Royal Decree: Gene Expression in Trans-Generationally Immune Primed Bumblebee Workers Mimics a Primary Immune Response

Invertebrates lack the cellular and physiological machinery of the adaptive immune system, but show specificity in their immune response and immune priming. Functionally, immune priming is comparable to immune memory in vertebrates. Individuals that have survived exposure to a given parasite are better protected against subsequent exposures. Protection may be cross-reactive, but demonstrations of persistent and specific protection in invertebrates are increasing. This immune priming can cross generations ("trans-generational" immune priming), preparing offspring for the prevailing parasite environment. While these phenomena gain increasing support, the mechanistic foundations underlying such immune priming, both within and across generations, remain largely unknown. Using a transcriptomic approach, we show that exposing bumblebee queens with an injection of heat-killed bacteria, known to induce trans-generational immune priming, alters daughter (worker) gene expression. Daughters, even when unexposed themselves, constitutively express a core set of the genes induced upon direct bacterial exposure, including high expression of antimicrobial peptides, a beta-glucan receptor protein implicated in bacterial recognition and the induction of the toll signaling pathway, and slit-3 which is important in honeybee immunity. Maternal exposure results in a distinct upregulation of their daughters' immune system, with a signature overlapping with the induced individual response to a direct exposure. This will mediate mother-offspring protection, but also associated costs related to reconfiguration of constitutive immune expression. Moreover, identification of conserved immune pathways in memory-like responses has important implications for our understanding of the innate immune system, including the innate components in vertebrates, which share many of these pathways

Analysis of a normalised expressed sequence tag (EST) library from a key pollinator, the bumblebee Bombus terrestris

<p>Abstract</p> <p>Background</p> <p>The bumblebee, <it>Bombus terrestris </it>(Order Hymenoptera), is of widespread importance. This species is extensively used for commercial pollination in Europe, and along with other <it>Bombus </it>spp. is a key member of natural pollinator assemblages. Furthermore, the species is studied in a wide variety of biological fields. The objective of this project was to create a <it>B. terrestris </it>EST resource that will prove to be valuable in obtaining a deeper understanding of this significant social insect.</p> <p>Results</p> <p>A normalised cDNA library was constructed from the thorax and abdomen of <it>B. terrestris </it>workers in order to enhance the discovery of rare genes. A total of 29'428 ESTs were sequenced. Subsequent clustering resulted in 13'333 unique sequences. Of these, 58.8 percent had significant similarities to known proteins, with 54.5 percent having a "best-hit" to existing Hymenoptera sequences. Comparisons with the honeybee and other insects allowed the identification of potential candidates for gene loss, pseudogene evolution, and possible incomplete annotation in the honeybee genome. Further, given the focus of much basic research and the perceived threat of disease to natural and commercial populations, the immune system of bumblebees is a particularly relevant component. Although the library is derived from unchallenged bees, we still uncover transcription of a number of immune genes spanning the principally described insect immune pathways. Additionally, the EST library provides a resource for the discovery of genetic markers that can be used in population level studies. Indeed, initial screens identified 589 simple sequence repeats and 854 potential single nucleotide polymorphisms.</p> <p>Conclusion</p> <p>The resource that these <it>B. terrestris </it>ESTs represent is valuable for ongoing work. The ESTs provide direct evidence of transcriptionally active regions, but they will also facilitate further functional genomics, gene discovery and future genome annotation. These are important aspects in obtaining a greater understanding of this key pollinator species.</p

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

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

No Evidence for Immune Priming in Ants Exposed to a Fungal Pathogen

There is accumulating evidence that invertebrates can acquire long-term protection against pathogens through immune priming. However, the range of pathogens eliciting immune priming and the specificity of the response remain unclear. Here, we tested if the exposure to a natural fungal pathogen elicited immune priming in ants. We found no evidence for immune priming in Formica selysi workers exposed to Beauveria bassiana. The initial exposure of ants to the fungus did not alter their resistance in a subsequent challenge with the same fungus. There was no sign of priming when using homologous and heterologous combinations of fungal strains for exposure and subsequent challenges at two time intervals. Hence, within the range of conditions tested, the immune response of this social insect to the fungal pathogen appears to lack memory and strain-specificity. These results show that immune priming is not ubiquitous across pathogens, hosts and conditions, possibly because of immune evasion by the pathogen or efficient social defences by the host

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

A depauperate immune repertoire precedes evolution of sociality in bees

Background Sociality has many rewards, but can also be dangerous, as high population density and low genetic diversity, common in social insects, is ideal for parasite transmission. Despite this risk, honeybees and other sequenced social insects have far fewer canonical immune genes relative to solitary insects. Social protection from infection, including behavioral responses, may explain this depauperate immune repertoire. Here, based on full genome sequences, we describe the immune repertoire of two ecologically and commercially important bumblebee species that diverged approximately 18 million years ago, the North American Bombus impatiens and European Bombus terrestris. Results We find that the immune systems of these bumblebees, two species of honeybee, and a solitary leafcutting bee, are strikingly similar. Transcriptional assays confirm the expression of many of these genes in an immunological context and more strongly in young queens than males, affirming Bateman’s principle of greater investment in female immunity. We find evidence of positive selection in genes encoding antiviral responses, components of the Toll and JAK/STAT pathways, and serine protease inhibitors in both social and solitary bees. Finally, we detect many genes across pathways that differ in selection between bumblebees and honeybees, or between the social and solitary clades. Conclusions The similarity in immune complement across a gradient of sociality suggests that a reduced immune repertoire predates the evolution of sociality in bees. The differences in selection on immune genes likely reflect divergent pressures exerted by parasites across social contexts

Unity in defence: honeybee workers exhibit conserved molecular responses to diverse pathogens

This is the final version of the article. Available from the publisher via the DOI in this record.Background: Organisms typically face infection by diverse pathogens, and hosts are thought to have developed specific responses to each type of pathogen they encounter. The advent of transcriptomics now makes it possible to test this hypothesis and compare host gene expression responses to multiple pathogens at a genome-wide scale. Here, we performed a meta-analysis of multiple published and new transcriptomes using a newly developed bioinformatics approach that filters genes based on their expression profile across datasets. Thereby, we identified common and unique molecular responses of a model host species, the honey bee (Apis mellifera), to its major pathogens and parasites: the Microsporidia Nosema apis and Nosema ceranae, RNA viruses, and the ectoparasitic mite Varroa destructor, which transmits viruses. Results: We identified a common suite of genes and conserved molecular pathways that respond to all investigated pathogens, a result that suggests a commonality in response mechanisms to diverse pathogens. We found that genes differentially expressed after infection exhibit a higher evolutionary rate than non-differentially expressed genes. Using our new bioinformatics approach, we unveiled additional pathogen-specific responses of honey bees; we found that apoptosis appeared to be an important response following microsporidian infection, while genes from the immune signalling pathways, Toll and Imd, were differentially expressed after Varroa/virus infection. Finally, we applied our bioinformatics approach and generated a gene co-expression network to identify highly connected (hub) genes that may represent important mediators and regulators of anti-pathogen responses. Conclusions: Our meta-analysis generated a comprehensive overview of the host metabolic and other biological processes that mediate interactions between insects and their pathogens. We identified key host genes and pathways that respond to phylogenetically diverse pathogens, representing an important source for future functional studies as well as offering new routes to identify or generate pathogen resilient honey bee stocks. The statistical and bioinformatics approaches that were developed for this study are broadly applicable to synthesize information across transcriptomic datasets. These approaches will likely have utility in addressing a variety of biological questions.This article is a joint effort of the working group TRANSBEE and an outcome of two workshops kindly supported by sDiv, the Synthesis Centre for Biodiversity Sciences within the German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, funded by the German Science Foundation (FZT 118). New datasets were performed thanks to the Insect Pollinators Initiative (IPI grant BB/I000100/1 and BB/I000151/1), with participation of the UK-USA exchange funded by the BBSRC BB/I025220/1 (datasets #4, 11 and 14). The IPI is funded jointly by the Biotechnology and Biological Sciences Research Council, the Department for Environment, Food and Rural Affairs, the Natural Environment Research Council, the Scottish Government and the Wellcome Trust, under the Living with Environmental Change Partnershi