate field sampling methods can differentiate the effects of fraudulent pesticide application by the organic farmer, from the results of both short-range and long-range spray-drift, and (II) for the specific case of aerial fungicide spraying in the banana industry, identify appropriate variables, which allow us to interpret the test results correctly for the purpose of this differentiation. More

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Ammonium and nitrate concentrations, and ammonium/nitrate ratioThe concentrations of ammonium in soil varied significantly due to fertilizer amendments (short rainy season: χ2 = 123.7, df = 2, p  More

Experiments using P. hysterophorus root exudate water samplesInsects for oviposition bioassayAnopheles gambiae mosquitoes (Mbita strain) used in this study were from a colony established in 2018 and maintained at the International Centre of Insect Physiology and Ecology (icipe), Duduville Campus, Nairobi, Kenya. The adults were reared using standard insectary conditions at 28 ± 2 °C, and 72% relative humidity (RH) under a photoperiod of 12 h: 12 h (L:D). For egg development, the mosquitoes were offered human blood through arm feeding, one to two times weekly and had ad libitum access to a 6% glucose solution (wt:vol). The blood fed females were kept together with males for 2 days and used on the third day for the oviposition experiment. Eggs were laid in oviposition cups (7 cm top diameter, 4 cm base diameter and 7 cm depth) lined with filter paper (Whatman 90 mm, GE Healthcare UK Ltd, Buckinghamshire, London, UK) and transferred to larval rearing trays (39 × 28 × 4 cm depth). Upon hatching, the larvae were separated into groups of ~ 500 larvae per rearing tray and fed on commercial Tetramin® fish food (Tetra, Germany). The rearing room was maintained at 32 ± 2 °C, and 52% RH during the day, and 24 ± 2 °C and 72% RH at night with a photoperiod of L:D 12:12 h15. The rearing water was changed after every 2 days. Pupae were transferred daily to emergence cups containing 15 mL water and placed in a new cage (15 × 15 × 15 cm). Emerged adults (1 day old) were maintained on 6% glucose solution as described above.Collection of root exudate water samples from wild growing P. hysterophorus
Root exudate water samples were collected from wild growing P. hysterophorus (~ 30, 60 and 90 cm tall) on the icipe campus and used in various biological assays (i.e., oviposition response, mosquito growth and development assays). Briefly, wild growing P. hysterophorus were transplanted into a plastic pot (23 cm top diameter, 12 cm base diameter and 22 cm depth; batches of ten plants per pot in garden soil from the icipe campus) and watered with rainwater to obtain root exudate water. The root exudate water was collected from the plants after 1 week: to allow the plants to stabilize and to eliminate possible contamination of it with defense compounds released in response to uprooting. Water obtained from potted soil (same volume as used for the potted P. hysterophorus) without P. hysterophorus plants but from the same site, served as control water.Oviposition response assay with root exudate waterRoot exudate water was evaluated for its ability to influence oviposition response of gravid females in a dual-choice assay (Fig. 1A). The gravid females (n = 12 as described in Dieter et al.18; electronic supplementary material, methods) were presented a choice between the treatment and distilled water contained in similar polypropylene cups and the number of eggs laid counted using a microscope (Leica M127, Switzerland) after every 24 h for four consecutive days7. The cups were placed in opposite corners of the experimental cages and their positions interchanged every 24 h to avoid positional bias. Each cup was lined with a filter paper (Whatman 90 mm, GE Healthcare UK Ltd, Buckinghamshire, London, UK) and filled with 30 mL of the test solution (to keep the filter paper moist; as previously described in19) or an equivalent volume of distilled water as the control. A similar experiment was performed using water obtained from soil without P. hysterophorus plants (Fig. 1A). The bioassays were performed in triplicate as previously described in Ilahi et al.20 and repeated once.Figure 1Effect of root exudate water on oviposition response and aquatic stage development of An. gambiae. (A) A schematic representation showing the setup of the oviposition experiment using root exudate water and soil water. (B) Oviposition activity of root exudate water and soil water. (C) Table summarizing median number of eggs laid and the range. (D) Egg hatch rates was low in root exudate water compared to soil water. (E) Time to pupation (days) for larvae exposed to root exudate water relative to soil water.Full size imageHeadspace collection and analysis of root exudate volatilesTo collect headspace volatiles, 500 mL of the root exudate water were placed in air-tight glass jars and activated charcoal-filtered and humidified air passed over it. The volatiles were collected for 24 h on three pre-cleaned (dichloromethane (DCM) and nitrogen-dried) Super Q adsorbent filters (30 mg each, Analytical Research System, Gainesville, Florida, USA) at a flow rate of 170 mL/min. The three Super-Q filters (each treatment), were each eluted with 200 µL GC-grade DCM (Sigma Aldrich, St. Louis, Missouri, USA) into 2 mL clear glass vials, each containing 250 µL conical point glass inserts (Supelco, Bellefonte, PA, USA) and immediately analyzed by coupled gas chromatography/mass spectrometry (GC/MS). Also, natural water from depressions harboring growing P. hysterophorus in the field was sampled during the rainy season (described under parthenin detection) to allow comparison of its volatiles with that of the root exudate water. Volatiles collected from distilled water served as negative controls. Compounds were identified by comparison of their mass spectral data with library data: Adams2, Chemeco and NIST11 and confirmed with those of authentic samples. The relative peak area (%) of each constituent as generated by the NIST11 software, following GC/MS analysis was used to determine the natural ratio of the volatiles in the component blend. The volatiles were stored in glass vials at − 80 °C until used for oviposition assays.Oviposition bioassay with identified volatilesDual choice bioassay was performed to determine the role of root exudate volatiles in egg laying behavior of An. gambiae. Of the volatiles identified, seven compounds: α-pinene, β-pinene, 3-carene, (E)-caryophyllene, camphor, α-phellandrene, β-phellandrene were selected and tested based on results obtained from Random Forest Analysis (RFA) (see electronic supplementary material, methods) and their commercial availability21. In dose–response assays, several tests were carried out: (i) a blend of the seven components (7-component blend) mimicking their naturally-occurring ratio in the root exudate volatile extract based on their GC/MS peak areas, (ii) single compounds, and (iii) a blend of the attractive components (5-component blend) guided by the positive attractive responses obtained from (ii). The compounds were dissolved in dimethyl sulfoxide (DMSO) and serially diluted from a stock solution to generate a concentration ranging from 0.25 to 4 µg/µL. Thereafter, for each concentration tested, 50 µL of each compound or blend solution were dispensed into the oviposition cup containing distilled (50 mL) water and monitored for egg laying. Each concentration was tested against a control (distilled water (50 mL) containing 50 µL of DMSO). The bioassays were performed in triplicate.Mosquito growth and development assaysThe root exudate water and the control water (water from soil) were used separately for mosquito rearing. First, mosquitoes were provided with root exudate water to lay eggs in a no-choice oviposition bioassay. Thereafter, a total of 200 eggs were counted and placed into each rearing tray (24 × 34 × 4 cm) and the number of hatched eggs was determined by counting the first instars larvae that emerged in each tray. The larvae were fed daily on a standardized regimen of ground fish food (Tetramin, Tetra, Germany) and water was changed every other day. Daily survival of larvae (from first instar (L1) to pupation) was recorded, and pupae transferred into cups in experimental cages (30 × 30 × 30 cm) until adult emergence. The conditions of the bioassay room were the same as that of the rearing room described above. All experiments were performed in four replicates.Experiments using partheninDetection of parthenin in root exudate waterOne liter (1 L) of water was collected from flooded depressions/open puddles in which wild P. hysterophorus was growing on the icipe campus and pooled (the field collection was repeated twice, 1 week apart). The habitat had other non P. hysterophorus plants such as grasses. The water was filtered using a muslin cloth and stored at − 80 °C overnight and then freeze-dried (VirTis SP scientific, Model Advantage EL-85) for 72 h to obtain 38 mg of root exudate. This was extracted with dichloromethane (DCM) and analyzed by GC/MS as described under chemical analysis. A similar analysis was carried out on the root exudate water (four replicates) obtained from potted plants described above.Preparation of parthenin stock solutionA sample of parthenin obtained from a methanolic extract of P. hysterophorus from a previous study conducted in our laboratory22, was used as a standard. A sample of this isolate (300 mg) was dissolved in 0.3 mL DMSO and then diluted to 300 mL with distilled water to obtain a stock solution of 1000 ppm.Oviposition response assay with partheninParthenin was evaluated at a concentration of 0.13 µg/µL corresponding to the estimated amount of the root exudate tested (described above). The amount of parthenin in the root exudate was estimated by comparing the relative peak area (%) of parthenin in the root exudate to that recorded for the standard parthenin of known concentration. The oviposition assays were performed as described for root exudate. However, the distilled water which served as a negative control was prepared in 0.1% DMSO. A similar experiment was performed using parthenin water spiked with 50 µL of headspace volatiles collected for 24 h from the plant root exudate. All experiments were performed in triplicates.Mosquito growth and development assays using partheninParthenin prepared as described for the oviposition bioassay was used for mosquito rearing. Mosquitoes reared on distilled water prepared in 0.1% DMSO served as the control group. The experiment was performed as described for root exudate water.To determine the effect of exposure to parthenin on mosquito adult survival, the female mosquitoes that emerged from parthenin treated water and controls were monitored separately in different cages. The mosquitoes were maintained on P. hysterophorus potted plant until their natural death (survival). The conditions in the bioassay rooms were the same as those of the rearing room described above.Chemical analysisGas chromatography/mass spectrometry (GC/MS)To detect parthenin in the root exudate, the sample was freeze-dried and prepared at a concentration of 600 ng/µL in dichloromethane (DCM) and dried over anhydrous Na2SO4 (Sigma Aldrich, St Louis, MO USA). The standard parthenin sample was prepared at a concentration of 300 ng/µL in DCM. For GC/MS analysis, 1 µL of each sample (parthenin and root exudate) was analyzed on a 7890B gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) linked to a 5977A mass selective detector under the following conditions: Inlet temperature 270 °C, transfer line temperature 280 °C, and column oven temperature programmed from 35 to 285 °C, with the initial temperature maintained for 5 min then 10 °C/min to 280 °C for 5.5 min and finally 5 °C/min to 285 °C for 34.9 min. The GC was fitted with a HP-5 MS low bleed capillary column (30 m × 0.25 mm i.d., 0.25 µm) (J&W, Folsom, CA USA). Helium at a flow rate of 1.2 mL/min served as the carrier gas. The mass selective detector was maintained at ion source temperature of 230 °C and a quadrupole temperature of 180 °C. Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV. Compounds were injected in the splitless mode using an auto-sampler 7693 (Agilent Technologies, Inc., Beijing, China). Fragment ions were analyzed over 38–550 m/z mass range in the full scan mode. The filament delay time was set at 3.0 min. Parthenin was identified based on its general fragmentation pattern and compared also with previously published results23. The root exudate samples were analyzed in triplicate, with each replicate collected from a different batch of plants.Statistical analysisThe oviposition activity index (OAI) for dual choice oviposition assay data was calculated according to the formula described in19;$$OAI=frac{mathrm{Nt}-mathrm{Nc}}{mathrm{Nt}+mathrm{Nc}}$$
where Nt is number of eggs laid in the treatment and Nc the number of eggs laid in the control. The OAI ranges from − 1 to + 1, with 0 indicating neutral response, positive value indicating an attraction towards the treatment and a negative value indicating the converse. The oviposition data was analyzed by generalized linear model using negative binomial. The model validity was assessed by inspection of residuals24: Number of eggs deposited served as the response variable while the treatments were used as the predictor variable.Hatch rate of mosquito eggs exposed to different treatments was calculated as follows:$$% hatchability=left(frac{number ; of ; hatched ; eggs}{total ;number ; of ; eggs}right) times 100$$The % hatchability data was also analyzed using generalized linear model with negative binomial error structure. The variation in survival was analyzed by the Kaplan-Meir method and statistical significance comparisons made using log-ranks test. All statistical analyses were performed using SPSS 23.0 software (IBM SPSS Statistics) and results considered significance at p  More

Field data and analysisField samplingWe captured adult juncos by mist net or potter trap at sites in Arizona (breeding season), Colorado (breeding), Illinois (non-breeding), Montana (breeding), New Mexico (non-breeding), New York (breeding), South Dakota (non-breeding), and Wyoming (breeding), spanning 16° in latitude and 37° in longitude (Fig. 1). This work was completed with approval from the U.S. Fish and Wildlife Service (MB84376B-1 to M.S.; MB01543B-0 to Z.A.C.; MB758442 to D.L.S.; MB06336A-4 to M.D.C.; MB757670-1 to David Winkler; and MB094297-0 to Christopher Witt,), the State of Arizona Game and Fish Department (SP590760 and SP707897 to D.L.S.), Colorado Parks and Wildlife (10TRb2030A15 to M.D.C.), the Illinois Department of Natural Resources (NH13.5667 to Z.A.C.), the Montana Department of Fish Wildlife and Parks (2016-013 and 2017-067-W to M.S.), the New Mexico Department of Game & Fish (3217 to Christopher Witt), the New York State Division of Fish, Wildlife, & Marine Resources (LCP 1477 to David Winkler), the State of South Dakota Department of Game, Fish, and Parks (06-03, 07-02, 08-03 to D.L.S.), Wyoming Game and Fish (754 to M.D.C.), and the Institutional Animal Care and Use Committees at Cornell University (2001-0051 to David Winkler), the University of Illinois (13385 to Z.A.C.), the University of Montana (010-16ZCDBS-020916 to Z.A.C.), the University of South Dakota (03-08-06-08B to D.L.S), and the University of Wyoming (A-3216-01 to M.D.C.). We classified individuals to taxonomic unit based on plumage (J. h. caniceps, J. h. hyemalis, J. h. mearnsi, J. h. oreganus group, and J. p. palliatus). The J. h. oreganus group encompasses seven subspecies (J. h. montanus, J. h. oreganus, J. h. pinosus, J. h. pontilis, J. h. shufeldti, J. h. thurberi, and J. h. townsendii) with similar plumages and overlapping nonbreeding ranges26,27. For this reason, we were unable to distinguish subspecies of nonbreeding individuals within this group, though all breeding individuals were collected from the J. h. montanus range.Field metabolic assaysBirds were transported from the site of capture to a nearby laboratory ( More