Ryan S. Schwarz

Bees brought to their knees: microbes affecting honey bee health

The biology and health of the honey bee Apis mellifera has been of interest to human societies for centuries. Research on honey bee health is surging, in part due to new tools and the arrival of colony-collapse disorder (CCD), an unsolved decline in bees from parts of the United States, Europe, and Asia. Although a clear understanding of what causes CCD has yet to emerge, these efforts have led to new microbial discoveries and avenues to improve our understanding of bees and the challenges they face. Here we review the known honey bee microbes and highlight areas of both active and lagging research. Detailed studies of honey bee-pathogen dynamics will help efforts to keep this important pollinator healthy and will give general insights into both beneficial and harmful microbes confronting insect colonies.

Direct effect of acaricides on pathogen loads and gene expression levels in honey bees Apis mellifera

The effect of using acaricides to control varroa mites has long been a concern to the beekeeping industry due to unintended negative impacts on honey bee health. Irregular ontogenesis, suppression of immune defenses, and impairment of normal behavior have been linked to pesticide use. External stressors, including parasites and the pathogens they vector, can confound studies on the effects of pesticides on the metabolism of honey bees. This is the case of Varroa destructor, a mite that negatively affects honey bee health on many levels, from direct parasitism, which diminishes honey bee productivity, to vectoring and/or activating other pathogens, including many viruses. Here we present a gene expression profile comprising genes acting on diverse metabolic levels (detoxification, immunity, and development) in a honey bee population that lacks the influence of varroa mites. We present data for hives treated with five different acaricides; Apiguard (thymol), Apistan (tau-fluvalinate), Checkmite (coumaphos), Miteaway (formic acid) and ApiVar (amitraz). The results indicate that thymol, coumaphos and formic acid are able to alter some metabolic responses. These include detoxification gene expression pathways, components of the immune system responsible for cellular response and the c-Jun amino-terminal kinase (JNK) pathway, and developmental genes. These could potentially interfere with the health of individual honey bees and entire colonies.

A unique T cell receptor discovered in marsupials

T cells recognize antigens by using T cell receptors (TCRs) encoded by gene segments, called variable (V), diversity (D), and joining (J), that undergo somatic recombination to create diverse binding specificities. Four TCR chains (α, β, γ, and δ) have been identified to date, and, as T cells develop in the thymus, they express exclusively either an αβTCR or a γδTCR heterodimer. Here, we show that marsupials have an additional TCR (TCRμ) that has V, D, and J that are either somatically recombined, as in conventional TCRs, or are already prejoined in the germ-line DNA in a manner consistent with their creation by retrotransposition. TCRμ does not have a known homolog in eutherian mammals but has features analogous to a recently described TCRδ isoform in sharks. TCRμ is expressed in at least two mRNA isoforms that appear capable of encoding a full-length protein, both of which are transcribed in the thymus and spleen. One contains two variable domains: a somatically recombined V and a prejoined V. This appears to be the dominant isoform in peripheral lymphoid tissue. The other isoform contains only the prejoined V and is structurally more similar to conventional TCR chains, however invariant. Unlike other TCRs, TCRμ uses prejoined gene segments and is likely present in all marsupials. Its similarity to a TCR isoform in sharks suggests that it, or something similar, may be present in other vertebrate lineages and, therefore, may represent an ancient receptor system.

Standard methods for molecular research in Apis mellifera

Summary From studies of behaviour, chemical communication, genomics and developmental biology, among many others, honey bees have long been a key organism for fundamental breakthroughs in biology. With a genome sequence in hand, and much improved genetic tools, honey bees are now an even more appealing target for answering the major questions of evolutionary biology, population structure, and social organization. At the same time, agricultural incentives to understand how honey bees fall prey to disease, or evade and survive their many pests and pathogens, have pushed for a genetic understanding of individual and social immunity in this species. Below we describe and reference tools for using modern molecular-biology techniques to understand bee behaviour, health, and other aspects of their biology. We focus on DNA and RNA techniques, largely because techniques for assessing bee proteins are covered in detail in Hartfelder et al. (2013). We cover practical needs for bee sampling, transport, and storage, and then discuss a range of current techniques for genetic analysis. We then provide a roadmap for genomic resources and methods for studying bees, followed by specific statistical protocols for population genetics, quantitative genetics, and phylogenetics. Finally, we end with three important tools for predicting gene regulation and function in honey bees: Fluorescence in situ hybridization (FISH), RNA interference (RNAi), and the estimation of chromosomal methylation and its role in epigenetic gene regulation.

Characterization of Two Species of Trypanosomatidae from the Honey Bee Apis mellifera: Crithidia mellificae Langridge and McGhee, † and Lotmaria passim n. gen., n. sp.

Trypanosomatids are increasingly recognized as prevalent in European honey bees (Apis mellifera) and by default are attributed to one recognized species, Crithidia mellificae Langridge and McGhee, 1967. We provide reference genetic and ultrastructural data for type isolates of C. mellificae (ATCC 30254 and 30862) in comparison with two recent isolates from A. mellifera (BRL and SF). Phylogenetics unambiguously identify strains BRL/SF as a novel taxonomic unit distinct from C. mellificae strains 30254/30862 and assign all four strains as lineages of a novel clade within the subfamily Leishmaniinae. In vivo analyses show strains BRL/SF preferably colonize the hindgut, lining the lumen as adherent spheroids in a manner identical to previous descriptions from C. mellificae. Microscopy images show motile forms of C. mellificae are distinct from strains BRL/SF. We propose the binomial Lotmaria passim n. gen., n. sp. for this previously undescribed taxon. Analyses of new and previously accessioned genetic data show C. mellificae is still extant in bee populations, however, L. passim n. gen., n. sp. is currently the predominant trypanosomatid in A. mellifera globally. Our findings require that previous reports of C. mellificae be reconsidered and that subsequent trypanosomatid species designations from Hymenoptera provide genetic support.

Single and mixed-species trypanosome and microsporidia infections elicit distinct, ephemeral cellular and humoral immune responses in honey bees.

Frequently encountered parasite species impart strong selective pressures on host immune system evolution and are more apt to concurrently infect the same host, yet molecular impacts in light of this are often overlooked. We have contrasted immune responses in honey bees to two common eukaryotic endoparasites by establishing single and mixed-species infections using the long-associated parasite Crithidia mellificae and the emergent parasite Nosema ceranae. Quantitative polymerase chain reaction was used to screen host immune gene expression at 9 time points post inoculation. Systemic responses in abdomens during early stages of parasite establishment revealed conserved receptor (Down syndrome cell adhesion molecule, Dscam and nimrod C1, nimC1), signaling (MyD88 and Imd) and antimicrobial peptide (AMP) effector (Defensin 2) responses. Late, established infections were distinct with a refined 2 AMP response to C. mellificae that contrasted starkly with a 5 AMP response to N. ceranae. Mixed species infections induced a moderate 3 AMPs. Transcription in gut tissues highlighted important local roles for Dscam toward both parasites and Imd signaling toward N. ceranae. At both systemic and local levels Dscam, MyD88 and Imd transcription was consistently correlated based on clustering analysis. Significant gene suppression occurred in two cases from midgut to ileum tissue: Dscam was lowered during mixed infections compared to N. ceranae infections and both C. mellificae and mixed infections had reduced nimC1 transcription compared to uninfected controls. We show that honey bees rapidly mount complex immune responses to both Nosema and Crithidia that are dynamic over time and that mixed-species infections significantly alter local and systemic immune gene transcription.

The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions

ABSTRACT As pollinators, bees are cornerstones for terrestrial ecosystem stability and key components in agricultural productivity. All animals, including bees, are associated with a diverse community of microbes, commonly referred to as the microbiome. The bee microbiome is likely to be a crucial factor affecting host health. However, with the exception of a few pathogens, the impacts of most members of the bee microbiome on host health are poorly understood. Further, the evolutionary and ecological forces that shape and change the microbiome are unclear. Here, we discuss recent progress in our understanding of the bee microbiome, and we present challenges associated with its investigation. We conclude that global coordination of research efforts is needed to fully understand the complex and highly dynamic nature of the interplay between the bee microbiome, its host, and the environment. High-throughput sequencing technologies are ideal for exploring complex biological systems, including host-microbe interactions. To maximize their value and to improve assessment of the factors affecting bee health, sequence data should be archived, curated, and analyzed in ways that promote the synthesis of different studies. To this end, the BeeBiome consortium aims to develop an online database which would provide reference sequences, archive metadata, and host analytical resources. The goal would be to support applied and fundamental research on bees and their associated microbes and to provide a collaborative framework for sharing primary data from different research programs, thus furthering our understanding of the bee microbiome and its impact on pollinator health.

Migration and differentiation potential of stem cells in the cnidarian Hydractinia analysed in eGFP-transgenic animals and chimeras

To analyse cell migration and the differentiation potential of migratory stem cells in Hydractinia, we generated animals with an eGFP reporter gene stably expressed and transmitted via the germline. The transgene was placed under the control of two different actin promoters and the promoter of elongation factor-1α. One actin promoter (Act-II) and the EF-1α promoter enabled expression of the transgene in all cells, the other actin promoter (Act-I) in epithelial and gametogenic cells, but not in the pluripotent migratory stem cells. We produced chimeric animals consisting of histocompatible wild type and transgenic parts. When the transgene was under the control of the epithelial cell specific actin-I promoter, non-fluorescent transgenic stem cells immigrated into wild type tissue, stopped migration and differentiated into epithelial cells which then commenced eGFP-expression. Migratory stem cells are therefore pluripotent and can give rise not only to germ cells, nematocytes and nerve cells, but also to epithelial cells. While in somatic cells expression of the act-I promoter was restricted to epithelial cells it became also active in gametogenesis. The act-I gene is expressed in spermatogonia, oogonia and oocytes. In males the expression pattern showed that migratory stem cells are the precursors of both the spermatogonia and their somatic envelopes. Comparative expression studies using the promoters of the actin-II gene and the elongation factor-1α gene revealed the potential of transgenic techniques to trace the development of the nervous system.

GENOMIC ANALYSIS OF EIMERIA SPP. POPULATIONS IN RELATION TO PERFORMANCE LEVELS OF BROILER CHICKEN FARMS IN ARKANSAS AND NORTH CAROLINA

Abstract The impact of coccidiosis outbreaks on the productivity of broiler chicken farms can be substantial, depending on the severity of disease caused by particular species and strains of Eimeria. We examined the genetic diversity of Eimeria species present in commercial broiler farms in relation to their performance level. Four groups of broiler chicken farms in Arkansas (AR) and North Carolina (NC), having either high or low performance levels, were sampled for Eimeria spp. oocysts. We amplified gDNA from oocysts by using genus-specific primers targeting 18S ribosomal RNA, the first and second internal transcribed spacer regions, and cytochrome c oxidase subunit I as the established species-specific primers. Eimeria spp. diversity was not homogenous among the 4 farm groups, with less-pathogenic species (E. mitis and E. mivati-like) associated with AR and NC high-performance farms, respectively, and a pathogenic species (E. brunetti) associated with AR low-performance farms. Sequence analyses identified multiple E. maxima and E. mitis genetic variants, from which 2 E. maxima variants were unique to low-performance farms. Distinct populations of sequences at the NC high-performance farms were identified as E. mivati-like, based on homology searches. Our study demonstrated the utility of analyzing multiple genomic loci to assess composition and polymorphisms of Eimeria spp. populations.

Early gut colonizers shape parasite susceptibility and microbiota composition in honey bee workers.

Significance Using a social insect model, we tested how supplementing young adult bees with a resident microbiota species affects host physiology and microbiome composition. This supplementation had significant consequences for host development and detoxification responses, parasite susceptibility, and microbiome community structure. Our results show that early perturbation of the microbiota composition can have sustained consequences for hosts. Additionally, this work provides a cautionary tale to the arbitrary use of probiotics in animal health management and highlights the importance of experimental research addressing factors that shape animal microbiome communities. Microbial symbionts living within animal guts are largely composed of resident bacterial species, forming communities that often provide benefits to the host. Gut microbiomes of adult honey bees (Apis mellifera) include core residents such as the betaproteobacterium Snodgrassella alvi, alongside transient parasites such as the protozoan Lotmaria passim. To test how these species affect microbiome composition and host physiology, we administered S. alvi and/or L. passim inocula to newly emerged worker bees from four genetic backgrounds (GH) and reared them in normal (within hives) or stressed (protein-deficient, asocial) conditions. Microbiota acquired by normal bees were abundant but quantitatively differed across treatments, indicating treatment-associated dysbiosis. Pretreatment with S. alvi made normal bees more susceptible to L. passim and altered developmental and detoxification gene expression. Stressed bees were more susceptible to L. passim and were depauperate in core microbiota, yet supplementation with S. alvi did not alter this susceptibility. Microbiomes were generally more variable by GH in stressed bees, which also showed opposing and comparatively reduced modulation of gene expression responses to treatments compared with normal bees. These data provide experimental support for a link between altered gut microbiota and increased parasite and pathogen prevalence, as observed from honey bee colony collapse disorder.