Neurospora crassa, relies on the development of conidiophores to produce new spores. Environmental, temporal, and genetic components of conidiophore development have been well characterized; however, little is known about their morphological variation. We explored conidiophore architectural variation in a natural population using a wild population collection of 21 strains from Louisiana, United States of America (USA). Our work reveals three novel architectural phenotypes, Wild Type, Bulky, and Wrap, and shows their maintenance throughout the duration of conidiophore development. Furthermore, we present a novel image-classifier using a convolutional neural network specifically developed to assign conidiophore architectural phenotypes in a high-throughput manner. To estimate an inheritance model for this discrete complex trait, crosses between strains of each phenotype were conducted, and conidiophores of subsequent progeny were characterized using the trained classifier. Our model suggests that conidiophore architecture is controlled by at least two genes and has a heritability of 0.23. Additionally, we quantified the number of conidia produced by each conidiophore type and their dispersion distance, suggesting that conidiophore architectural phenotype may impact N. crassa colonization capacity.The vegetative life cycle in the model filamentous fungus,  Neurospora crassa propagates asexually through the dissemination of haploid spores, conidia, which develop via specialized aerial structures called conidiophores [fluffy (fl), a necessary regulator of conidiophore development [N. crassa have been well characterized [iophores . Macrocoiophores . Since miophores . After eiophores . It is telopment ,6. Whilecterized , little Aspergillus niger following deletion of velvetA, directly impacting both spore dispersion and colonization capacity of the organism [acropetal locus of Magnaporthe grisea modifies arrangement of the developing spores in a conidiophore, resulting in spores that are nonpathogenic [N. crassa, estimate heritability of these phenotypes, and examine their potential impact on sporulation. To do this, we employed a wild population collection of 21 N. crassa strains collected from Louisiana, United States of America (USA) [In other filamentous fungal species, conidiophore architecture has been shown to directly impact the ability of an organism to disseminate throughout an environment and infect host tissues. For example, conidiophore morphology is altered in organism . Additiothogenic . Given tca (USA) . These ica (USA)  and charca (USA) ,13. Our ca (USA)  specificWild Louisiana isolates were obtained from the Fungal Genetics Stock Center   and are Cultures for time-course images were grown following the same protocol with nitrocellulose membranes removed at 20, 25, or 35 h for imaging of conidiophores at different stages of development.Crosses were performed in the dark on cornmeal crossing medium, after which ascospores were plated on sorbose + fructose + glucose (SFG) media. Colonies were subsequently picked to isolate random ascospore progeny. Strains selected for crossing were FGSC8872 (WT) × FGSC3943 (Bulky), FGSC8872 (WT) × FGSC8876 (Wrap), and FGSC2229 (Bulky) × FGSC8876 (Wrap). Crosses were conducted in duplicate with 25 progeny selected from each. To isolate and image conidiophores in a high-throughput manner while preventing fusion of different progeny sharing a plate, each strain was inoculated on a 1 mL 1.5% agar droplet containing 1.8% glucose/1.8% fructose with 1× Vogel’s media and recommended biotin and trace element supplements. Each 150 × 15 mm petri dish contained eight agar droplets evenly spaced roughly 2.5 cm apart. Each droplet was inoculated with progeny conidia and incubated at 30 °C for 20 h to allow sufficient mycelial growth without hyphal fusion between droplets. Each droplet was then harvested onto a separate nitrocellulose membrane with 0.45 µm pore size. Each membrane was inverted onto a new agar droplet as described above and placed under light for aerial hyphae to penetrate the membrane. After 25 h, membranes were removed from the agar and secured on a flat surface for imaging of conidiophores.Nitrocellulose membranes containing conidiophores were visualized on an inverted microscope  at 20× magnification and brightfield images were taken with a charge-coupled device (CCD) camera . Multiple z-slices were captured and overlaid in ImageJ  to conveResidual nets (ResNet) was used to classify brightfield images into the three phenotypic classes by transfer learning . After sWe balanced the three classes of conidiophore phenotypes and separated the dataset into training (543), validation (66), and test sets (66). An external test set generated from progeny conidiophore images was collected in a separate batch and used for further validation . The training set was used to train the neural network and update its parameters. The validation set was used for updating learning rates and finding the final model to alleviate overfitting . The tesFeature importance was also evaluated on the test set. Features were evaluated on an integrated gradient , and thehttps://github.com/michaelSkaro/image_classification/tree/master/src).The codes for training and visualization are shared on GitHub  were inoculated on 1.8% glucose/1.8% fructose/1.5% agar plates with 1× Vogel’s media, biotin, and trace element supplements and incubated at 30 °C for 30 h. Three biological replicates of each strain were performed. Cultures were then harvested onto an 82 mm diameter nitrocellulose membrane and inverted onto a new agar plate as previously described above. Plates were placed under light for 32 h to allow penetration of aerial hyphae, development of conidiophores, and subsequent sporulation. Nitrocellulose membranes were then removed, and conidia were suspended into 50 mL of water and counted and sized with the Cellometer Auto 2000  ,25.To compare dispersal distance traveled by spores of each phenotype, the same representative strains listed above were inoculated onto the same growth medium. After 20 h at 30 °C, cultures were harvested onto a 60 mm diameter nitrocellulose membrane, inverted onto a new plate, and set under light for 30 h. Membranes were then removed and placed at the center of a 245 mm square bioassay dish, where SFG medium surrounded a 60 mm diameter blank space now occupied by the membrane. The dish was placed under light for 48 h to allow for sporulation and subsequent colonial growth. At this time, pictures of each plate were taken with an iPhone XS, and contrast enhancement was performed in ImageJ. ImageJ was used to measure distance from the center of each nitrocellulose membrane to the center of each colony. Two biological replicates were conducted for each strain.N. crassa  of the phenotypic classes using the guidelines described above . Many stTo ensure the three phenotypes were not just representations of different time points along the same developmental trajectory, we captured images of strains strongly representing each phenotype at 20, 25, 30, and 35 h after transferring cultures to the nitrocellulose membrane, with 30 h as the initial reference point for mature conidiophore images. It is important to note that each time point was collected from a separate set of cultures, and these are not time-series images. WT, Bulky, and Wrap phenotypes were presented as early as 20 h and maintained until sporulation captured at 35 h, suggesting that the three architectural phenotypes are not a result of temporal disparity along the same developmental trajectory .2 = 4.9196, df = 10, p = 0.8965) (n = 3) were collected from sugarcane, although WT and Bulky strains were also found on this substrate. WT strains were found on all six substrates , and Bulky strains were found on sugarcane, grass burn, and pine burn. We also did not find any clear relationship between most prominent phenotype and the town from which each strain was collected, as reported by FGSC   (chi-squ 0.8965) . Interes by FGSC .Manually categorizing conidiophores into phenotypic classes is time-consuming and introduces potential bias. To streamline the future classification of additional conidiophore images, an automated classification process was developed. We demonstrated that an accurate image classifier could be constructed based on the limited conidiophore image dataset of 543 training samples and presents reasonable performances .Images from both the test and the validation set can be accurately classified at rates of 76% and 79%, respectively . TestingThree crosses were performed between representative strains for each architectural phenotype: FGSC8872 × FGSC8876 (WT × Wrap), FGSC8876 × FGSC2229 (Wrap × Bulky), and FGSC 8872 × FGSC3943 (WT × Bulky). A total of 50 progeny were selected from each cross, from which conidiophores were isolated and imaged. For efficiency, these 1932 images were each assigned a phenotype by automatic classification as described above. These phenotype counts were then used to estimate an inheritance model for this discrete complex trait, described below .Emericella (Aspergillus) nidulans, at least three genes control conidiophore development [ij, sum to 1. The data tabled below are the multinomial counts of progeny from each of the three crosses  are linear in the parameters of the model, such as the allelic effects; thus, the model is sometimes referred to as a loglinear model [A denotes an allele associated with the WT phenotype, B denotes an allele associated with the Wrap phenotype, and C denotes an allele associated with the Bulky phenotype. These counts are stored in a 9 × 1 vector ar model . Much ase models .Each model was fitted by the Method of Maximum Likelihood using iteratively reweighted least squares (IRLS) ,30. UndeThe fitting of this model with seven parameters or a simplification of this inheritance model was obtained from solving the below weighted least squares problem where Y = S + NAX−8. The process is initialized by solving the multiple regression problem with 2. The only thing to be changed for simplified models is the X matrix by removing the appropriate column(s) to eliminate a parameter or parameters not in a simplified model. The results for fitting the models with IRLS [−8 after nine iterations of IRLS are summarized in the table below.The matrix A and vector Y are evaluated with the current provisional parameter estimate ith IRLS  computed2 = 9.96, df = 5, p = 0.08). The conclusion was that at least two genes control conidiophore phenotype. An additive model and the model without gene effects was then used to assess the heritability. The heritability of H2 was typical for quantitative traits [The full epistatic model with three genes fitted the ratios from three crosses . The onle traits , namely,The maximum likelihood estimates of parameters for the inheritance model are tabled . The twoThe allelic and epistatic effects are fairly stable between the two models. The C (Bulky) gene appears to have the largest effect both additively and epistatically with both B (Wrap) and A (WT). There also appears to be a strong epistatic interaction between B (Wrap) and C (Bulky) alleles.Aspergillus has shown that conidiophore architecture impacts the colonization capability of the organism [N. crassa, we quantified the average number of conidia produced by each conidiophore phenotype, using a representative strain for each. There was no statistically significant difference in the number of conidia produced by each phenotype when suspended in liquid culture (Previous work conducted in organism . To inve culture . Additio culture .n = 334) developed compared to Wrap and Bulky , suggesting a difference in sporulation behavior in an aerial versus aqueous environment. We quantified the distance from the center of each nitrocellulose membrane to the center of each colony. The distribution of these distances are referred to as the spore shadow. Two replicate datasets were combined, as there was no significant difference between replicates of a phenotype following a two-sample Kolmogorov–Smirnov test [p = 3.101 × 10−5) (p = 0.02849) and Wrap and Bulky  by two-sample Kolmogorov–Smirnov tests  . No signov tests .N. crassa has been thoroughly documented over decades of study [N. crassa collection of Louisiana isolates provides a convenient tool for characterizing variation in a natural population and has previously been used to describe local adaptations and reveal novel gene functions [Conidiophore development in of study . Geneticof study ,31; howeunctions ,12,13. Functions . It is eBy utilizing this population set of 21 wild strains to explore morphological variation in the conidiophore, we identified three novel architectural phenotypes: Wild Type, Bulky, and Wrap. These three phenotypes are consistently displayed throughout conidiophore development and are likely not the result of temporal disparity along the same growth trajectory. Interestingly, no clear dependence of phenotype on collection location or substrate was observed, suggesting a genetic component to this variation. To further explore this, we conducted crosses between representative strains for each phenotype. By quantifying the subsequent progeny conidiophores of these crosses, we were able to fit a model for inheritance. Our resulting model suggests that at least two genes control conidiophore architectural phenotype with an estimated heritability of 0.23.con genes [attenuated (at) knockout mutant, where conidia form in dense aggregates [at mutants. Identifying and characterizing additional genes possibly underlying WT, Bulky, and Wrap phenotypes would provide greater understanding of conidiophore development through a morphological lens and perhaps lend insight into the natural population structure of these wild isolates.Future work should seek to identify these genetic variants and characterize transcriptional profiles for conidiophores of different architectural phenotypes. Many key genetic players of conidiophore development have already been identified and thoroughly characterized, most notably the on genes , and traon genes . Interesgregates . HoweverTo investigate a potential environmental impact of conidiophore architecture, we evaluated the effect of conidiophore phenotype on sporulation in both an aqueous suspension and aerial environment. No significant effect was detected on the number of conidia or average conidium diameter when suspended in water, nor was there any difference in wettability of conidia while washing off a plate. This indicates that the conidiophore architectural phenotype is not due to difference in hydrophobicity of the conidia. In an aerial environment, fewer conidia from Wrap conidiophores germinated compared to Bulky. Additionally, spore shadows by conidia from Wrap conidiophores fit a different distribution compared to that of both WT and Bulky groups. This suggests that conidiophore architectural phenotype may impact colonization capacity of the organism. Sporulation experiments should be conducted at a larger scale to more effectively determine the maximum dispersal distance of each conidiophore phenotype and, hence, effective population size and neighborhood size ,36. FurtIt remains a challenge to relate complex traits describing growth and form to their underlying genes . Doing s


The use of molecular markers can support the management of endangeredpopulations and should be combined with appropriate breeding strategies to improveproductive traits avoiding the decline of the breed. The genetic variability at 10microsatellite loci were investigated in a sample of 100 unrelated Markhoz goats. The investigated population was reared at the Sanandaj Markhozgoat Performance Testing Station in Kurdistan, Iran. Markhoz goat, a multipurpose breed,is one of the most valuable genetic resources in Iran. All the studied loci were found tobe polymorphic and a total number of 52 alleles were identified with an average number ofalleles of 5.2. Moreover, some population genetic indices, such as observed and expectedheterozygosity, observed and expected number of alleles, Shannon's index, Nei's expectedheterozygosity, and polymorphism information content were also calculated. Despite thedecreasing population size, Markhoz goat genetic diversity is still conserved. The breedseems to have a good level of genetic variability and, as a consequence, a potentialmargin of adaptability to environment and for future genetic improvement. Goats are able to provide high-quality productsunder diverse climatic conditions, being resilient to extreme and capricious environments. Moreover, local goat breeds playa significant role in the sociocultural life of the rural community and in preservingmarginal areas. .Undoubtedly, the effective management of genetic resources of farm animalsrequires comprehensive knowledge about breed characteristics, data on thesize and structure of the populations, and the geographicaldistribution of genetic diversity within and between breeds .Markhoz goat breed is one of the most valuable genetic resources in Iran. This breed iswell adapted to the environmental conditions of arid and semi-arid areas where it is usedas a multipurpose animal for the production of milk, meat, hair and hide . Markhoz goat has a coat called mohair being the only mohair goat breed inKurdistan province where it represents the main economic income for goat producers.Twenty years ago, about 25 000 Markhoz goats were raised in Kurdistan province of Iran.Latest census data reported a total population size of 1669 does and bucks . This rapid number decrease is due to many different reasons such asdiscouragement in youth and family members, shortage of shepherds, nutrition problems,existence of haired goat breeds with higher income, low income of goat raising and lowfinancial feasibility for herders. Although Markhoz goat is not at risk of extinction,when considering the downward trend, this breed may face the risk of genetic erosion dueto natural disasters and epidemic diseases .Therefore, given the importance of this breed and the need to identify geneticvariability in the Markhoz goat population, it is necessary to assess the geneticdiversity within this breed. In fact, genetic diversity is fundamental for long-termsurvival of populations providing the raw material for adaptation and evolution,especially when environmental conditions have changed . Thus, thegenetic characterization of native breeds is the first step for their conservation. Theuse of highly variable molecular genetic markers, such as microsatellites, is one of themost powerful tools to study genetic diversity because of its high polymorphic degree,random distribution across genome and neutrality with respect to selection . Therefore, the present study aimed to genetically characterize the Markhoz goatbreed using specific microsatellite markers to investigate the genetic diversity withinthis population and to improve future breeding strategies and conservation programmes.2Individual blood samples (10 mL each) were collected in K3-EDTA tubes and stored atTen microsatellite markers, previously described as polymorphic in goat, wereinvestigated: BM1312, LSCV06, ILSTS082, IDVGA64, LSCV25, BMS1248, BM2830,IL2RA, LSCV36 and MAP2. The studied microsatellites are located onchromosomes 1, 2, 5, 13 and 19 (Table 1).PCR amplifications were carried out in a 25 Population genetic indices, namely observed and expected heterozygosity, observed and expected number of alleles, Shannon's index andNei's expected heterozygosity  were performed by POPGENE32software version 1.32 . Moreover, polymorphism informationcontent (PIC) was calculated according to Botstein et al. (1980).3Microsatellite markers used in this study were previously assessed in different goatbreeds . Table 2 shows the measuresof genetic diversity at each microsatellite locus in Markhoz goat population. All 10microsatellite loci were successfully amplified and found to be polymorphic. A total of52 different alleles were identified with observed allele numbers ranging from 4 to 6 anda mean of 5.2 alleles per locus, being similar to findings obtained by Ganai and Yadav(2001), Gour et al. (2006) and Ramamoorthi et al. (2009) in some Indian goat breeds. Theinvestigated microsatellites showed a good level of polymorphism. The average effectivenumber of alleles was 4.12 varying from 3.46 (BMS1248) to 5.36 (LSCV25). Similar resultswere found by Dixit et al. (2011) who observed a mean effective number of alleles of 4.2in Kannai Adugoat; Kumar etal. (2009) who reported a value of 4.7 in Gohilwadi goat and Verma etal. (2010) who found a mean effective allele number of 4.0 in Sangamneri goat breed.Considering that at least four alleles were detected for each microsatellite locus in ourpopulation, this is in line with the selective standard of the microsatellite loci givenby the Secondary Guidelines for Development of National Farm Animal Genetic ResourcesManagement Plans using reference microsatellitegiven by FAO (2004) for proficient judgment of genetic distance between breeds. Thus, allstudied microsatellite markers exhibited sufficient polymorphism for evaluatingintra-population genetic variability in Markhoz goat breed.Heterozygosity is a good measure of the genetic variability within a population. In oursample, the most polymorphic locus was LSCV25 with six alleles and the highest value ofexpected heterozygosity was 0.817. On the other side, MAP2 was the least polymorphic withfour alleles and expected heterozygosity of 0.721. The average expected heterozygosityobtained was 0.756, being lower than the observed one (0.982). This slight excess ofheterozygosity was also reported by Ramamoorthi et al. (2009) in Barbari goats. Thediscrepancy between the observed and expected heterozygosity can be attributed to therandom mating among the individuals of the population. However, the available literaturegenerally reports lower values of observed heterozygosity . Considering the higher observedheterozigosity in the present population and the lack of allelic fixation at theinvestigated loci, it is possible to state that Markhoz breed has a wide margin ofgenetic improvement.PIC is a parameter indicative of the degree of informativeness of a marker. The PIC valuemay range from 0 to 1. In the studied population, the average PIC value was 0.719 rangingfrom 0.653 (ILSTS082) to 0.789 (BM1312). According to the classification of PIC , this populationpossessed high genetic diversity. PIC values calculated in the present investigation werecomparable with those reported by Ramamoorthi et al. (2009) (0.686) in Barbari goats,Serrano et al. (2009) (0.743) in Spanish Guadarrama breed, Verma et al. (2010) (0.711) inSangamneri goats, Dixit et al. (2012) (0.770) in Indian goat breeds and by Zhang etal. (2012) (0.781) in Chinese goat breeds. More recently, Radhika et al. (2015) reportedan average PIC value of 0.77 in Indian native and crossbred goats, and Wang et al. (2017)stated a mean PIC of 0.716 in Chinese dairy goats. Conversely, lower PIC values werereported in Indian and Pakistani goat breeds , in goats from Korea and China  and in SouthAfrican Angora goats .The variability in PIC values found in literature may be due to different mating methodsused in the studied populations. In the present study, the high PIC values prove that themicrosatellite markers used are highly polymorphic and can be well utilized for molecularcharacterization of goat germplasm.Shannon's index measures the biodiversity level in a population  and itresulted in sufficiently high values for the studied goats with a mean value of 1.50,ranging from 1.32 (MAP2) to 1.73 (LSCV25).Nei's expected heterozygosity indicates the level of gene diversity within the population. In the Markhoz breed it ranged from 0.62 (LSCV25) to 0.81 (LSCV06 andILSTS082) with a mean value of 0.73. These values were within the range of 0.3–0.8 asdetermined by Takezaki, and Nei (1996) for markers to be useful to assess geneticvariation in a population. Similar results were reported by Rout et al. (2008) in someIndian goat breeds, Dixit et al. (2011) in Kanniadu goats, Verma et al. (2010) inSangamneri breed and by Rout et al. (2012) in Jamunapari goat.It is important to underline that it is quite difficult to compare results from otherstudies as different microsatellite sets have been investigated by different scientists.Thus, comparisons may only give suggestive indication of diversity in a population.Nevertheless, to study the genetic variability in a population plays an important role indeveloping rational breeding strategies for domestic species.4Genetic diversity is the main component of the adaptive evolution mechanism because ofits preeminent role for the long-term survival probability of all species. Despite thedecreasing population size, Markhoz goat genetic diversity seems still conserved. Infact, considering that the sampled population included unrelated animals, the breed seemsto have a good level of genetic variability and, as a consequence, a potential margin ofadaptability to environment and for future genetic improvement. Nevertheless, furtherstudies should be conducted to deeply evaluate the genetic variability of the Markhozgoat breed.


Metarhizium robertsii fungus and avermectins on the midgut physiology of Colorado potato beetle larvae. We analyzed changes in the bacterial community, immunity- and stress-related gene expression, reactive oxygen species (ROS) production, and detoxification enzyme activity in response to topical infection with the M. robertsii fungus, oral administration of avermectins, and a combination of the two treatments. Avermectin treatment led to a reduction in microbiota diversity and an enhancement in the abundance of enterobacteria, and these changes were followed by the downregulation of Stat and Hsp90, upregulation of transcription factors for the Toll and IMD pathways and activation of detoxification enzymes. Fungal infection also led to a decrease in microbiota diversity, although the changes in community structure were not significant, except for the enhancement of Serratia. Fungal infection decreased the production of ROS but did not affect the gene expression of the immune pathways. In the combined treatment, fungal infection inhibited the activation of detoxification enzymes and prevented the downregulation of the JAK-STAT pathway caused by avermectins. The results of this study suggest that fungal infection modulates physiological responses to avermectins and that fungal infection may increase avermectin toxicosis by blocking detoxification enzymes in the gut.Fungal infections and toxicoses caused by insecticides may alter microbial communities and immune responses in the insect gut. We investigated the effects of  Different types of interactions may be observed between bacterial symbionts and insect pathogenic ascomycetes , which infect their hosts primarily through integuments and, less frequently, through the gut. These fungi may interact with host bacteria directly on the cuticle surface or in the gut. In these cases, bacteria inhibit fungal growth and differentiation, thereby acting as a component of the defense system against fungal infections6. In contrast, indirect interactions between fungi and symbiotic bacteria may be mediated by host immune responses. These more complicated interactions include the spatial relocation of physiological reactions, which may lead to synergy between the microorganisms and accelerated mortality. For example, topical infection of the mosquito Anopheles with the fungus Beauveria bassiana led to the proliferation of bacteria Serratia marcescens in the gut and hemocoel, thereby promoting fungal infection7. Similar effects were observed for the bark beetle Dendroctonus valens8 and the wax moth Galleria mellonella9.Insect microbial associates play important roles in the development of diseases caused by entomopathogens11. Various approaches for integrated pest control have been developed on this basis18. Researchers usually explain synergy between pathogenic fungi and insecticides by altering the immune response to fungal infections, as well as developmental and behavioral disorders. Importantly, xenobiotics may lead to changes in microbial communities in different ways. For example, certain insecticides cause different dysfunctions in the gut, such as alterations in peristaltic intensity, pH, and gut tissue damage19. Moreover, different toxicants may affect the immune and stress reactions in the gut. These effects may lead to changes in the bacterial community and, consequently, to changes in the susceptibility to fungi20.In natural and agricultural habitats, insects are exposed to a broad range of entomopathogenic microorganisms and various toxicants, such as plant secondary metabolites, synthetic and natural insecticides, and parasitoid venoms. It is well-known that these toxicants may significantly modulate the susceptibility of hosts to fungal pathogens21. The level of ROS is dictated by the Mesh-DUOX system22. It was proposed that DUOX generates superoxides outside the cells, which are unstable and are rapidly converted to H2O223. In addition, the DUOX peroxidase homology domain may serve to convert H2O2 to HOCl24. All of these compounds are powerful oxidants and exhibit microbicidal activity24. It was shown for Drosophila and mosquito species that the Mesh-DUOX system is essential for both resistance to pathogens and the management of commensal microbial populations26. The inhibition of the Mesh-DUOX system leads to uncontrolled proliferation of bacteria in the gut and a reduction in insect survival22. Interestingly, downregulation of DUOX in the mosquito gut was observed after topical infection with the entomopathogenic fungus B. bassiana, as shown by Wei et al.7. However, contradictory results were obtained by Ramirez et al.27. AMP production is controlled by three major immune-signaling pathways: Toll, IMD and JAK-STAT. The Toll pathway is involved in the defense against gram-positive bacteria and fungi, the IMD pathway is primarily involved in the defense against gram-negative bacteria, and the JAK-STAT pathway is involved in the defense against viruses but also against fungi28 and bacteria29. Changes in the expression levels of the transcription factors Dif/Dorsal, Relish and Stat (or their orthologs) indicate the activation/inhibition of the Toll, IMD and JAK-STAT pathways, respectively27. Topical fungal infection may both downregulate and upregulate the expression of these transcription factors and AMPs in the gut, depending on certain models of pathogenesis27.Homeostasis in the gut bacterial community is determined by immune-physiological reactions, with many of these reactions producing reactive oxygen species (ROS) and antimicrobial peptides (AMPs)31. These proteins participate in the stabilization and transport of proteins and are therefore involved in cell repair, immune-signaling pathways and other vital processes32. Antioxidants and detoxification enzymes participate in the inactivation of insecticides and microbial metabolites in various organs and tissues of insects, including the gut33. Esterases (ESTs) function against a broad range of insecticides34, bacterial toxins35 and metabolites formed under mycoses36. Glutathione-S-transferases (GSTs) participate in ROS inactivation and are involved in the metabolism of insecticides from different classes34 and are likely involved in the metabolism of fungal toxins37. The abovementioned immune responses in the insect gut during the development of mycoses or toxicoses caused by insecticides have not been fully elucidated, particularly when the fungi and insecticides are combined.In insects, stress responses may play important roles in the response to fungi, bacteria and insecticides. In particular, heat-shock proteins are activated under various stress conditions, including infections and toxicosesLeptinotarsa decemlineata (Say) is an important pest in most potato-producing regions around the world. The rapid development of resistance to various chemical insecticides requires the development of integrated approaches to the control of L. decemlineata populations38. One of these approaches may be employing a combination of insecticides and entomopathogens40. Previously, we studied the immunophysiological mechanisms of the synergy between the fungus Metarhizium robertsii and the macrocyclic lactones avermectins after a combined treatment to control the Colorado potato beetle40. We have shown that avermectins cause a delay in larval development and inhibit cellular immunity, which disrupts the immune response against fungal infection. In this study, we attempted to determine how these agents influence the gut microbiota and gut immunity and whether these changes contribute to the synergy between the fungus and avermectins. We measured the changes in the microbiota structure, ROS production, detoxification system activity, and gene expression involved in the immune response and stress management in the Colorado potato beetle midgut under the oral administration of avermectins, topical infection with M. robertsii and combined treatment with these agents.The Colorado potato beetle M. robertsii, avermectins and a combination of the two led to significant differences in the survival dynamics . At 10 days after treatment, the control larvae survived at a rate of 93.3%, whereas oral administration of avermectin and topical infection with fungus resulted in 61.1% and 43.3% survival, respectively, and the combined treatment resulted in 6.9% survival. Larvae that died after treatment with the fungus alone were primarily mummified (86%). Larvae that died after the combined treatment were mummified (39%) or decomposed by bacteria (61%), which was manifested by decay of cadavers with the appearance of an ammonia smell.Treatment of Colorado potato beetle larvae with Enterobacter and Klebsiella. The second most predominant OTU, OTU-20, showed high similarity (99.8%) only with Citrobacter strains. Spiroplasma (OTU-3) was identical (100%) only with Spiroplasma leptinotarsae (strain LD-1B), which is an obligate symbiont of the Colorado potato beetle41. In addition, relatively high abundances of OTUs were identified as Serratia (Enterobacteriaceae), Acinetobacter (Moraxellaceae), Pseudomonas (Pseudomonadaceae) and Lactococcus (Streptococcaceae).An analysis of 16S rRNA gene sequencing in the whole experiment demonstrated the presence of 171 operative taxonomical units (OTUs) that belonged to bacteria from 63 families, 21 classes and 11 phyla , as well as a 1.9- to 2.2-fold decrease in the Chao-1 index . Notably, the combined treatment did not lead to a stronger decrease in diversity compared to the single treatments . Based on changes in the Shannon index, significant effects were not detected .Fungal infection and avermectin toxicosis led to a reduction in the diversity of the midgut bacterial community Fig. . In partSpiroplasma and increased abundance of unclassified Enterobacteriaceae and Serratia were observed . Avermectins caused a strong elevation of unclassified Enterobacteriaceae and decrease in Spiroplasma relative abundance . In addition, an increasing trend for Pseudomonas was observed after treatment with avermectins . Factor interactions (fungus × avermectins) in the changes of the predominant group of bacteria were not observed . The structure of bacterial communities was similar in the larvae treated with avermectins and the combination of avermectins and fungus. It is important to note that fungal infection and avermectin toxicosis led to a decrease in the relative abundance of chloroplast DNA by 10- and 100-fold, respectively . The transcription factor Stat of the JAK-STAT pathway exhibited a different pattern of expression. Stat was significantly downregulated after treatment with avermectins alone . However, the combined treatment and fungal infection alone did not result in changes in Stat expression compared with the control. The fungus × avermectins interaction was determined to be significant . Thus, fungal infection prevented the downregulation of Stat caused by avermectins.We determined the upregulation of the transcription factors Att34) significantly increased after treatment with avermectins  and insignificantly increased with fungal infection , whereas the combined treatment upregulated this gene by sevenfold (P = 0.02 compared to the control). Hsp90 was significantly downregulated under the influence of avermectins  and was insignificantly upregulated after treatment with the fungus alone (P = 0.16 compared to the control); however, a significant effect of the interaction between factors on Hsp90 expression was not observed . Expression of Mesh did not exhibit any significant effects, although a trend of decreased expression was observed under the influence of mycosis .The expression of the AMP Attacin 34 . The combined treatment did not lead to additional suppression of ROS production compared to individual treatments . Positive correlations between the level of ROS production and diversity of the gut microbiota, especially based on the OTU count and Chao-1 index, were demonstrated . Moreover, the level of ROS production was positively correlated with Mesh expression , although the correlation was not significant (P = 0.20).Analysis of ROS production using electron paramagnetic resonance (EPR) spectroscopy demonstrated that 1,71 = 26.4, P < 0.001, EST—F1,70 = 7.4, P = 0.001, Fig. We demonstrated significant factor interactions in the activities of GST and EST in the Colorado potato beetle midgut after treatment with fungus and avermectins  only on day 5 after treatment . An analogous weak effect was detected between M. robertsii and S. nematodiphila. No synergy was observed between M. robertsii and S. quinivorans. The total mortality at day 10 after treatment for all combinations and fungal infections alone was 76–81%. It should be noted that individual administration of S. liquefaciens and S. nematodiphila led to a 19–23% increase in mortality compared to the control, and the differences were significant . Individual administration of S. marcescens did not cause mortality of larvae .Since we observed an increase in the proliferation of the opportunistic pathogen sis Fig. , we idenAppendix . Weak efAppendix , Fig. S1Spiroplasma. Fungal infection also led to a decrease in the midgut microbiota diversity; however, changes in the community structure were less pronounced and were largely insignificant. Importantly, under the combined treatment (M. robertsii + avermectins), fungal infection inhibited enterobacterial proliferation and impeded the activation of the detoxification system in the midgut caused by avermectins. Thus, fungal infection may modulate the physiological response of insects to toxicosis caused by the insecticide in the midgut.This work demonstrated dramatic changes in the Colorado potato beetle midgut bacterial community after treatment with avermectins, as indicated by a reduction in diversity, proliferation of enterobacteria, and decrease in the relative abundance of 45, including data for the West Siberian population of the beetle, where Enterobacteriaceae and Spiroplasma predominated46. In the present study, a decrease in bacterial diversity under both avermectin toxicosis and mycosis clearly had two causes: (1) interruption of feeding, which is evidenced by the decreased abundance of chloroplast 16S DNA, and (2) elevation of enterobacteria abundance. The first finding is consistent with our previous work40, where we showed that the same concentrations of avermectins reduced the amount of food consumed threefold during the first day after treatment. Moreover, a trend toward a decrease in larval weight was observed after infection with the fungus M. robertsii40, which may also indicate interruption of feeding. An increase in Proteobacteria (including Enterobacteriaceae) proliferation under different pathological states has also been reported by other authors48 and may be explained by degenerative changes in the gut tissue, pH alterations, peristaltic disturbances and aeration changes. Enterobacteria are facultative anaerobes; therefore, a deceleration of peristalsis may increase their abundance. A shift in the bacterial community structure toward enterobacteria predomination was demonstrated in the wax moth gut after treatment with H. hebetor venom, which leads to a cessation of peristalsis9. A decrease in the relative abundance of obligate intracellular S. leptinotarsa may be explained by the elevation of enterobacteria, as well as by the destruction of midgut epithelial cells after treatment with avermectins. Damage to the gut tissue by avermectins was demonstrated for different insects, especially for mosquitoes19 and Colorado potato beetles .Overall, the structure of the Colorado potato beetle gut microbiota was consistent with earlier published data46, we observed slight changes in the structure of the bacterial community and total bacterial load during the development of prolonged mycoses caused by different strains of M. robertsii, which is consistent with the results of the present study. However, in the present work, we documented an increase in the relative abundance and CFU count of Serratia during fungal infection, as well as during avermectin toxicosis. Previous studies showed that the oral administration of enterobacteria  that proliferate in the gut under mycoses and other pathologies caused different insect taxa to be more susceptible to fungi9. In this study, oral administration of different Serratia cultures to Colorado potato beetle larvae had a weak effect on mycosis development , destruxins were detected in negligible concentrations in insects50. The decrease in ROS production in the midgut was likely caused by prioritization of the immune response between the integuments and midgut. Insignificant effects of fungal infection on gene expression in the IMD and Toll and JAK-STAT pathways in Colorado potato beetle midgut may have been caused by the initial stage of mycosis, when the main action of fungal metabolites is the hydrolysis of integuments. Alterations in the expression of the genes in the gut were primarily identified at the latter stages of mycoses, as shown for mosquitoes27.With fungal infection, we observed a trend of decreasing Mesh expression, and decreased ROS generation was only observed as a trend (P = 0.08). However, avermectin treatment led to a stronger perturbation of the bacterial community and the strongest increase in the bacterial load compared to fungal infection. Clearly, these changes were caused by pathological processes directly in the gut. Upregulation of transcriptional factors in the Toll and IMD pathways and the AMP attacin after avermectin treatment may be considered to be a response to the elevated bacterial load. Interestingly, avermectin treatment caused a weak reaction or inhibition of stress- and ROS-related systems in the gut. This insecticide affects the glutamate-gated chloride channel (GluCl) and is associated with the ɣ-aminobutyric acid (GABA) receptor, and it also disrupts intracellular Ca2+ homeostasis52, which leads to the failure of the ion-exchange function of cellular membranes, disruption of the receptors containing G-protein (GPCRs)53, and inactivation of mitogen-activated protein kinases (MAPК)54. It is possible that these processes block or downregulate the expression of stress- and ROS-related genes (Mesh and Hsp90), as well as the JAK-STAT immune-signaling pathway. However, the IMD and Toll pathways are activated through the PGRP or TLR (Toll-like receptor) and PGRP receptors55, and they continue to function. Therefore, we observed the upregulation of the Dorsal-like and NF-kB-like genes. Activation of the IMD and Toll pathways is likely to be less dependent on Ca2+ homeostasis.Avermectin toxicosis did not lead to changes in Galleria mellonella by the parasitoid H. hebetor led to the downregulation of stress- and ROS-related genes, which was followed by the strong upregulation of AMP genes9. In cultured cells of Mamestra brassicae, induction of Hsp90, Hsp70, Hsp20.7, and Hsp19.7 was caused by halogenated pyrrole chlorfenapyr but not by other insecticides from different groups, such as pyrethroids, insect growth regulators, organophosphates, carbamates and trithians56. In contrast, Nazir et al.57 and Yoshimi et al.58 demonstrated significant induction of Hsp70 expression in guts of D. melanogaster and in whole bodies of Chironomus yoshimatsui after treatment with organophosphate pesticides and pyrethroids. Chen et al. 59 showed that neonicotinoid imidacloprid caused insignificant upregulation of Hsp70 in whole bodies of Colorado potato beetle larvae under optimal temperature (25 °C) and slight downregulation in response to the insecticide under high temperature (43 °C). Thus, the change in expression of HSPs may depend on the insecticide group, insect taxa and environment. However, it appears that neurotoxic insecticides do not cause significant change in expression of HSPs in Colorado potato beetles.It should be noted that similar patterns in gene expression in insect midguts were observed after treatment with other toxicants. In particular, envenomation of the wax moth Hsp90, Dorsal-like and NF-kB-like gene expression levels, although a stronger enhancement of Att34 gene expression was observed. Interestingly, fungal infection prevented the downregulation of Stat caused by avermectins  did not lead to an additional decrease in microbiota diversity and increase in the bacterial load compared to the single treatments. In contrast, the fungus inhibited the elevation of the enterobacteria load with the combined treatment Fig. . Joint tins Fig. . It is lDrosophila29. Upregulation of JAK-STAT pathway genes was observed in the gut of Bombyx mori after infection with different gram-negative and gram-positive bacteria61. In addition, JAK-STAT is involved in the insect antifungal response28. A decrease in the bacterial load under the combined action of fungus and avermectins  may have been caused by preventing JAK-STAT downregulation by fungus. We hypothesize that this effect may be caused by a systemic response of larvae to fungal infection, as demonstrated for mosquitoes by Ramirez et al. 27. To generate such evidence, it is necessary to analyze JAK-STAT gene expression in different tissues  after fungal infection of Colorado potato beetle, as well as investigating the effects of JAK-STAT silencing on bacterial communities.The JAK-STAT pathway is activated by cytokines produced after damage to the gut epithelium, and this pathway participates in AMP production, as shown in 39. However, we observed another effect in which fungal infection impeded the detoxification response to avermectins. In particular, M. robertsii infection prevented the activation of GST and EST in the midgut in response to avermectin treatment. EST and GST are involved in resistance to avermectins. Increased GST and EST levels were observed in arthropod lines that demonstrated resistance to this insecticide64, and inhibition of the detoxification enzymes led to an increase in susceptibility to abamectin67. In particular, direct silencing of GSTz2 expression increased the susceptibility of the fruit fly Bactrocera dorsalis to abamectin67. In Colorado potato beetle larvae, inhibition of esterases by S,S,S-tributyl phosphorotrithioate led to a fivefold increase in susceptibility to abamectin, and inhibition of GST by diethyl maleate caused a twofold increase in susceptibility to this insecticide68. Moreover, the inhibition of esterase activity may increase insect susceptibility to bacterial toxins35. Thus, blocking detoxification enzyme activation in Colorado potato beetle gut by the fungus may lead to increased toxic effects of avermectins, which may lead to general physiological consequences, such as a delay in development, weakening of cellular immunity and changes in the thickness and biochemical properties of integuments69. These disruptions are crucial to the susceptibility of Colorado potato beetle to Metarhizium and Beauveria70. It is likely that both direct and inverse effects between fungal infection and avermectins may occur in the investigated system and may lead to the formation of a "vicious circle" in which avermectins promote fungal infection (in the cuticle and hemolymph), and this infection increases the toxic effect of avermectins (in the gut). These interactions may cause a synergistic effect between the fungus and the avermectins. It should be noted that similar alterations of the detoxification enzyme activities under the combined action of M. robertsii and avermectins were established on mosquito Aedes aegypti larvae at the initial stages of mycosis and toxicosis71, which is in keeping with our hypothesis.In this study, considering the processes that occur in the midgut, we did not find direct confirmation that avermectins may promote fungal infection, as indicated in studies on the hemolymph and cuticle of the beetleSerratia proliferation occurred in the midgut of beetle larvae after topical treatment with fungus, which is consistent with the results obtained for mosquitoes7 and supports the hypothesis that immune reactions are prioritized between the integuments and gut under the development of fungal infections. Avermectin toxicosis led to a significant shift in bacterial communities toward enterobacterial prevalence and activation of the Toll and IMD immune-signaling pathways. However, downregulation of stress-regulated genes (Hsp90) and the JAK-STAT pathway was observed after treatment with this insecticide, which may be attributable to the disruption of receptors containing G-protein. Significant interactions between fungal infection and avermectin toxicosis were demonstrated for the gut immune responses and bacterial load. In particular, fungal infection impeded the elevation of enterobacteria, downregulation of STAT and activation of detoxification enzymes caused by avermectins. Blocking the detoxification response by fungi may be one of the mechanisms governing the synergy between M. robertsii and avermectins. Further research may investigate the temporal-spatial distribution of immune reactions in Colorado potato beetles under mycoses and toxicoses.In conclusion, this study is the first to show alterations in the gut immune reactions and bacterial community of Colorado potato beetle after treatment with fungal infection, insecticide and both. We demonstrated that a decrease in ROS production and elevation of M. robertsii (GenBank no. KP172147) from the microorganism collection of the Institute of Systematics and Ecology of Animals, Siberian Branch of Russian Academy of Science (SB RAS) was used in the experiments. Conidia were grown on autoclaved millet as described previously69. For the infection, conidia were suspended in a water-Tween 20 solution (0.03%) to a final concentration 106 conidia/mL. The concentration was determined using a Neubauer hemocytometer. The viability of the conidia was checked by plating on Saburoad dextrose agar. The germination of the conidia was > 95%. The industrial product Actarophyt 0.2%  with a complex of natural avermectins produced by Streptomyces avermitilis (http://enzim.biz) was used. Actarophyt dissolved in distilled water to 0.0054%  was used for the treatment. Selected concentrations of conidia and avermectins enable synergy between these agents40.Colorado potato beetle larvae were collected from private potato fields in the steppe zone of Western Siberia  in July 2018. Strain P-72 Solanum tuberosum leaves treated with either the avermectin solution or distilled water. The following four treatments were set up: Control, Fungus, Avermectins and Fungus + Avermectins. Larvae were maintained in 300 mL ventilated plastic containers (10 larvae in each) with potato foliage (6 g per container daily). To prevent plant desiccation, leaf footstalks were inserted in 2.5 mL tubes that were plugged with damp cotton. Containers were exposed at 25–26 °C, 25–30% RH and 14:10 h photoperiod. Insect mortality was registered daily for 10 days. At least 60 larvae of each treatment were used for the bioassay.Larvae (4–6 h after molting at the fourth instar) were infected with fungus by being dipped for 15 s into a water-Tween suspension of conidia. Control larvae were treated with a conidia-free water-Tween solution. After inoculation, the larvae were immediately placed on 72. The 16S libraries were sequenced with 2 × 300 bp paired-ends reagents on the MiSeq platform (Illumina) at the SB RAS Genomics Core Facility .At 48 h after treatment, the larvae midguts with contents were isolated and frozen in liquid nitrogen (5 midguts per sample) and stored at − 80 °C. Total DNA was extracted using the DNeasy PowerSoil Kit (QIAGEN). Bead beating was performed using a TissueLyser II (QIAGEN) for 10 min at 30 Hz. The V3-V4 region of the 16S rRNA genes was amplified with the primer pair 343F and 806R as described early73 using USEARCH v11.0.667. The UPARSE pipeline included paired read merging, read quality filtering, length trimming, identical read merging (dereplication), discarding singleton reads, removing chimeras and OTU clustering using the UPARSE algorithm74. The OTU sequences were assigned a taxonomy using the SINTAX75 and 16S RDP training set v16 as a reference76. The OTUs related to chloroplast 16S rDNA were removed from the final data set and analyzed separately.Raw sequences were analyzed with the UPARSE pipeline77 and showed trends approaching the saturation plateau, which indicated that enough reads were included  (see Appendix Appendix . Alpha d–2–5 × 10–5) were inoculated onto the surface of selective media. The CFUs of Lactococcus were determined by plating onto bile esculin azide agar M493 ; those of Enterobacteriaceae (excluding Serratia) were determined by plating onto endo agar M029; and those of Serratia were determined by plating onto Serratia differential medium M1288 (same manufacturer). Petri dishes with bacteria were incubated at 26 °C for 48 h. Then, the bacterial colonies were counted based on the morphological characteristics. At least 5 replicates for each treatment were used for analysis.Colorado potato beetle midguts with contents (1 sample = 3 midguts) were homogenized and suspended in sterile 150 mM NaCl solution. Aliquots of 100 µL suspension  according to the manufacturer’s instructions. qPCR analysis was performed in three technical replicates under the following conditions: 95 °C for 3 min followed by 40 cycles of 94 °C for 15 s and annealing and elongation at 62 °C for 30 s; and generation of melting curves (70–90 °C). Two genes of Leptinotarsa decemlineata were used as reference genes: 60S ribosomal protein L4 (Rp4) and ADF-ribosylation factor-like protein 1 (Arf1). These genes and primer sequences were from the work of Shi et al. 78 and were more stable in our investigated system. The relative expression of six genes of interest  was assayed. Primer sequences are provided in Appendix http://eu.idtdna.com/calc/analyzer).Colorado potato beetle midguts were isolated at 48 h after treatment, dissected in PBS and cleaned and washed to remove the contents of the gut lumen. All procedures were performed according to previously described techniques79. CP-H rapidly reacts with oxygen-centered free radicals, including superoxide, and some molecular oxidants to generate stable nitroxide that can be detected by EPR. In contrast, the reaction with oxygen, hydrogen peroxide and organic peroxides is very slow80.Spin trapping using 1-hydroxy-3-carboxy-pyrrolidine (CP-H) has been used to measure the rate of ROS formation79 with modifications to the sample preparation. Colorado potato beetle midguts were isolated at 48 h after treatment, dissected in PBS and cleaned and washed to remove the contents of the gut lumen. The sample contained 5 guts homogenized in 50 μL sodium phosphate buffer , and 1 mM CP-H was placed in a 50 μL glass capillary tube for EPR measurement. Ten samples were analyzed in each treatment.ROS measurement was performed as previously described81 and Prabhakaran and Kamble82 with minor modifications. In brief, one sample included 2 guts that were homogenized in 100 μL sodium phosphate buffer (0.1 M pH 7.2) (PBS) with phenylthiourea (4 mg/mL) and centrifuged at 10,000g for 10 min. The GST activity was estimated by measuring the generation of 5--glutathione at a wavelength of 340 nm. The EST level was estimated according to p-nitrophenyl acetate hydrolysis at 410 nm. The protein concentration was estimated using the Bradford method83. Bovine serum albumin was used for the calibration curve. At least 15 samples were analyzed in each treatment.To measure GST and EST activity, we utilized the technique of Habig et al.9 by sequencing a 1308-bp fragment of the 16S rRNA gene using primers 27F 5′-AGAGTTTGATCMTGGCTCAG-3′ and 1492R 5′-CCCTACGGTTACCTTGTTACGACTT-3. The obtained 16S rRNA gene sequences were compared against GenBank-available sequences from the type material  or distilled water (controls) and dried at 30 m under 25 °C. Larvae were dipped in the suspension of M. robertsii (106 conidia/mL) or conidia-free water-Tween solution and immediately placed on treated or untreated potato leaves. The following four treatments were used: Control, Serratia, Fungus, and Serratia + Fungus. Larvae were maintained as described above. Mortality was measured for 10 days. At least 40 larvae (1 replicate = 10 larvae) were used in each treatment.For the bioassay, potato leaves were dipped in suspensions of 84, STATISTICA 8 , SigmaStat 3.1 , and AtteStat 12.585. Data were checked for a normal distribution using the Shapiro–Wilk W test. Normally distributed data were analyzed by two-way ANOVA, followed by Tukey’s post-hoc test. For abnormally distributed data, we used a nonparametric equivalent of the two-way ANOVA Scheirer–Ray–Hare test86, followed by Dunnʼs post-test. The synergistic effects in mortality were detected by comparing the expected and observed mortality as described by Robertson and Preisler87. In addition, the log-rank test was applied for estimating differences in the mortality dynamics. Data on the plots are presented as the arithmetic means and standard errors.Data analyses were conducted using PAST 3Supplementary Information 1.Supplementary Information 2.


Panaqolus albomaculatus, Panaqolus gnomus, Panaqolus nocturnus, and Panaque bathyphilus, as well as that of submerged wood on which they feed. The gut bacterial community did not significantly vary across three gut regions  for any catfish species, although interspecific variation in the gut microbiome was significant, with magnitude of interspecific difference generally reflecting host phylogenetic proximity. Further, the gut microbiome of each species was significantly different to that present on the submerged wood. Inferring the genomic potential of the gut microbiome revealed that the majority of wood digesting pathways were at best equivalent to and more often depleted or nonexistent within the catfish gut compared to the submerged wood, suggesting a minimal role for the gut microbiome in wood digestion. Rather, these fishes are more likely reliant on fiber degradation performed by microbes in the environment, with their gut microbiome determined more by host identity and phylogenetic history.Neotropical wood‐eating catfishes (family Loricariidae) can occur in diverse assemblages with multiple genera and species feeding on the same woody detritus. As such, they present an intriguing system in which to examine the influence of host species identity on the vertebrate gut microbiome as well as to determine the potential role of gut bacteria in wood digestion. We characterized the gut microbiome of two co‐occurring catfish genera and four species:  We describe the gut microbiomes of four co‐existing freshwater catfish species as well as that of the submerged wood on which they feed. Bacterial communities did not vary by gut region for any catfish species, although they were all different to those identified on submerged wood. Wood digesting pathways were functionally predicted for the wood bacterial microbiome, but were less predicted for the catfish gut microbiome supporting the hypothesis that wood‐eating catfishes do not rely on their gut microbiota to digest wood. While microbiome research on nonhuman vertebrates has increased in recent years, an ongoing question is the influence of diet or environmental factors on the gut microbiome in comparison to host identity or evolutionary history  and ~24 described species  and Panaque  were collected in August 2006 (dry season) by electrofishing among partially submerged dead logs and branches in middle reaches of the Marañon River, a tributary of the upper Amazon River, in Condorcanqui Province, Amazonas Department, northern Peru. Specimens were collected from a single coarse woody debris dam at approximately 4°35′22″S, 77°51′10″W , sterilized cutting board. Following German and Bittong  before being measured (standard length) and dissected following the procedures of German and Bittong . Guts fr Bittong , the intn = 4) of the submerged coarse woody debris on which the fishes were found were also collected by scraping to a depth of ~1 cm with a razorblade and were immediately frozen, processed, transported, and stored as for gut samples.Samples (2.2DNA was extracted from the contents of a 5‐cm section of each frozen gut sample using a MoBio PowerSoil DNA extraction kit (MoBio). After an initial step in which the gut section was thawed (37°C) and vortexed for 2 min in 200 µl of the first lysis buffer to disperse gut contents, DNA was extracted following the manufacturer's specifications. Frozen woody debris samples were scrubbed with sterile toothbrushes into the first lysis buffer solution and DNA extracted from that suspension. DNA from all extractions was amplified using a dual index barcoding approach that targets the V4 regions of the 16S rRNA gene . Sequences were aligned to the SILVA 16S rRNA database release 132 and sequences that did not align with the V4 region or contained homopolymers >8 bp were discarded. Aligned sequences were classified to the Ribosomal Database Project release version 16 database, and any sequences that classified as nonbacterial were removed. Valid bacterial sequences were clustered into operational taxonomic units (OTUs) based on 97% sequence similarity and used for subsequent analyses of alpha and beta diversity. OTUs represented by just a single sequence in the dataset were excluded from diversity analyses, and those analyses occurred following subsampling  to a standardized number of 10,000 sequence reads per sample. Linear mixed models were conducted on the alpha diversity indices, treating the gut region as the fixed effect, with significant differences determined using Tukey's HSD test. The beta diversity index (Bray–Curtis dissimilarity) was exported from mothur into R version 3.6.1  relationships between taxa and exported into Cytoscape version 3.7.2.Analysis of 16S rRNA gene sequence data was conducted in mothur  yielded low sequence counts  despite multiple attempts and was removed from the dataset. Thus, the final dataset consisted of 940,168 sequences, which was represented by 56,655 unique sequence types, with a mean of 24,106 sequences per sample .Across all samples, a total of 940,950 valid 16S rRNA gene sequences were obtained after removal of sequences that were potentially chimeric or of nonbacterial origin, for a mean of >24,000 sequence reads per sample. One sample . Within Proteobacteria, the majority of sequences belonged to the Alphaproteobacteria (38.5%) and Gammaproteobacteria (29.4%), with Deltaproteobacteria (14.8%), Betaproteobacteria (6.7%), Epsilonproteobacteria (0.1%), and unclassified Proteobacteria (10.5%) constituting the remaining sequences.Three bacterial phyla accounted for 50.7% of the sequence reads: Proteobacteria 26.9%), Firmicutes (12.4%), and Planctomycetes (11.4%), and 28.68% of sequences were unclassified at the phylum level  and was similar in abundance across wood and catfish tissues, while a third OTU identified as a member of the Planctomycetaceae  was more abundant in Pqe. bathyphilus tissues than in Pqs. albomaculatus, Pqs. nocturnus or on wood . Of the remaining dominant OTUs, two members of the order Rhizobiales  were significantly more abundant in Pqe. bathyphilus and Pqs. nocturnus while an OTU classified as being within the class Clostridiales (1.3% of reads) was more abundant in Pqs. nocturnus than other samples , than any region of gut tissue , while for Pqe. bathyphilus the distal gut community was significantly richer than the proximal . Further, there were no significant differences in Shannon's index of diversity between species, or between gut regions for any species , and there were no significant differences between any gut regions within catfish species. Pqs. albomaculatus and Pqe. bathyphilus shared a similar number, but not identity, of core OTUs, comprising approximately 6% of their entire bacterial sequences  specific clusters identified included a cluster of wood samples and clusters of multiple individuals of each catfish species  but there were exceptions  in the gut microbiomes of all the wood‐eating catfishes compared to the bacterial assemblage on wood , the abundance counts for 20 genes were below the cutoff of 97% identity, indicating very low counts and so were excluded from the analysis. Of the remainder, 42 orthologs differed significantly in their relative abundance between catfishes and on wood Appendix . For twoAppendix . Just fi4Our results indicate that host identity, and perhaps phylogenetic history, plays important roles in determining the gut microbiome of wood‐eating catfishes in the Amazon basin of South America, not supporting our hypothesis of microbiome similarity among co‐occurring fishes. The gut bacterial communities of different individuals in the study generally clustered according to their taxonomic identity, and each of the four species in this study had a gut microbiome that differed from that of all other species, with a distinct core microbiome. Moreover, the bacterial community of each catfish species was different from that of the wood on which they were feeding, whereas bacterial samples from different gut regions and different pieces of wood showed few to no differences. This, in congruence with previous work on captive wood‐eating catfishes ; Formal analysis ; Visualization ; Writing‐original draft ; Writing‐review & editing . Donovan German: Conceptualization ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Writing‐original draft ; Writing‐review & editing . Nathan K Lujan: Conceptualization ; Funding acquisition ; Investigation ; Methodology ; Visualization ; Writing‐original draft ; Writing‐review & editing . Colin R Jackson: Conceptualization ; Funding acquisition ; Investigation ; Methodology ; Project administration ; Writing‐original draft ; Writing‐review & editing .DPG, NKL, and CRJ: Designing the study based on past field sampling by DPG and NKL. CRJ: Conducting laboratory assays. CRJ and MM: Conducting bioinformatics and data analysis. MM, DPG, NKL, and CRJ: Manuscript writing.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.Appendix S3Click here for additional data file.Supplementary MaterialClick here for additional data file.


Garlic is considered to have a strong positive effect on the growth and yield of receptors under soil cultivation conditions. However, how this positive promotion is produced by changing the growth environment of the receptors or directly acting on the receptors is still not very clear. The direct influence of co-culturing with different quantities of garlic plants  on the growth and biochemical processes of cucumber plants was studied using a hydroponic co-culture system. Different numbers of garlic bulbs inhibited the growth of cucumber plants and increased the production and induction of reactive oxygen species, which accompanied the enhancement of lipid peroxidation and oxidative damage to cucumber. This allelopathic exposure further reduced the chlorophyll contents and photosynthesis rate, and consequently impaired the photosynthetic performance of photosystem II (PSII). Garlic root exudates increased the leaves’ carbohydrates accumulation, such as soluble sugar contents and sucrose levels by regulating the activities of metabolismic enzymes; however, no such accumulation was observed in the roots. Our results suggested that garlic root exudates can mediate negative plant–plant interactions and its phytotoxic influence on cucumber plants may have occurred through the application of oxidative stress, which consequently imbalanced the source-to-sink photo-assimilate flow.  Chemical-mediated plant–plant interference is an important allelopathic mechanism by which certain plant species produce and release organic compounds into the growth environment through leaching, volatilization, root exudation, and passive discharge through decomposition, which have beneficial and harmful effects from the donor plant on the receptor plant . Many plThe negative response of receptors to allelochemicals manifests as growth retardation and tissue destruction, which results in oxidative damage to the plant system, where oxidative stress is thought to be a putative mode of action of allelochemicals induced by the overproduction of reactive oxygen species, and consequently causes lipid peroxidation . The mosCucumber is an important economic vegetable mostly grown under greenhouse cultivation in China. However, consecutive continuous cropping obstacles and allelopathic autotoxicity of this crop have become severe threats to cucumber production, which is accredited to the accumulation of phytotoxic phenolic compounds in the substrates used for long-time cultivation ,15. GarlHowever, some bioassay experiments advocated that garlic produced a direct negative and dose-dependent effect on receptors under controlled laboratory environmental conditions ,20. Thesp < 0.05), and the magnitude of this inhibition was also markedly apparent for the total dry mass of the plant. The SPAD  value of each treatment in all measurement periods was lower than that of the control  ratios when compared to the control, as shown in  control . HoweverGarlic root exudates exerted a differential oxidative stress in cucumber leaves and the intensity of oxidative damage caused by malondialdehyde (MDA) and proline accumulation was remarkable, showing a clear dose–response relationship . After c2O2 content in cucumber tissues were more obvious and had relatively significant effects on superoxide anions . The data showed that the effects on He anions . Dramati 36 days B. The activity of three kinds of leaf cucumber cell protective enzymes showed an upward trend at each stage of assessment . HoweverThe experiment revealed that the investigated cucumber seedlings exposed with 2:0, 2:5, 2:10, 2:15, and 2:20 ratios had a significant observed difference in ascorbate (ASA) and glutathione (GSH) synthesis compared with the control . However2 assimilation rate changed with plant age in cucumber leaves but was not statistically significant throughout the whole experiment.The effect of garlic root exudates on photosynthetic gas exchange parameters is shown in p < 0.05) than that of the control and the extent of NPQ progress increased with increased concentration only after 20 days of exposure. The chlorophyll fluorescence analysis indicated that the photosynthetic performance of the cucumber plants was dramatically impaired by the garlic bulb infusion . The sigIn order to investigate the garlic root exudate’s effect on the cucumber carbohydrate metabolism, we determined the total soluble sugar, hexose, starch, and sucrose in the root and leaf samples . VariatiGarlic root exudates with different concentrations reduced the plant acid invertase (AI) and sucrose synthetase–cleavage (SS-c) activities . The droRoot exudate is one of the important ways to release plant allelochemicals; however, the complexity of its action environment has been difficult to study and understand. Different substances in the root exudate play different functions and have different effects; therefore, the extraction of a single substance for research has certain limitations ,22. For Our results indicate that garlic species display a strong allelopathic activity, which significantly inhibits the growth of cucumber plants by imposing oxidative stress; its root exudates can release some growth inhibitors or allelochemicals, which can reduce the growth of target plants. This further confirmed our previous hypothesis that garlic root exudates not only impaired the morphological indices of pepper plant, but also altered the plant physiology by indirectly increasing the incidence of oxidative damage . AqueousIn this study, the significant growth difference among different treatments could be attributed to the garlic root exudates involved in the endogenous hormonal imbalance by producing allelochemical stress, which strongly affected the cucumber growth due to the growth environment and nutrient concentration during the whole growth period for cucumber plants that were apparently identical . HoweverAfter the phenotypic malformation of plants begins, a further imbalance of plant biochemical and physiological entities may maturate through the imposition of a wide range of oxidative damage and osmotic stress. Stressed plants respond according to biotic and abiotic fluctuations; however, variable resistance or adaptation mechanisms depend on the stress tolerance degree, growth, exposure duration, and different developmental stages, which increase their complexity . The oxi2O2, MDA, and proline are signs of lipid peroxidation and the induction of oxidative stress [In this study, we measured the oxidative stress by means of lipid peroxidation and proline synthesis, and the results showed the cucumber’s sensitivity to oxidative and osmotic damage. The MDA appeared as a primary stress indicator that reflects the amount of membrane damage in cucumber caused by garlic root exudates, and the degree of damage was dose-dependent. There were quantitative and co-culture time differences, and the amplitude of the membrane lipid peroxidation increased linearly with the increasing quantity of garlic bulbs A. Numeroe stress ,37. The e stress .2− and H2O2) in cucumber leaves, which increased the membrane lipid peroxidation and caused cellular damage. Consistent with these results, the plant response after exposure to allelochemicals was equivalent to that for allelopathic stress, which increased the accumulation of oxygen derivatives (ROS), such as the superoxide radical O2– anion and H2O2 in plant cells [2O2 and OH− production might be correlated with the lipid peroxidation observed in cucumber leaves [Intracellular origins of reactive oxygen species (ROS) and modification of the redox state or redox homeostasis in the stressful plant community was identified as a common allelopathic reaction . In manynt cells . The incr leaves .2O2-degrading enzyme that produces O2 and H2O molecules, and the higher activity of CAT suggested the ability of the cells to defend ROS by scavenging H2O2 molecules. However, the regulatory mechanism by CAT may not be efficient at fine-tuning the sensitive redox balances with low H2O2 concentrations [2—induced radical damage through catalyzing the dismutation of the superoxide free radical to O2 and H2O2. Increased SOD and POD activities induced by the garlic root exudates could provide more appropriate protection against the oxidative damage caused by O2− radicals [−1 garlic bulb extract, the SOD activity drastically decreased and the malondialdehyde (MDA) content increased [When the ROS-mediated oxidative stress becomes severe and prolonged, plant cells respond against the adversarial ecosystem. The defensive metabolic systems are armed with the antioxidant machinery consisting of enzymatic components and non-enzymatic antioxidants, which play an important role in ROS detoxification. ROS detoxification and increases of ROS-scavenging enzyme activities accompanied by allelochemicals has been observed in cucumber roots . Figure trations . SOD andradicals . Interesradicals ; similarncreased . To explThe defense mechanism against ROS damage was further evaluated by the activation of non-enzymatic molecules, such as ascorbate and glutathione components. Ascorbate (ASA) and glutathione (GSH) are well-described stress indicators that have an interaction with a cysteinyl thiol group and are typically engaged in maintaining the complex regulation of several physiological processes. Through exposure to multifarious eco-stresses in plants, ASA and GSH responses have been reported regarding the detoxification of unfavorable ROS forms, stress tolerance, intracellular redox signaling, and organ development . Our res2 concentration, and stomatal conductance was frequently observed under allelopathic stress, and photo inhibition investigations indicated significantly greater physiological stress [2 assimilation rate; in contrast, a reduced stomatal conductance and an increased intracellular CO2 concentration is characterized by a non-stomatal limitation. In our study, we found a decrease in stomatal conductance with a decrease in intracellular CO2 concentration, suggesting the decrease in photosynthetic rate induced by allelochemicals was at least partly due to stomatal closure [The best-characterized phytotoxic mechanisms induced by allelochemicals are the inhibition of photosynthesis and oxygen evolution through interactions with components of photosystem II . The inhl stress ,44. Meanl stress . During  closure . Pn was  closure . Our res closure . It is n closure .Evaluation and serial monitoring of the photosynthetic performance during abiotic stress is an important component of photochemistry. Under such circumstances, chlorophyll fluorescence is a useful tool to assess the physiological status of plants, as well as abrupt changes in photosystem II (PSII) . PhotoinThe partitioning of the metabolism of carbohydrates is strongly influenced by stress-related stimuli or other environmental cues, which can have deep developmental effects. The complex regulatory mechanism in the plant life cycle determines the allocation of carbohydrates between the different plant organs, reflecting the source–sink transition . In thisStarch is a significant form of a carbohydrate reservoir. We observed starch depletion in both the root and leaf samples for the final stages, where the decrease in the amount of starch in the leaves was relatively higher than in the root organs D. StarchSucrose is also an important photo-assimilate that triggers essential metabolic events in response to diverse environmental signals , where aIn our study, the activities of both sucrose-cleaving enzymes showed a downward regulation pattern that may indicate higher sucrose biosynthesis at the source site rather than its greater degradation in sink tissues A,C. In g2 assimilation can involve the accumulation of carbohydrate metabolites and alter the source-to-sink translocation pattern in plants receiving an allelochemical treatment [2 availability.”However, the regulatory effects of allelopathic agents on the metabolism of carbohydrates in oxidative-stress-sensitive plant organs are still poorly understood and is still a matter of debate. However, one possibility suggests that the reduction of COreatment . To veriThe garlic cv. G064 was obtained from the horticulture department of Northwest A&F University, Yangling, Shaanxi, China. Fresh and uniform-sized garlic bulbs  were selected and cultivated in sterilized perlite in plastic pots (40 cm × 30 cm × 12 cm) and incubated at 20 °C to yield a rooting culture. After completion of the specified growing duration to make sure maximum root development occurred, the garlic bulbs were uprooted at the fifth leaf stage at which its plantlets attained an approximately 5-cm root length and 50 cm in height. Harvested bulbs were rinsed with tap water to remove soil, sterilized with 0.1% potassium permanganate for 10 min, thoroughly rinsed with distilled water, and grown hydroponically in Hoagland nutrient solution for 30 days with cucumber seedlings.Cucumis sativus L. cv. BoNai 13B) seeds were sterilized in hot water at 55 °C, stirred for twenty minutes, and finally rinsed three times with distilled water. The plant-growing media was commercially procured and contained peat moss for optimum growth and was autoclaved (125 Pa/121 °C) for 30 minutes and then poured into plastic pots (length × width × depth: 8 cm × 8 cm × 10 cm). The seeds were sowed in these pots, followed by shower irrigation, and then transferred to the growth chamber with a 25 °C/20 °C day/night temperature, 70% humidity level, and 12 h light period. Every two to three days, a shower irrigation was performed to ensure the uniform growth of seedlings. When the fourth leaves were fully expanded, the uniform-sized seedlings were transplanted to the hydroponic culture medium. These selected cucumber seedlings were rinsed with tap water to remove perlite, sterlized with 0.05% potassium permanganate for 20 min, and finally rinsed with distilled water and grown hydroponically in Hoagland nutrient solution for 30 days with garlic plants.The cucumber , College of Horticulture, Northwest A&F University, Yangling, Shanxi province, China. Each hydroponic system was mainly composed of a hard culture plastic pot of 45 cm × 35 cm × 15 cm (length × width × depth) and an air pump  with a time switch. The air was periodically recycled by the pump (20 min/h). A total five treatments were proposed in the experiment in which two cucumber plants were co-cultured with 0, 5, 10, 15, and 20 garlic plants per pot, respectively. Therefore, the cucumber/garlic ratios (here after presented as C/G ratio) were 2:0 (control), 2:5, 2:10, 2:15, and 2:20 . The SPAD value of the cucumber leaves were measured using a SPAD-502 The oxidative stress-related parameters MDA and proline content in the leaves were determined according the procedures followed by Bu et al. . The pro2, 25 °C, 70% relative humidity, and light intensity of 1000 μmol m−2 s−1. The stomatal limitation (Ls) was calculated using Ls = 12Ci/Ca .Gas exchange parameters were measured using a portable photosynthesis system . The photosynthetic rate was measured at 800 μmol/mol CO0) was measured under a weak pulse of modulating light  after a dark adaptation for 30 min, and the maximal fluorescence (Fm) was induced using a saturating flash  applied over 0.8 s. The maximal quantum efficiency of PSII was determined using Fv/Fm, where Fv is the difference between F0 and Fm. An actinic light source (600 μmol m−2 s−1) was then applied to achieve a steady-state photosynthesis and to obtain the Fs (steady-state fluorescence yield), after which, a second saturation pulse was applied for 0.7 s to obtain F’m (light-adapted maximum fluorescence). Fluorescence parameters were measured using a PAM-2500 based on the dark-adapted and light-adapted fluorescence measurements. The quantum efficiency of PSII (ΦPSII) and the efficiency of the excitation capture by open PSII centers were calculated using (F’m ± Fs)/F’m and F’v/F’m, respectively [0) [Chlorophyll fluorescence was measured using the second fully expanded leaf from the apex at 25 °C using a chlorophyll fluorometer . The minimal fluorescence of leaves (Fectively . Photochvely [0) .v/v) and the supernatant was analyzed for hexose, sucrose, and total sugars. The residue was boiled for 3 h in 10 mL 2% HCl (v/v) and the supernatant was analyzed for starch content.Freeze-dried samples were used for the determination of the carbohydrate content. Sucrose, starch, and hexose (glucose and fructose) content were determined using a modified phenol-sulphuric acid method. One hundred milligrams of sample was extracted overnight in 25 mL 80% ethanol  and cleavage (SS-c) directions using the method of Lowellet [Sucrose phosphate synthase (SPS 2.4.1.14) and sucrose synthase (SS 2.4.1.13) were extracted at 0 ± 4 °C in accordance with Lowell et al. . SPS actLowellet . ReactioLowellet . ActivitLowellet .p < 0.05 using STATISTICA 8.0 software . The experiment was accomplished by using a completely randomized design (CRD). Values for all data were represented as the mean ± SE of four independent replicates. Analysis of variance (ANOVA) was performed to determine significant differences according to Fisher’s least significant difference (LSD) multiple range test at The incorporation of garlic root exudates in a small fraction contributed as growth regulators, as previously defined in our study, provided new allelopathic insight into exploring their quantitative strength by focusing on possible cropping obstacles in cucumber production. Present findings revealed that garlic root exudates triggered the oxidative stress due to severe lipid peroxidation, as well as proline and ROS induction, in cucumber during certain developmental stages. The result of these effects appeared in the reduction of photosynthetic performance that led to a constitutive accumulation of carbohydrates in the leaves and a decreased assimilate export to the roots, resulting an in imbalance in the source-to-sink photo-assimilate flow, which may account for the feedback inhibition of photosynthesis. We suggested that a high proportion of garlic root exudates in the cropping system might perform as strong allelopathic agents, which directly or indirectly affect the cucumber morpho-physiological indices. These findings may have practical implications for understanding the allelopathic inhibition mechanisms and further guide the development of new sustainable measures in the mitigation of alleloapthic stress.