Ecology

Surface active agents (surfactants) are a group of chemicals that have a polar hydrophilic headgroup and a non-polar lipophilic hydrocarbon tail group1. This structure of surfactants allows them to be used in households and industries to increase the solubility of non-water-soluble substances, such as cleaning agents and emulsifiers. Global production of synthetic surfactants was 7.2 million tons in 20002; since 2006, this value has risen to 12.5 million tons3 and these numbers will grow with the growth of the detergent and cosmetics industry. After use, the residual surfactants are discharged into the sewage system or directly into surface water, resulting in an increase in the level of surfactants in the environment and a significant impact on the ecosystem1.The toxicity of surfactants to organisms is well known4 and depends on the physico-chemical properties of the surfactants themselves. They are generally classified into anionic, cationic, amphoteric and nonionic, depending on the charge of their headgroup. Among the groups listed above, the anionic surfactants are the most common in everyday and industrial uses and are toxic to both humans and the environment. In particular, anionic surfactants can bind to peptides, enzymes and DNA and alter their spatial layout (folding) and surface charge5. Such interactions can change the biological functions of biomolecules. Sodium dodecyl sulfate (SDS) is one of the most commonly used anionic surfactants, producing more than 3.8 million tons globally for industrial applications in cosmetics, clothing, food, fuel, and medicine6. Such mass production and use of SDS results in releases to the environment, with a semi-lethal concentration of not more than 45 μg/ml7 for algae, fish and crustaceans. In addition, it is known that surfactants can accumulate in the human body and cause autoimmune diseases, brain, liver, kidney and lung damage8,9. Besides the permissible limits for surfactants is 1 mg/l in water and at 0.5 mg/l for potable water10. In order to prevent negative environmental and human impacts of anionic surfactants (in particular SDS) in a timely manner, methods are needed to detect this surfactant in both wastewater and surface waters and in the soil11, food9, dust12,13, etc.Spectrophotometric and potentiometric methods are the most common means of determining anionic surfactant, and chromatography is often used to concentrate and separate complex surfactant mixtures14. Most often, the ionometric determination of the surfactant is carried out using ionic electrodes, which makes it possible to determine the concentration of the substance under investigation in a short time (up to 30 min). However, this method has low sensitivity (280–600 μg/ml)15 and low selectivity, which does not allow the determination of surfactants in relatively complex samples. Spectrophotometric methods are also labour-free (10–30 min) and have a high sensitivity of 0.001 μg/ml16,17. The main disadvantage is the low specificity and dilution of the sample to the measuring limit of 0.01 μg/ml, which complicates the measurement procedure. These defects are corrected by chromatography, which allows separating the studied mixture and increasing the concentration of the required substance, but this procedure requires a minimum of several hours15.There is therefore a need to develop a fast, low-cost method for determining anionic surfactant with high selectivity (specificity) that allows measurements to be made at environmentally toxic concentrations (10–50 μg/ml). Therefore, a quick semi-quantitative or qualitative determination of the substance by means of various rapid tests, such as paper tests, is sufficient for a number of practical tasks to determine the surfactants before applying a more precise and labour-intensive method systems, tracer powders, fabrics, polymer films, tablets18,19,20,21. In particular, Dmitrienko’s work with co-authors presents a method based on adsorption of a red-colored polyurethane foam (PUF) complex of an anionic surfactant with cation 1,10-fenantrolinate iron complex(II)22. This method allows the determination of anionic surfactants between 1 and 30 μg/ml. But all these systems have a common disadvantage—the need to use toxic reagents.Thus, we propose a non-toxic diagnostic system based on polyelectrolyte microcapsules for quick, cheap and highly selective qualitative and semi-quantitative determination of SDS in the medium. More

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Current and past scenariosThe Arbutus unedo distribution presence database and eight environmental variables were used to predict the species potential distribution under past, current, and future climatic conditions. The current fitted model predicted the A. unedo suitable habitat with good performance, when evaluated using AUC (AUC = 0.92)31.The results suggest that the species distribution was mainly determined by the following attributes: (1) mean temperature of the driest quarter (BIO9), (2) annual mean temperature (BIO1), (3) slope and (4) temperature annual range (BIO7), summing up to 78.6% of the model’s total contributions (Table 1). In a recent study for the same species in Portugal, the authors considered that the variables precipitation seasonality (BIO15) and slope had a net significant influence on the species’ habitat suitability16. The variable slope was expected to influence the A. unedo distribution, since this tree is ecologically adapted to rocky slopes, which are edaphically unsuitable for woodland, where the vegetation is limited to shrub communities. The Arbutus unedo is particularly intolerant to shade, and this high light requirement is particularly needed for fruit production, restricting this species to settlement with open habitats21. This aspect was confirmed in a study about a woodland disturbance, where the A. unedo widespread initially, and, afterward, the species declined as canopy woodland re-developed35.The Arbutus unedo was adapted to a wide range of climatic conditions considering the response curves analysis (Fig. 1). According to Santiso24, this species developed a conservative strategy in the use of nutrients and water, when they are scarce. The Arbutus unedo plasticity and evolvability explained the current species persistence throughout its distribution range, which will be decisive in future response to climate change. The species distribution prediction for the present is in agreement with the species distribution according to Caudullo et al.38 (Fig. 6). Indeed, the A. unedo is widespread in Portugal, Galicia, and southwest of Spain, occupying the coastal belt from Tunisia to Morocco along the North of Africa, and from the Iberian Peninsula to Turkey across southern Europe, yet occurring in the northern Iberian Peninsula, western France, and south-western Ireland. The results showed that MaxEnt fitted model suitability prediction was high in those regions, which was further supported by the high observed AUC. Furthermore, it is important to stress the current potential distribution meaningful accuracy, and, therefore, the ability to extrapolate past and future prediction scenarios for A. unedo distribution in the Mediterranean Basin. Not surprisingly, the highest predicted suitability for the species occurs mostly in its native range, in regions where the mean January temperature is above 4ºC, a limit required for the species’ survival23. Unfortunately, this variable is only available in the WorldClim database for the present, but not for the future or past periods. Nevertheless, the species’ current spatial distribution was overlapped to the areas with mean January temperature higher than 4ºC, and a high overlap is reckoned, thus confirming this factor ecological importance17,23. The analysis of the species’ predicted suitability show that high-suitability areas [0.8–1.0] are more frequent in current conditions, compared to other scenarios, and thus confirming the suitability of climate current conditions for the species’ development (Fig. 2d).Figure 6Arbutus unedo L. distribution area and occurrences. Black dots represent the species’ occurrences (Supplementary Table S1), and the green area represents the species native distribution range for A. unedo downloaded from the data sets in38. Equal-area projection EPSG:3035. The map was made using the sf 1.0–3 and the terra 1.4–14 (https://rspatial.org/terra/) packages in R 4.1.032,33.Full size imageThe predicted habitat suitability for the LGM and MH scenarios differed, as expected, from the obtained for the current climatic conditions. The climatic conditions in the MH, were more suitable than during the LGM, due to climate warming, thus allowing the species expansion. According to the LGM projections, the species occurred in the Mediterranean islands (Sicily, Sardinia, and Balearic), North of Italy, south and eastern coast of Spain, Catalonia, North Africa, and spots in Portugal, Greece, and Turkey (Fig. 2a). These were suitable putative cryptic refugia areas that remained during the LGM. Keppel et al.39 defined refugia as locations to which species retreated during periods of adverse climate, and could potentially expand from, when environmental conditions turn out to be more suitable for the species. These locations/habitats were responsible for the species’ survival under changing environmental conditions for millennia. Identifying and characterizing climate refugia provides an important context for understanding the modern species distribution development, traits, and local adaptation40. The Iberian, Italian and Balkan peninsulas, which remained relatively ice-free during the ice ages were identified as the main glacial tree refugia areas in Europe41,42.The Betic mountains, in the Iberian Peninsula (IP), were sought to be a species’ refuge from the last ice age, nearby the sea (Valencia region, Spain), where fossil pollen pieces of evidence were found in the Canal de Navarrés peat deposit4, with 34 ka, and in the Siles lake (ca. 19 ka) located in the Segura mountains of southern Spain43 (Fig. 5, numbers 1 and 4, respectively). These mountains were ice-free during the Late Pleistocene, and the persistence of a mild climate could explain that other thermophilic species have prevailed in this particular region during the Full Glacial, proved by high genetic diversity levels44, and with the pollen spectra suggesting the region as a glacial refugium for temperate and Mediterranean trees9,13,43.The Iberian Peninsula border could have been a refuge for the species, due to the importance of the sea as a temperature regulator. Indeed, pollen evidence was found in the Basque mountains (northern IP, Fig. 5, number 3: 21 ka)45. Despite the information given by the MaxEnt modelling, the A. unedo reconstruction for the LGM was observed in other areas, since paleo-evidences were found in unexpected northern sites (Donatella Magri, personal communication). Certainly, fossil charcoal pieces of evidence were found in the central region of Portugal (Serra do Sicó), nearby the sea, supporting the presence of thermophilous taxa, including A. unedo, during the Full Glacial (24 ka, Fig. 5, number 2), and the presence of other Mediterranean taxa, such as olive tree and Pistacia lentiscus L., suggested that this may have been refuge zones for thermophilous plants46. Corroboration of the species expansion later during the warm and humid period of the Holocene (see Fig. 3b; green area) was confirmed by pollen remains (Fig. 5), particularly inland and northwardly, in the IP, and, also, in a molecular population genetics study made in Portugal, with northward haplotype migration47.Santiso et al.17 in a study about the A. unedo phylogeography, concluded that this species had two clades, separated during the late Pleistocene, before the LGM, suggesting that it may have coincided with the hardest glaciations recorded in the Quaternary. One clade occupied the Atlantic Iberia and, possibly, North Africa, while the other occurred in the western Mediterranean Basin in Spain. Besides, the A. unedo possibly persisted in the late Quaternary in the western Mediterranean, based on chloroplast DNA observations, and the results from the current study supported this interpretation, and by the fossil evidence (Supplementary Table 3). The same strong genetic differentiation between the western and eastern Mediterranean Basin was also found in olive lineages, and this pattern is congruent in other Mediterranean shrubs and tree species9,13,15. In another species (maritime pine), Bucci et al.44 confirmed the existence of a genetic divide between eastern and western lineages, also previously described by Burban and Petit48 based on mitochondrial DNA. Additionally, maximum haplotypic diversity for this species was found in south-eastern and central Spain, which, therefore, may be considered a biodiversity hotspot and a strong signal for refuge, as long-term populations tend to harbour more diversity than recently expanding ones41. In their study with A. unedo in Portugal using cpSSR, Ribeiro et al.47 found signals of two putative refugia in southern and central littoral in the country, also supported by macrofossil and pollen remains. Furthermore, according to Médail and Diadema41, several regions in the Western Mediterranean (large Mediterranean islands, North Africa and Catalonia), could have played a role in the case of A. unedo, and sought as refugia locations.The Arbutus unedo suitability maps obtained for past conditions support the claim that MH climatic conditions were more suitable for the species expansion than the LGM ones (Fig. 2a,b, and d). Consequently, spatial distribution changes analysis between past and current scenarios (Fig. 3) showed that A. unedo potential distribution expanded extensively from the LGM-current and the MH-current periods, indicating that more suitable areas were available for the species at present. Pollen records during the Early-Middle Holocene supported the Mediterranean presence of this species, in particular, nearby the sea (Fig. 5), which is under the MaxEnt predicted eastward dispersion (Fig. 3a, b). In another species bird-dispersed, the Myrtus communis L. (myrtle), a spread from west to east was verified, following genetic differentiation during the Pleistocene15, since the western region of the Mediterranean Basin had a milder climate, compared to the eastern one, during the LGM.The Arbutus unedo had, probably, a considerable ability to disperse, migrating over thousands of kilometres and even crossing sea stretches, allowing the species expansion during the MH, when the climatic conditions became more favourable17. In the future, A. unedo migration possibility will depend on climatic change pace and, also, on seed dispersal, particularly long-distance dispersal events49, since long-distance migration fitness will be useful in future change scenarios, allowing species to progress to newly suitable areas, as happened in the past. Nevertheless, from past evidence in Ireland, Britain, and across Europe, trees migrated faster than would be expected, during the warming period at the beginning of the Holocene, and that biotic and/or abiotic vectors must have been involved in dispersal50. This is the case with A. unedo, the seeds are dispersed by different type of birds that eat the soft fruit and, some of them are migratory species, as the European robin (Erithacus rubecula L.)51.Future scenariosThe impact of climate change on the A. unedo potential distribution was assessed under two representative concentration pathways (RCP 4.5 and RCP8.5) for the years 2050 and 2070. The results showed that climate change, under both moderate and high emission scenarios, will affect the species distribution range. A decrease in the predicted suitable areas was, generally, observed, since the climate becomes less favourable for the species in the future.According to the results, under the RCP 4.5 scenario, the potential distribution area will increase up to 2050, and decrease afterward according to the prediction for 2070 (Fig. 2d, e, f). Considering the RCP 8.5 scenario, the suitable area will exhibit a net decrease from the present up to 2050 and continue this trend until 2070. It is also expected that he medium–high suitable areas will gradually decrease from the present to the future, especially for scenario RCP 8.5. These results are in agreement with those obtained by other authors, confirming that Mediterranean species (e.g. Quercus sp.) distribution areas will be negatively affected by future climate change52,53.Several studies concluded that global warming will influence species distributions by causing expansions, contractions, or shifts in the species ranges2. As expected, the results from the current study showed that suitable areas will contract under future climate scenarios when compared to the current conditions, though suitable areas will emerge. These effects’ impact will depend on the climate change scenario severity. Despite contraction and expansion effects, the presence of the species will gradually decrease from the 2050s to the 2070s. Moreover, a species’ shift toward the North will be verified, because of suitable areas emergence, observed mainly in the RCP 8.5 scenario. Those areas will mostly emerge in the North of France, South of the United Kingdom, and Ireland, implying species’ latitudinal migration. The species’ northward displacement is consistent with climate change studies’ results obtained by several authors, including A. unedo16,20,54,55. Nevertheless, according to Gerassis et al.20, under climate change, the expected habitat disruption and fragmentation could lead to very adverse conditions for A. unedo survival in the future, which could undoubtedly conduct to a possible species’ presence decline in most of the current distribution area. Moreover, distribution models that predict climate‐induced range shifts do not account for spatial dispersal variation56, but adaptive dispersal evolution always reduced neutral genetic diversity across the species’ range. This means that the species’ genetic pool might be erased, depending on the climate change velocity, amongst other conditions, like landscape fragmentation and competition with other species/crops16,57. Additionally, the species ability to migrate mainly through seeds, dispersed by migratory frugivorous birds, bird abundance, and the velocity of climate change, are key issues for future species survival23,58.These results suggested that future changes in environmental conditions may lead to suitable habitat loss in areas where the species had persisted and with a possible range shift towards the North. These findings also revealed that with continuous future climate warmth, the current potential distribution A. unedo areas will become unsuitable or contract, leading to significant changes in the species’ current distribution pattern and putative presence loss. The possibility of species’ migration will ultimately depend on its capability to keep pace with the changing conditions and the velocity to adapt to environmental changes, such as those presented by habitat and climate modifications56.The Mediterranean Basin is one of the most vulnerable climate change hotspots in the world, thus understanding how future climate changes will disturb Mediterranean plant species distribution will be key for tree management planning and conservation design. Nevertheless, further investigation is needed for species well adapted in this region to assess the impacts of climate change in their current and future potential distributions. Those studies including past climate impact on species distribution should be complemented with phylogeographic methods and paleoclimate reconstructions to locate refuges. Other species distribution models, besides MaxEnt, could be tested, although the lack of absence records data limits considerably the modelling approach. Additionally, the human influence magnitude on the predicted ecological niche should be further studied in detail, including urbanization, industrialization and putative tourism pressure, particularly in coastal areas. More

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Forest fires are disasters that cause extensive damage to the entire world in economic, ecological, and environmental ways. These fires can be caused by natural reasons, such as high temperatures that can create spontaneous combustion of dry fuel such as sawdust, leaves, lightning, etc., or by human activities, such as unextinguished campfires, arson, inappropriately burned debris, etc1. According to research, 90% of the world’s forest fire incidents have occurred as a result of the abovementioned human carelessness1. The increase in carbon dioxide levels in the atmosphere due to forest fires contributes to the greenhouse effect and climate change. Additionally, ash destroys much of the nutrients in the soil and can cause erosion, which may result in floods and landslides.
At earlier times, forest fires were detected using watchtowers, which were not efficient because they were based on human observations. In recent history and even the present day, several forest fire detection methods have been implemented, such as watchtowers, satellite image processing methods, optical sensors, and digital camera-based methods2, although there are many drawbacks, such as inefficiency, power consumption, latency, accuracy and implementation costs. To address these drawbacks, a forest fire detection system using wireless sensor networks is proposed in this paper.Wireless sensor networks (WSNs) are self-configured and infrastructure-free wireless networks that help monitor physical or environmental conditions and pass these data through the network to a designated location or sink where the data can be observed and analyzed3. Efficiency and low power consumption are the major advantages of a WSN. In the proposed detection system, wireless sensor nodes are deployed according to cellular architecture to cover the entire area with sensors to monitor temperature, relative humidity, light intensity level, and carbon monoxide (CO) level using a microcontroller, transceiver module, and power components. The power supply to the sensor node is provided using batteries as the primary power supply, and solar panels are used as the secondary power supply. These sensor nodes are specially designed with a spherical shape to withstand damage caused by environmental conditions as well as animals.The sensor readings for each parameter are checked with a preset threshold ratio and a ratio that is calculated continuously in the node in real time, and only the ratios that exceed the preset ratio are sent from the sensor node to the base station for further analytical processing. The network utilized for this transmission is in the architecture of tree topology considering facts such as low power consumption, reduced latency, less complexity, etc. Cluster heads are used in this network to gather data from several sensor nodes and pass them on to the base station or the gateway node. The gateway node is an interface that connects the network with the secondary analysis process.For the analysis process, a machine learning regression model was used along with threshold ratio analysis to enhance detection accuracy. For the training and testing process of the model, data were collected during the fire and no fire situations in different areas and under different climatic conditions. During the data collection process, 7000 data samples were collected, where a data sample included temperature, relative humidity, light intensity level, and CO level at a particular time. Eighty percent of the collected data were randomly used as training data for the model, and the remaining 20% were used as test data.If the outcome of the machine learning model indicates a fire in a specific area, a text message will be sent to the mobile phone numbers of the authorized officers in responsible units. As this process is designed with a minimum delay, the fire can be detected within the initial stage, and the responsible parties can take necessary actions in a shorter period, which will minimize the damage.Related workForest fire detection has been a focus of many researchers for the last decade because of increased forest fire case reports from all over the world due to severe damage to society and the environment. Many methods have been proposed to detect forest fires, such as camera-based systems, WSN-based systems, and machine learning application-based systems, with both positive and negative aspects and performance figures of detection. Due to the higher probability of accurate and early detection due to the use of multiple sensor sources and deployment of sensor nodes in areas not visible to satellites, wireless sensor networks have a more positive outlook, and they have become the more applicable technology in many fields4.Many researchers have focused on environmental parameters, such as air temperature, relative humidity, barometric pressure, sound, light intensity, soil moisture, and wind speed and direction, along with gases, such as CO, CO2, methane, H2, and hydrocarbons apart from smoke, to detect forest fire conditions by considering the variations in these parameters during a fire5,6, and sensors have been selected according to the range, sensitivity, power consumption, and cost7,8.As supplying power to a sensor node is a challenging task in forested areas, utilizing only battery options is difficult because they do not last long, and distributing power using a wire would require a higher cost to deploy in a large forest. Therefore, many researchers have proposed solar-powered systems as secondary power sources along with rechargeable batteries as the main power source4,6, while some researchers have proposed solar batteries because they last longer9. To reduce the power consumption of sensor nodes, techniques such as keeping selected components active while others are deactivated have been proposed10,11,12.Most WSN-based detection systems are centered around a base station due to the memory and processing limitations of the nodes. Important and partially processed data are transferred to the base station through wireless media for processing and enabling relevant actions, while the base station also acts as the gateway between the sensor nodes and the system user4,9.When constructing a WSN, communicating data among the relevant entities is the main objective, and star topology and mesh topology-based networks have been proposed in many papers because of the different attributes in their systems. A mesh topology was chosen over a star topology because of its ability to self-organize, self-configure, and automatically establish among nodes in a network13. As a smaller number of nodes involved for transmission results in minimum energy consumption, concepts based on cluster heads have been used14. To minimize the loss of energy and data packages during transfer, a cluster-tree network topology structure was proposed15. Considering the sensing range of a node, fault tolerance, and energy consumption, a paper has proposed applying the on-demand k-coverage technique that provides event detection using static nodes with variable sensing ranges. This technique utilizes the maximum detection performance with the minimum power consumption for an event16. A survey on rare event detection has mentioned many event detection strategies that deliver maximized detection capability, minimized detection delay and low energy consumption, such as duty cycle, component deactivation, overpopulation/node redundancy, collaboration, and energy harvesting17.To reduce deployment cost and power consumption, a paper proposes a novel localization scheme that divides the whole forest area into different grids and allocates them to respective zones with another 8 neighboring grids. One centroid node from those grids, which is called the initiator node, predicts whether the zone is highly active (HA), medium active (MA), and low active (LA). Here, HA zones send data continuously to the base node through the interior node, MA zones send data periodically, and LA zones do not transfer data in the status that manages power consumption effectively18. Another author proposed obtaining data from the sensors every 2 min if there is the potential of a forest fire or obtaining data every 15 min otherwise to reduce the energy wastage19.To place sensor nodes in the most effective configuration to detect fire conditions, a sensor node was proposed at three different heights to perform different parameter measurements, while some authors have suggested covering sensor nodes to avoid direct sunlight exposure and minimize the false alarm rate4,10. As the network connectivity of service providers in forest areas is not robust, communication techniques that use dedicated network paths such as LoRa, ZigBee, and XBee have been used as the communication infrastructure. When considering attributes such as transmission range, high security, low power consumption (LPWAN protocol), and other relevant configurations, most papers have suggested using the LoRa module for transmission13,19.Most papers have suggested having threshold value-based fire detection on a sensor node, and if the exceeded threshold has remained the same, then a sink determines the location and will send an alarm to the fire department11. Because of the environmental parameter variations according to the place and time, threshold values are configured by the user considering geographic situations, climatic changes, seasonal changes, etc. after sensors obtain the data from the surrounding10.A fusion information process was proposed, where information from multiple sources is considered in making the final decision, which is better than using those sources individually, and two algorithms based on the threshold ratio method and Dempster-Shafer theory were used4. To enhance the detection accuracy, machine learning applications have been proposed in many papers based on different machine learning approaches, such as support vector machine (SVM) classification14 and regression techniques, such as logistic regression. However, applying machine learning techniques to fire detection systems has many limitations, such as the limited amount of energy, the energy required for data processing, the short range of communication and limited computations, the complexity of ML algorithms when executing on sensor nodes, and the difficulty of being distributed on every sensor node20,21. More

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Symbiont diversity and distributionThe present study provides the first evidence of symbiosis in R. alisae, a species of nudibranchs. This is the most multiple symbiosis that have ever been recorded for marine invertebrates. While many organisms establish an exclusively one-on-one relationship with a single microbial species or microbes belonging to the same functional group5,12, there are also organisms that harbor multiple microbial species, in which symbiont–symbiont and host–symbiont interactions occur. Six phylotypes of chemoautotrophic bacteria were reported for mussel Idas sp. from a cold seep area11 and five extracellular symbionts for the gutless oligochaete worm Olavius algarvensis34. However, in these cases, symbioses involving bacteria and marine invertebrates are either endosymbiotic microbes co-occurring inside the host bacteriocytes5,11 or ectosymbiotic microbes associated with the external surfaces of the animals3,4,9,15,34, with the exception of scaly-foot snail from hydrothermal vents having partnerships simultaneously with epi- and endosymbiontic microbes35.Bacterial symbionts in R. alisae have appeared to be more diverse than was previously known for marine invertebrates. It is evident that the detected symbiont phylotypes differ greatly from all other known symbionts found in marine invertebrates. Labrenzia (Rodobacteriales) and Maritalea (Rhizobiales) have not been recorded as forming symbiotic associations with invertebrates or plants so far, although other members of the families Rodobacteriales and Rhizobiales are well known symbionts14. Strains of Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas are reported as symbionts of plants, interacting with a vast majority of nodulating legume species and efficient in biological nitrogen fixation36. This may be important when considering the nature of these symbionts in the nudibranch. Symbioses between cyanobacteria and marine organisms are commonly found among marine plants, fungi, sponges, ascidians, corals, and protists37,38. Synechococcus, identified as dominant symbiont clones of R. alisae (Table S2), is a unicellular cyanobacterium common in the marine environment, providing a range of beneficial functions including photosynthesis, nitrogen fixation, UV protection, and production of defensive toxins8,9,37. Symbiotic interactions between actinobacteria and their host have been observed in insects, human, animals, and plants, where the bacteria provide the host with protection against pathogens and produce essential nutrients39. However, none of the members of the clade Actinobacteria recorded in R. alisae are known to live symbiotically.Arrangement of symbiotic associationDespite the high diversity of bacteria, they are well organized in the host. Dense clusters of rod-shaped bacteria, Labrenzia, Maritalea, Bradyrhizobium, Burcholderia, Achromobacter, and Stenotrophomonas, were found within host-derived vacuoles, referred to as bacteriocytes, inside epithelial cells of R. alisae (Fig. 3). Although such arrangement differs from that typical of bacteriocytes, which are usually considered as specialized cells of the hosts for harboring bacteria, it resembles that reported for scaly-food snail from hydrothermal vents, which harbor symbionts in the esophageal gland35. Bacteriocytes in the gastropod Lurifax vitreus found near hydrothermal vents also constitute a portion of the mantle epithelium; they have large vacuoles containing many live and dividing bacteria40. Each bacteriocyte was densely packed with certain symbionts, and the bacteriocytes were randomly distributed within the epithelium cells. A distinctly regular distribution pattern was observed in the gill epithelium of the mussel Bathymodiolus sp.: the thiotrophic symbionts occupy the apical region, and the methanotrophic symbionts are more abundant in the basal region of bacteriocytes4. In the mussel Idas sp., however, there is no spatial pattern of the six distinct bacterial phylotypes, and the symbionts are mixed within bacteriocytes11.Synechococcus dominated the cytoplasm of intestinal epithelium and, more rarely, epidermis cells, mainly as specialized cell type referred to as nitrogen-fixing heterocysts. They are visually similar to cyanobacteria from corals and sponges8,37.The phylogenetic diversity and the spatial organization of the symbiotic community in R. alisae were determined by the 16S rRNA analysis, which was consistent with the results of FISH and TEM. Unlike most symbioses of marine invertebrates when bacteria house specialized host cells5,11 or cover epidermis7,15, symbiotic association of R. alisae exhibited spatial partitioning between symbionts, which were unevenly distributed between the tissues (Table S2). It has been established that different members of the microbial community can complement each other in acquisition of various restrictive nutrients, confirming the importance of the functional diversity of symbionts41. Thus, Stenotrophomonas rhizophila and Bradyrhizobium build a beneficial association in the rhizosphere and can act synergistically on promoting growth and nutrient uptake of soybean36. Cyanobacteria can interact synergistically with beneficial members from the endophytic microbiome of rice seedlings42. The location of bacterium in the organism of R. alisae may, in fact, depend on the specific metabolic and ecological roles that the symbionts play, and also on the interaction with bacterium belonging to different physiological groups.Nature of symbiosisSymbiotic associations between microbes and invertebrates are acquired mainly in a nutrient-depleted environment where symbionts usually provide their hosts with essential nutrients and high-energy compounds1. In contrast to known symbioses between microbes and gutless invertebrates, which obtain nutrients exclusively from the bacteria, R. alisae, like most nudibranch species, is a sponge-eating predator. However, due to the lack of adipose tissue, sponges are distinguished by a low lipid content (0.4 to 1.5% of wet weight)43 and also by specific proteinaceous spongin fibers and chitin, a polysaccharide similar to cellulose that can be indigestible for some predators, which together indicate their low nutritional value. Furthermore, R. alisae feeds exclusively on the sponge O. pennata; therefore, in habitats with low prey availability, this nudibranch has to survive starvation while searching for sponge assemblages. We suppose that symbiotic bacteria of R. alisae contribute to the utilization of low-quality food, similarly to symbiotic bacteria from the genera Rhodobacter, Burkholderia, and Aeromonas associated with the detritivorous isopod Asellus aquaticus44.A fatty acid analysis, as a useful approach to clarifying the nature of symbiosis5,20,32, has confirmed the trophic interaction between symbionts and the nudibranch host (Table S2). Among the fatty acids of symbiotic bacteria in R. alisae, OBFA are a major acyl constituent of membranes in Stenotrophomonas45 and also in Actinobacteria, Arthrobacter, Iamia, Ilumatobacter, and Kocuria46. Cis-vaccenic acid is a major component of Maritalea30. Omega-cyclohexyl tridecanoic acid (cyclo19:0) is specific to Bradyrhizobium47, Burkholderia, and Achromobacter48. Linoleic acid is produced by cyanobacteria including marine species of Synecoccocus33; in nudibranch, it obviously serves as a precursor in the synthesis of arachidonic acid (20:4n-6), thus, providing additional evidence for the transfer of fatty acids from symbionts to the host. Mollusks are capable of converting linoleic acid to arachidonic acid, since they have enzymes required for its synthesis21. The presence of these bacteria-specific markers and the abundance of arachidonic acid confirm the metabolic role of symbionts in the nudibranch host.Among nutrients, biologically available nitrogen can be considered a restrictive nutrient for the sponge-eating R. alisae, which can be acquired with the help of nitrogen-fixing symbionts, also referred to as diazotrophs. R. alisae harbors Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas that are efficient in biological nitrogen fixation previously found to be associated with nodulating legume species36. Symbiotic nitrogen fixers are known to be associated with a variety of marine invertebrates such as wood-boring bivalves, corals, sponges, sea urchins, tunicates, and polychaetes7,8,37. Moreover, the protection of the enzyme nitrogenase that catalyzes N2 fixation against oxygen is an important physiological requirement in bacteria such as symbiotic Bradyrhizobium, Burkholderia, Achromobacter, and Stenotrophomonas that are located in bacteriocytes and provide this protection. Synechococcus is known as a nitrogen-fixer37,49. It performs N2 fixation in heterocysts where nitrogenase is restricted under oxic conditions. Indeed, heterocysts of Synechococcus are abundant in the intestine cells of R. alisae (Fig. 5B–D).Nitrate assimilation is one of the major processes of nitrogen acquisition by many heterotrophic bacteria and cyanobacteria50,51. Symbionts of R. alisae can play an important role in the process of nitrate utilization through denitrification, dissimilatory nitrate reduction, and assimilatory nitrate reduction as a nitrogen source and synthesize it into organic nitrogen. The nitrate reducers, Labrenzia52, Stenotrophomonas53, Maritalea30, and Rhodobacteraceae29 are widely represented in R. alisae. Synechococcus also utilizes nitrate, nitrite, or ammonium for growth50. Thus, symbiotic bacteria may play a significant role in the N-budget of the nudibranch mollusk.The symbiotic bacteria of R. alisae, including Bradyrhizobium, Maritalea, Labrenzia, Burkholderia, Achromobacter, Stenotrophomonas, Arthrobacter, Iamia, Ilumatobacter, and Kocuria, are known as carboxydotrophic or carbon monoxide (CO) oxidizers54,55. Despite the toxicity of CO for multicellular organisms, numerous aerobic and anaerobic microorganisms can use CO as a source of energy and/or carbon for cell growth56. The marine worm Olavius algarvensis establishes symbiosis with chemosynthetic bacteria using CO, a substrate previously not known to play a role in symbiotic associations with marine invertebrates, as an energy source57. We do not rule out that the R. alisae symbionts also might exploit CO as carbon and energy source. Despite this, assumption may seem impossible taking in account the CO toxicity, but, since many invertebrates (mollusks, tube worm, etc.) use toxic sulfate, thiosulfate, and methane as an energy source1,15, this hypothesis is worth to be addressed.An important component of skeleton in marine sponges of the family Microcionidae, including O. pennata, is the structural polysaccharide chitin58. Some bacteria are capable of hydrolyzing chitin via the activity of chitinolytic enzymes and can utilize chitin as a source of carbon, nitrogen, and/or energy59. Chitinase activity was documented for strains of Labrenzia60, Burkholderia61, Arthrobacter62, Achromobacter63, Stenotrophomonas64, Alcaligenes65, and actinobacteria59 associated with R. alisae. Thus, these bacteria can work synergistically to digest chitin and spongin, contributing to feeding success of the host nudibranch which depends solely on low-quality, nitrogen- and carbon-deficient food available.Furthermore, direct evidence has confirmed that many bioactive compounds from invertebrates are produced by symbiotic microorganisms66,67. Many biologically active compounds including toxic and deterrent secretions have been identified in nudibranchs of the family Discodorididae68. Symbiotic bacteria may exhibit toxic activity to provide the host nudibranch with chemical defense against predators and environment. Bacteria, especially actinobacteria, living in a symbiotic relationship with R. alisae may help the host in defense, since nudibranch lack a shell, and secondary metabolites of bacteria can provide them with chemical defense against predators and environment, as has been reported for some marine invertebrates2,9,10.In complex associations, the integration and coexistence of symbionts depend on supplementary partnerships and mutual contribution to the host’s metabolism41. The most intensively studied cases are highly specialized associations, where both partners can only exist in close relationship with one another. The relatively high diversity of microbes in R. alisae complicates understanding the complex pattern of molecular and cellular interactions between the host and its symbionts. More

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