A simulated natural heatwave perturbs bumblebee immunity and resistance to infection.

As a consequence of ongoing climate change, heatwaves are predicted to increase in frequency, intensity, and duration in many regions. Such extreme events can shift organisms from thermal optima for physiology and behaviour, with the thermal stress hypothesis predicting reduced performance at temperatures where the maintenance of biological functions is energetically costly. Performance includes the ability to resist biotic stressors, including infectious diseases, with increased exposure to extreme temperatures having the potential to synergise with parasite infection. Climate change is a proposed threat to native bee pollinators, directly and through indirect effects on floral resources, but the thermal stress hypothesis, particularly pertaining to infectious disease resistance, has received limited attention. We exposed adult Bombus impatiens bumblebee workers to simulated, ecologically relevant heatwave or control thermal regimes and assessed longevity, immunity, and resistance to concurrent or future parasite infections. We demonstrate that survival and induced antibacterial immunity are reduced following heatwaves. Supporting that heatwave exposure compromised immunity, the cost of immune activation was thermal regime dependent, with survival costs in control but not heatwave exposed bees. However, in the face of real infections, an inability to mount an optimal immune response will be detrimental, which was reflected by increased trypanosomatid parasite infections following heatwave exposure. These results demonstrate interactions between heatwave exposure and bumblebee performance, including immune and infection outcomes. Thus, the health of bumblebee pollinator populations may be affected through altered interactions with parasites and pathogens, in addition to other effects of extreme manifestations of climate change.

Experimental evolution under varying sex ratio and behavioral plasticity in response to perceived competitive environment independently affect calling effort in male crickets.

The operational sex ratio (OSR) is a key component influencing the magnitude of sexual selection driving the evolution of male sexual traits, but males often also retain the ability to plastically modulate trait expression depending on the current environment. Here we employed an experimental evolution approach to determine whether the OSR affects the evolution of male calling effort in decorated crickets, a costly sexual trait, and whether plasticity in calling effort is altered by the OSR under which males have evolved. Calling effort of males from 2 selection regimes maintained at different OSRs over 18-20 generations (male vs. female biased) was recorded at 2 different levels of perceived competition, in the absence of rivals or in the presence of an experimentally muted competitor. The effect of the OSR on the evolution of male calling effort was modest, and in the opposite direction predicted by theory. Instead, the immediate competitive environment strongly influenced male calling effort as males called more in the presence of a rival, revealing considerable plasticity in this trait. This increased calling effort came at a cost, however, as males confined with a muted rival experienced significantly higher mortality.

Divergence and convergence of gut microbiomes of wild insect pollinators.

Pollination services provided by wild insect pollinators are critical to natural ecosystems and crops around the world. There is an increasing appreciation that the gut microbiota of these insects influences their health and consequently their services. However, pollinator gut microbiota studies have focused on well-described social bees, but rarely include other, more phylogenetically divergent insect pollinators. To expand our understanding, we explored the insect pollinator microbiomes across three insect orders through two DNA sequencing approaches. First, in an exploratory 16S amplicon sequencing analysis of taxonomic community assemblages, we found lineage-specific divergences of dominant microbial genera and microbiota community composition across divergent insect pollinator genera. However, we found no evidence for a strong broad-scale phylogenetic signal, which we see for community relatedness at finer scales. Subsequently, we utilized metagenomic shotgun sequencing to obtain metagenome-assembled genomes and assess the functionality of the microbiota from pollinating flies and social wasps. We uncover a novel gut microbe from pollinating flies in the family Orbaceae that is closely related to Gilliamella spp. from social bees but with divergent functions. We propose this novel species be named Candidatus Gilliamella eristali. Further metagenomes of dominant fly and wasp microbiome members suggest that they are largely not host-insect adapted and instead may be environmentally derived. Overall, this study suggests selective processes involving ecology or physiology, or neutral processes determining microbe colonization may predominate in the turnover of lineages in insect pollinators broadly, while evolution with hosts may occur only under certain circumstances and on smaller phylogenetic scales. IMPORTANCE Wild insect pollinators provide many key ecosystem services, and the microbes associated with these insect pollinators may influence their health. Therefore, understanding the diversity in microbiota structure and function, along with the potential mechanisms shaping the microbiota across diverse insect pollinators, is critical. Our study expands beyond existing knowledge of well-studied social bees, like honey bees, including members from other bee, wasp, butterfly, and fly pollinators. We infer ecological and evolutionary factors that may influence microbiome structure across diverse insect pollinator hosts and the functions that microbiota members may play. We highlight significant differentiation of microbiomes among diverse pollinators. Closer analysis suggests that dominant members may show varying levels of host association and functions, even in a comparison of closely related microbes found in bees and flies. This work suggests varied importance of ecological, physiological, and non-evolutionary filters in determining structure and function across largely divergent wild insect pollinator microbiomes.

Consequences of microsporidian prior exposure for virus infection outcomes and bumble bee host health.

Host-parasite interactions do not occur in a vacuum, but in connected multi-parasite networks that can result in co-exposures and coinfections of individual hosts. These can affect host health and disease ecology, including disease outbreaks. However, many host-parasite studies examine pairwise interactions, meaning we still lack a general understanding of the influence of co-exposures and coinfections. Using the bumble bee Bombus impatiens, we study the effects of larval exposure to a microsporidian Nosema bombi, implicated in bumble bee declines, and adult exposure to Israeli Acute Paralysis Virus (IAPV), an emerging infectious disease from honey bee parasite spillover. We hypothesize that infection outcomes will be modified by co-exposure or coinfection. Nosema bombi is a potentially severe, larval-infecting parasite, and we predict that prior exposure will result in decreased host resistance to adult IAPV infection. We predict double parasite exposure will also reduce host tolerance of infection, as measured by host survival. Although our larval Nosema exposure mostly did not result in viable infections, it partially reduced resistance to adult IAPV infection. Nosema exposure also negatively affected survival, potentially due to a cost of immunity in resisting the exposure. There was a significant negative effect of IAPV exposure on survivorship, but prior Nosema exposure did not alter this survival outcome, suggesting increased tolerance given the higher IAPV infections in the bees previously exposed to Nosema. These results again demonstrate that infection outcomes can be non-independent when multiple parasites are present, even when exposure to one parasite does not result in a substantial infection.

Gene expression in bumble bee larvae differs qualitatively between high and low concentration imidacloprid exposure levels.

Neonicotinoid pesticides negatively impact bumble bee health, even at sublethal concentrations. Responses to the neonicotinoid imidacloprid have been studied largely at individual adult and colony levels, focusing mostly on behavioral and physiological effects. Data from developing larvae, whose health is critical for colony success, are deficient, particularly at the molecular level where transcriptomes can reveal disruption of fundamental biological pathways. We investigated gene expression of Bombus impatiens larvae exposed through food provisions to two field-realistic imidacloprid concentrations (0.7 and 7.0 ppb). We hypothesized both concentrations would alter gene expression, but the higher concentration would have greater qualitative and quantitative effects. We found 678 genes differentially expressed under both imidacloprid exposures relative to controls, including mitochondrial activity, development, and DNA replication genes. However, more genes were differentially expressed with higher imidacloprid exposure; uniquely differentially expressed genes included starvation response and cuticle genes. The former may partially result from reduced pollen use, monitored to verify food provision use and provide additional context to results. A smaller differentially expressed set only in lower concentration larvae, included neural development and cell growth genes. Our findings show varying molecular consequences under different field-realistic neonicotinoid concentrations, and that even low concentrations may affect fundamental biological processes.

Transcriptomic responses of females to consumption of nuptial food gifts as a potential mediator of sexual conflict in decorated crickets.

Nuptial food gift provisioning by males to females at mating is a strategy in many insects that is thought to be shaped by sexual conflict or sexual selection, as it affords males access to a female's physiology. While males often attempt to use these gifts to influence female behaviour to their own advantage, females can evolve counter mechanisms. In decorated crickets, the male's nuptial gift comprises part of the spermatophore, the spermatophylax, the feeding on which deters the female from prematurely terminating sperm transfer. However, ingested compounds in the spermatophylax and attachment of the sperm-containing ampulla could further influence female physiology and behaviour. We investigated how mating per se and these two distinct routes of potential male-mediated manipulation influence the female transcriptomic response. We conducted an RNA sequencing experiment on gut and head tissues from females for whom nuptial food gift consumption and receipt of an ejaculation were independently manipulated. In the gut tissue, we found that females not permitted to feed during mating exhibited decreased overall gene expression, possibly caused by a reduced gut function, but this was countered by feeding on the spermatophylax or a sham gift. In the head tissue, we found only low numbers of differentially expressed genes, but a gene co-expression network analysis revealed that ampulla attachment and spermatophylax consumption independently induce distinct gene expression patterns. This study provides evidence that spermatophylax feeding alters the female post-mating transcriptomic response in decorated crickets, highlighting its potential to mediate sexual conflict in this system.

Investigating the influence of diet diversity on infection outcomes in a bumble bee (Bombus impatiens) and microsporidian (Nosema bombi) host-pathogen system.

Diet can have an array of both direct and indirect effects on an organism's health and fitness, which can influence the outcomes of host-pathogen interactions. Land use changes, which could impact diet quantity and quality, have imposed foraging stress on important natural and agricultural pollinators. Diet related stress could exacerbate existing negative impacts of pathogen infection. Accounting for most of its nutritional intake in terms of protein and many micronutrients, pollen can influence bee health through changes in immunity, infection, and various aspects of individual and colony fitness. We investigate how adult pollen consumption, pollen type, and pollen diversity influence bumble bee Bombus impatiens survival and infection outcomes for a microsporidian pathogen Nosema (Vairimorpha) bombi. Experimental pathogen exposures of larvae occurred in microcolonies and newly emerged adult workers were given one of three predominantly monofloral, polyfloral, or no pollen diets. Workers were assessed for size, pollen consumption, infection 8-days following adult-eclosion, survival, and the presence of extracellular microsporidian spores at death. Pollen diet treatment, specifically absence of pollen, and infection independently reduced survival, but we saw no effects of pollen, pollen type, or pollen diet diversity on infection outcomes. The latter suggests infection outcomes were likely already set, prior to differential diets. Although infection outcomes were not altered by pollen diet in our study, it highlights both pathogen infection and pollen availability as important for bumble bee health, and these factors may interact at different stages of bumble bee development, at the colony level, or under different dietary regimes.

Induction of Multiple Immune Signaling Pathways in Gryllodes sigillatus Crickets during Overt Viral Infections.

Despite decades of focus on crickets (family: Gryllidae) as a popular commodity and model organism, we still know very little about their immune responses to microbial pathogens. Previous studies have measured downstream immune effects (e.g., encapsulation response, circulating hemocytes) following an immune challenge in crickets, but almost none have identified and quantified the expression of immune genes during an active pathogenic infection. Furthermore, the prevalence of covert (i.e., asymptomatic) infections within insect populations is becoming increasingly apparent, yet we do not fully understand the mechanisms that maintain low viral loads. In the present study, we measured the expression of several genes across multiple immune pathways in Gryllodes sigillatus crickets with an overt or covert infection of cricket iridovirus (CrIV). Crickets with overt infections had higher relative expression of key pathway component genes across the Toll, Imd, Jak/STAT, and RNAi pathways. These results suggests that crickets can tolerate low viral infections but can mount a robust immune response during an overt CrIV infection. Moreover, this study provides insight into the immune strategy of crickets following viral infection and will aid future studies looking to quantify immune investment and improve resistance to pathogens.

Consuming sunflower pollen reduced pathogen infection but did not alter measures of immunity in bumblebees.

Certain diets can benefit bee health by reducing pathogens, but the mechanism(s) driving these medicinal effects are largely unexplored. Recent research found that sunflower (Helianthus annuus) pollen reduces the gut pathogen Crithidia bombi in the common eastern bumblebee (Bombus impatiens). Here, we tested the effects of sunflower pollen and infection on two bee immune metrics to determine whether sunflower pollen diet drives changes in host immunity that can explain this medicinal effect. Bees were infected with C. bombi or not and given either sunflower or wildflower pollen. Subsequently, bees received a benign immune challenge or were left naive to test the induced and constitutive immune responses, respectively. We measured haemolymph phenoloxidase activity, involved in the melanization cascade, and antibacterial activity. Sunflower pollen reduced C. bombi infection, but we found no significant pollen diet effect on either immune measure. Phenoloxidase activity was also not affected by C. bombi infection status; however, uninfected bees were more likely to have measurable constitutive antibacterial activity, while infected bees had higher induced antibacterial activity. Overall, we found that sunflower pollen does not significantly affect the immune responses we measured, suggesting that the mechanisms underlying its medicinal effect do not involve these bee immune parameters. This article is part of the theme issue 'Natural processes influencing pollinator health: from chemistry to landscapes'.

Genetic covariance in immune measures and pathogen resistance in decorated crickets is sex and pathogen specific.

Insects are important models for studying immunity in an ecological and evolutionary context. Yet, most empirical work on the insect immune system has come from phenotypic studies meaning we have a limited understanding of the genetic architecture of immune function in the sexes. We use nine highly inbred lines to thoroughly examine the genetic relationships between a suite of commonly used immune assays (haemocyte count, implant encapsulation, total phenoloxidase activity, antibacterial zone of inhibition and pathogen clearance) and resistance to infection by three generalist insect pathogens (the gram-negative bacterium Serratia marcescens, the gram-positive bacterium Bacillus cereus and the fungus Metarhizium robertsii) in male and female Gryllodes sigillatus. There were consistent positive genetic correlations between haemocyte count, antibacterial and phenoloxidase activity and resistance to S. marcescens in both sexes, but these relationships were less consistent for resistance to B. cereus and M. robertsii. In addition, the clearance of S. marcescens was genetically correlated with the resistance to all three pathogens in both sexes. Genetic correlations between resistances to the different pathogen species were inconsistent, indicating that resistance to one pathogen does not necessarily mean resistance to another. Finally, while there is ample genetic (co)variance in immune assays and pathogen resistance, these genetic estimates differed across the sexes and many of these measures were not genetically correlated across the sexes, suggesting that these measures could evolve independently in the sexes. Our finding that the genetic architecture of immune function is sex and pathogen specific suggests that the evolution of immune function in male and female G. sigillatus is likely to be complex. Similar quantitative genetic studies that measure a large number of assays and resistance to multiple pathogens in both sexes are needed to ascertain if this complexity extends to other species.

Active and Covert Infections of Cricket Iridovirus and Acheta domesticus Densovirus in Reared Gryllodes sigillatus Crickets.

Interest in developing food, feed, and other useful products from farmed insects has gained remarkable momentum in the past decade. Crickets are an especially popular group of farmed insects due to their nutritional quality, ease of rearing, and utility. However, production of crickets as an emerging commodity has been severely impacted by entomopathogenic infections, about which we know little. Here, we identified and characterized an unknown entomopathogen causing mass mortality in a lab-reared population of Gryllodes sigillatus crickets, a species used as an alternative to the popular Acheta domesticus due to its claimed tolerance to prevalent entomopathogenic viruses. Microdissection of sick and healthy crickets coupled with metagenomics-based identification and real-time qPCR viral quantification indicated high levels of cricket iridovirus (CrIV) in a symptomatic population, and evidence of covert CrIV infections in a healthy population. Our study also identified covert infections of Acheta domesticus densovirus (AdDNV) in both populations of G. sigillatus. These results add to the foundational research needed to better understand the pathology of mass-reared insects and ultimately develop the prevention, mitigation, and intervention strategies needed for economical production of insects as a commodity.

Testing the multiple stressor hypothesis: chlorothalonil exposure alters transmission potential of a bumblebee pathogen but not individual host health.

Numerous threats are putting pollinator health and essential ecosystem pollination services in jeopardy. Although individual threats are widely studied, their co-occurrence may exacerbate negative effects, as posited by the multiple stressor hypothesis. A prominent branch of this hypothesis concerns pesticide-pathogen co-exposure. A landscape analysis demonstrated a positive association between local chlorothalonil fungicide use and microsporidian pathogen (Nosema bombi) prevalence in declining bumblebee species (Bombus spp.), suggesting an interaction deserving further investigation. We tested the multiple stressor hypothesis with field-realistic chlorothalonil and N. bombi exposures in worker-produced B. impatiens microcolonies. Chlorothalonil was not avoided in preference assays, setting the stage for pesticide-pathogen co-exposure. However, contrary to the multiple stressor hypothesis, co-exposure did not affect survival. Bees showed surprising tolerance to Nosema infection, which was also unaffected by chlorothalonil exposure. However, previously fungicide-exposed infected bees carried more transmission-ready spores. Our use of a non-declining bumblebee and potential higher chlorothalonil exposures under some scenarios could mean stronger individual or interactive effects in certain field settings. Yet, our results alone suggest consequences of pesticide co-exposure for pathogen dynamics in host communities. This underlies the importance of considering both within- and between-host processes when addressing the multiple stressor hypothesis in relation to pathogens.

Genus-Wide Characterization of Bumblebee Genomes Provides Insights into Their Evolution and Variation in Ecological and Behavioral Traits.

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary life-cycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships, whereas incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. Our study reveals how bumblebee genes and genomes have evolved across the Bombus phylogeny and identifies variations potentially linked to key ecological and behavioral traits of these important pollinators.

Male and female genotype and a genotype-by-genotype interaction mediate the effects of mating on cellular but not humoral immunity in female decorated crickets.

Sexually antagonistic coevolution is predicted to lead to the divergence of male and female genotypes related to the effects of substances transferred by males at mating on female physiology. The outcome of mating should thus depend on the specific combination of mating genotypes. Although mating has been shown to influence female immunity in diverse insect taxa, a male-female genotype-by-genotype effect on female immunity post mating remains largely unexplored. Here, we investigate the effects of mating on female decorated cricket baseline immunity and the potential for a male-genotype-by-female-genotype interaction affecting this response. Females from three distinct genotypic backgrounds were left unmated or singly mated in a fully reciprocal design to males from the same three genotypic backgrounds. Hemocytes and hemocyte microaggregations were quantified for female cellular immunity, and phenoloxidase, involved in melanization, and antibacterial activity for humoral immunity. In this system, female cellular immunity was more reactive to mating, and mating effects were genotype-dependent. Specifically, for hemocytes, a genotype-by-mating status interaction mediated the effect of mating per se, and a significant male-female genotype-by-genotype interaction determined hemocyte depletion post mating. Microaggregations were influenced by the female's genotype or that of her mate. Female humoral immune measures were unaffected, indicating that the propensity for post-mating effects on females is dependent on the component of baseline immunity. The genotype-by-genotype effect on hemocytes supports a role of sexual conflict in post-mating immune suppression, suggesting divergence of male genotypes with respect to modification of female post-mating immunity, and divergence of female genotypes in resistance to these effects.

Macronutrient intake and simulated infection threat independently affect life history traits of male decorated crickets.

Nutritional geometry has advanced our understanding of how macronutrients (e.g., proteins and carbohydrates) influence the expression of life history traits and their corresponding trade-offs. For example, recent work has revealed that reproduction and immune function in male decorated crickets are optimized at very different protein:carbohydrate (P:C) dietary ratios. However, it is unclear how an individual's macronutrient intake interacts with its perceived infection status to determine investment in reproduction or other key life history traits. Here, we employed a fully factorial design in which calling effort and immune function were quantified for male crickets fed either diets previously demonstrated to maximize calling effort (P:C = 1:8) or immune function (P:C = 5:1), and then administered a treatment from a spectrum of increasing infection cue intensity using heat-killed bacteria. Both diet and a simulated infection threat independently influenced the survival, immunity, and reproductive effort of males. If they called, males increased calling effort at the low infection cue dose, consistent with the terminal investment hypothesis, but interpretation of responses at the higher threat levels was hampered by the differential mortality of males across infection cue and diet treatments. A high protein, low carbohydrate diet severely reduced the health, survival, and overall fitness of male crickets. There was, however, no evidence of an interaction between diet and infection cue dose on calling effort, suggesting that the threshold for terminal investment was not contingent on diet as investigated here.

Global Trends in Bumble Bee Health.

Bumble bees (Bombus) are unusually important pollinators, with approximately 260 wild species native to all biogeographic regions except sub-Saharan Africa, Australia, and New Zealand. As they are vitally important in natural ecosystems and to agricultural food production globally, the increase in reports of declining distribution and abundance over the past decade has led to an explosion of interest in bumble bee population decline. We summarize data on the threat status of wild bumble bee species across biogeographic regions, underscoring regions lacking assessment data. Focusing on data-rich studies, we also synthesize recent research on potential causes of population declines. There is evidence that habitat loss, changing climate, pathogen transmission, invasion of nonnative species, and pesticides, operating individually and in combination, negatively impact bumble bee health, and that effects may depend on species and locality. We distinguish between correlational and causal results, underscoring the importance of expanding experimental research beyond the study of two commercially available species to identify causal factors affecting the diversity of wild species.

Dynamic Changes of Gut Microbial Communities of Bumble Bee Queens through Important Life Stages.

Bumble bees are important pollinators in natural and agricultural ecosystems. Their social colonies are founded by individual queens, which, as the predominant reproductive females of colonies, contribute to colony function through worker production and fitness through male and new queen production. Therefore, queen health is paramount, but even though there has been an increasing emphasis on the role of gut microbiota for animal health, there is limited information on the gut microbial dynamics of bumble bee queens. Employing 16S rRNA amplicon sequencing and quantitative PCR, we investigate how the adult life stage and physiological state influence a queen's gut bacterial community diversity and composition in unmated, mated, and ovipositing queens of Bombus lantschouensis We found significant shifts in total gut microbe abundance and microbiota composition across queen states. There are specific compositional signatures associated with different stages, with unmated and ovipositing queens showing the greatest similarity in composition and mated queens being distinct. The bacterial genera Gilliamella, Snodgrassella, and Lactobacillus were relatively dominant in unmated and ovipositing queens, with Bifidobacterium dominant in ovipositing queens only. Bacillus, Lactococcus, and Pseudomonas increased following queen mating. Intriguingly, however, further analysis of unmated queens matching the mated queens in age showed that changes are independent of the act of mating. Our study is the first to explore the gut microbiome of bumble bee queens across key life stages from adult eclosion to egg laying and provides useful information for future studies of the function of gut bacteria in queen development and colony performance.IMPORTANCE Bumble bee queens undergo a number of biological changes as they transition through adult emergence, mating, overwintering, foraging, and colony initiation including egg laying. Therefore, they represent an important system to understand the link between physiological, behavioral, and environmental changes and host-associated microbiota. It is plausible that the bumble bee queen gut bacteria play a role in shaping the ability of the queen to survive environmental extremes and reproduce, due to long-established coevolutionary relationships between the host and microbiome members.

An interaction between host and microbe genotypes determines colonization success of a key bumble bee gut microbiota member.

There has been a proliferation of studies demonstrating an organism's health is influenced by its microbiota. However, factors influencing beneficial microbe colonization and the evolution of these relationships remain understudied relative to host-pathogen interactions. Vertically transmitted beneficial microbes are predicted to show high levels of specificity in colonization, including genotype matching, which may transpire through coevolution. We investigate how host and bacterial genotypes influence colonization of a core coevolved microbiota member in bumble bees. The hindgut colonizing Snodgrassella alvi confers direct benefits, but, as an early colonizer, also facilitates the further development of a healthy microbiota. Due to predominantly vertical transmission promoting tight evolution between colonization factors of bacteria and host lineages, we predict that genotype-by-genotype interactions will determine successful colonization. Germ-free adult bees from seven bumble bee colonies (host genotypic units) were inoculated with one of six genetically distinct strains of S. alvi. Subsequent colonization within host and microbe genotypes combinations ranged from 0 to 100%, and an interaction between host and microbe genotypes determined colonization success. This novel finding of a genotype-by-genotype interaction determining colonization in an animal host-beneficial microbe system has implications for the ecological and evolutionary dynamics of host and microbe, including associated host-fitness benefits.

Intestinal B cells in the red-eared slider turtle, Trachemys scripta: Anatomical distribution and implications for ecological interactions with pathogenic microbes.

Disease is a significant threat in the global decline of reptile species. Many aquatic reptiles live in habitats with high levels of opportunistic microbial pathogens, yet little is known about their immune system. Gut-associated lymphoid tissue is vital for protection against ingested pathogens and maintenance of normal gut microbiota. In mammals, gut mucosal immunity is well-characterized and mucosal surfaces are coated in protective antibodies. However, reptiles lack lymph nodes and Peyer's patches, which are the major sites of mammalian B cell responses. The presence or distribution of mucosal B cells in reptiles is unknown. In this study, we first set out to determine if B cells could be detected in intestinal tissues of red-eared slider turtles, Trachemys scripta. Using whole-mount immunochemistry and a primary antibody to turtle antibody light chains, we identified widely distributed B cell aggregates within the small intestine of hatchling turtles. These aggregates appeared similar to isolated lymphoid follicles (ILFs) in mammals and the frequency was much higher in distal intestinal sections than in proximal sections. To determine if these structures were inducible in the presence of microbes, we introduced an enteric Salmonella species through oral gavage. Analysis of intestinal tissues revealed that hatchlings exposed to Salmonella exhibited significantly more of these aggregates when compared with those that did not receive bacteria. These studies provide the first evidence for B cell-containing ILF-like structures in reptiles and provide novel information about gut immunity in nonmammalian vertebrates that could have important implications for ecological interactions with pathogens.