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A Varroa destructor protein atlas reveals molecular underpinnings of developmental transitions and sexual differentiation*

  • Alison McAfee
    Affiliations
    From the Department of Biochemistry & Molecular Biology, Michael Smith Laboratories, University of British Columbia, 2125 East Mall, Vancouver, British Columbia, Canada V6T 1Z4;
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  • Queenie W.T. Chan
    Affiliations
    From the Department of Biochemistry & Molecular Biology, Michael Smith Laboratories, University of British Columbia, 2125 East Mall, Vancouver, British Columbia, Canada V6T 1Z4;
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  • Jay Evans
    Affiliations
    Bee Research Laboratory, Beltsville Agricultural Research Center—East, U.S. Department of Agriculture, Beltsville, MD, USA 20705-0000
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  • Leonard J. Foster
    Correspondence
    To whom correspondence should be addressed: Tel.:+1 (604) 822-8311.
    Affiliations
    Bee Research Laboratory, Beltsville Agricultural Research Center—East, U.S. Department of Agriculture, Beltsville, MD, USA 20705-0000
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  • Author Footnotes
    * This work was supported by the Natural Sciences and Engineering Research Council.
    This article contains supplemental material.
Open AccessPublished:September 03, 2017DOI:https://doi.org/10.1074/mcp.RA117.000104
      Varroa destructor is the most economically damaging honey bee pest, weakening colonies by simultaneously parasitizing bees and transmitting harmful viruses. Despite these impacts on honey bee health, surprisingly little is known about its fundamental molecular biology. Here, we present a Varroa protein atlas crossing all major developmental stages (egg, protonymph, deutonymph, and adult) for both male and female mites as a web-based interactive tool (http://foster.nce.ubc.ca/varroa/index.html). We used intensity-based label-free quantitation to find 1,433 differentially expressed proteins across developmental stages. Enzymes for processing carbohydrates and amino acids were among many of these differences as well as proteins involved in cuticle formation. Lipid transport involving vitellogenin was the most significantly enriched biological process in the foundress (reproductive female) and young mites. In addition, we found that 101 proteins were sexually regulated and functional enrichment analysis suggests that chromatin remodeling may be a key feature of sex determination. In a proteogenomic effort, we identified 519 protein-coding regions, 301 of which were supported by two or more peptides and 169 of which were differentially expressed. Overall, this work provides a first-of-its-kind interrogation of the patterns of protein expression that govern the Varroa life cycle and the tools we have developed will support further research on this threatening honey bee pest.
      The Varroa destructor mite is the most devastating pest for Western honey bees (Apis mellifera) (
      • Cornman S.R.
      • Schatz M.C.
      • Johnston S.J.
      • Chen Y.P.
      • Pettis J.
      • Hunt G.
      • Bourgeois L.
      • Elsik C.
      • Anderson D.
      • Grozinger C.M.
      • Evans J.D.
      Genomic survey of the ectoparasitic mite Varroa destructor, a major pest of the honey bee Apis mellifera.
      ,
      • Le Conte Y.
      • Ellis M.
      • Ritter W.
      Varroa mites and honey bee health: Can Varroa explain part of the colony losses?.
      ,
      • Dietemann V.
      • Pflugfelder J.
      • Anderson D.
      • Charrière J.-D.
      • Chejanovsky N.
      • Dainat B.
      • de Miranda J.
      • Delaplane K.
      • Dillier F.-X.
      • Fuch S.
      • Gallmann P.
      • Gauthier L.
      • Imdorf A.
      • Koeniger N.
      • Kralj J.
      • Meikle W.
      • Pettis J.
      • Rosenkranz P.
      • Sammataro D.
      • Smith D.
      • Yañez O.
      • Neumann P.
      Varroa destructor: Research avenues towards sustainable control.
      ). This obligate parasite feeds on honey bee hemolymph (blood), simultaneously weakening its host, suppressing the innate immune system, and transmitting debilitating viruses (see Rosenkranz et al. (
      • Rosenkranz P.
      • Aumeier P.
      • Ziegelmann B.
      Biology and control of Varroa destructor.
      ) for a comprehensive review on Varroa biology). Varroa's natural host is the Eastern honey bee (A. cerana), and millions of years of coevolution have led A. cerana to develop various tolerance mechanisms, thereby minimizing the mite's negative impact on these colonies (
      • Rath W.
      Co-adaptation of Apis cerana Fabr., and Varroa jacobsoni Oud.
      ,
      • Lin Z.
      • Page P.
      • Li L.
      • Qin Y.
      • Zhang Y.
      • Hu F.
      • Neumann P.
      • Zheng H.
      • Dietemann V.
      Go east for better honey bee health: Apis cerana is faster at hygienic behavior than A. mellifera.
      ,
      • Boot W.J.
      • Calis J.N.
      • Beetsma J.
      • Hai D.M.
      • Lan N.K.
      • Toan T.V.
      • Trung L.Q.
      • Minh N.H.
      Natural selection of Varroa jacobsoni explains the different reproductive strategies in colonies of Apis ceranaApis mellifera.
      ). However, in the mid-1900s, the mite jumped hosts to A. mellifera—the bee species that is most commonly used for active crop pollination today—which is less effective at defending itself (
      • Rosenkranz P.
      • Aumeier P.
      • Ziegelmann B.
      Biology and control of Varroa destructor.
      ,
      • Lin Z.
      • Page P.
      • Li L.
      • Qin Y.
      • Zhang Y.
      • Hu F.
      • Neumann P.
      • Zheng H.
      • Dietemann V.
      Go east for better honey bee health: Apis cerana is faster at hygienic behavior than A. mellifera.
      ). Managed A. mellifera colonies infested with Varroa have shorter lifespans than uninfested colonies unless they are actively treated with miticides (
      • Fries I.
      • Imdorf A.
      • Rosenkranz P.
      Survival of mite infested (Varroa destructor) honey bee (Apis mellifera) colonies in a Nordic climate.
      ,
      • Korpela S.
      • Aarhus A.
      • Fries I.
      • Hansen H.
      Varroa jacobsoni Oud. in cold climates: Population growth, winter mortality and influence on the survival of honey bee colonies.
      ), causing serious negative economic impacts (
      • Currie R.
      • Gatien P.
      Timing acaricide treatments to prevent Varroa destructor (Acari: Varroidae) from causing economic damage to honey bee colonies.
      ,
      • Vanengelsdorp D.
      • Meixner M.D.
      A historical review of managed honey bee populations in Europe and the United States and the factors that may affect them.
      ,
      • Guzman-Novoa E.
      • Eccles L.
      • Calvete Y.
      • McGowan J.
      • Kelly P.G.
      • Correa-Benitez A.
      Varroa destructor is the main culprit for the death and reduced populations of overwintered honey bee (Apis mellifera) colonies in Ontario, Canada.
      ).
      Despite being responsible for significant colony losses, very little is known about the molecular biology of the Varroa mite. Since the egg, protonymph, and deutonymph life stages (Fig. 1) only exist when the foundress mite (reproductive female) is actively reproducing within capped honey bee brood comb (
      • Rosenkranz P.
      • Aumeier P.
      • Ziegelmann B.
      Biology and control of Varroa destructor.
      ), they are seldom observed and are tedious to sample. Furthermore, male mites (even as adults) die soon after the adult honey bee emerges, so even though they are obviously important factors in mite reproduction, our knowledge of their basic molecular biology is extremely limited. Research on Varroa has focused on its role as a vector for viruses (
      • Francis R.M.
      • Nielsen S.L.
      • Kryger P.
      Varroa-virus interaction in collapsing honey bee colonies.
      ,
      • Levin S.
      • Sela N.
      • Chejanovsky N.
      Two novel viruses associated with the Apis mellifera pathogenic mite Varroa destructor.
      ,
      • Erban T.
      • Harant K.
      • Hubalek M.
      • Vitamvas P.
      • Kamler M.
      • Poltronieri P.
      • Tyl J.
      • Markovic M.
      • Titera D.
      In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.
      ,
      • Di Prisco G.
      • Pennacchio F.
      • Caprio E.
      • Boncristiani Jr., H.F.
      • Evans J.D.
      • Chen Y.
      Varroa destructor is an effective vector of Israeli acute paralysis virus in the honeybee, Apis mellifera.
      ,
      • Gisder S.
      • Aumeier P.
      • Genersch E.
      Deformed wing virus: Replication and viral load in mites (Varroa destructor).
      ,
      • Ryabov E.V.
      • Wood G.R.
      • Fannon J.M.
      • Moore J.D.
      • Bull J.C.
      • Chandler D.
      • Mead A.
      • Burroughs N.
      • Evans D.J.
      A virulent strain of deformed wing virus (DWV) of honeybees (Apis mellifera) prevails after Varroa destructor-mediated, or in vitro, transmission.
      ), their response to pheromone cues (
      • Ziegelmann B.
      • Lindenmayer A.
      • Steidle J.
      • Rosenkranz P.
      The mating behavior of Varroa destructor is triggered by a female sex pheromone.
      ,
      • Del Piccolo F.
      • Nazzi F.
      • Della Vedova G.
      • Milani N.
      Selection of Apis mellifera workers by the parasitic mite Varroa destructor using host cuticular hydrocarbons.
      ,
      • Nazzi F.
      • Le Conte Y.
      Ecology of Varroa destructor, the major ectoparasite of the western honey bee, Apis mellifera.
      ), attempts to control it via RNAi (
      • Garbian Y.
      • Maori E.
      • Kalev H.
      • Shafir S.
      • Sela I.
      Bidirectional transfer of RNAi between honey bee and Varroa destructorVarroa gene silencing reduces Varroa population.
      ,
      • Campbell E.M.
      • Budge G.E.
      • Bowman A.S.
      Gene-knockdown in the honey bee mite Varroa destructor by a non-invasive approach: Studies on a glutathione S-transferase.
      ,
      • Campbell E.M.
      • Budge G.E.
      • Watkins M.
      • Bowman A.S.
      Transcriptome analysis of the synganglion from the honey bee mite, Varroa destructor and RNAi knockdown of neural peptide targets.
      ), and host shifts (
      • Andino G.K.
      • Gribskov M.
      • Anderson D.L.
      • Evans J.D.
      • Hunt G.J.
      Differential gene expression in Varroa jacobsoni mites following a host shift to European honey bees (Apis mellifera).
      ). At the time of writing, there have only been two previous Varroa proteomic investigations, one of which focused on viral proteins (
      • Erban T.
      • Harant K.
      • Hubalek M.
      • Vitamvas P.
      • Kamler M.
      • Poltronieri P.
      • Tyl J.
      • Markovic M.
      • Titera D.
      In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.
      ) and the other identifying fewer than 700 proteins within one developmental stage (
      • Surlis C.
      • Carolan J.C.
      • Coffey M.F.
      • Kavanagh K.
      Proteomic analysis of Bayvarol® resistance mechanisms in the honey bee parasite Varroa destructor.
      ). Global protein expression changes associated with developmental transitions and sexual differentiation are yet unknown.
      Figure thumbnail gr1
      Fig. 1Schematic representation of the mite life cycle. All stages were included in this study (n = 3 for all) except the phoretic stage. For egg and protonymph stages, males and females are visually indistinguishable, so for these stages, sexes were pooled. Colors indicate melanization of the cuticle and sizes are proportional.
      The Varroa genome was first sequenced in 2010 (
      • Cornman S.R.
      • Schatz M.C.
      • Johnston S.J.
      • Chen Y.P.
      • Pettis J.
      • Hunt G.
      • Bourgeois L.
      • Elsik C.
      • Anderson D.
      • Grozinger C.M.
      • Evans J.D.
      Genomic survey of the ectoparasitic mite Varroa destructor, a major pest of the honey bee Apis mellifera.
      ) and was accompanied by a provisional gene annotation that will be updated shortly. Gene annotations are living databases and, particularly with newly sequenced species, they undergo continuous refinement as more omic data become available. Unfortunately, the more evolutionarily distant a species is from well-annotated species typically used for orthology delineation and gene prediction training sets, the less accurate the predictions become. Such is the case for Varroa. Proteogenomics (
      • McAfee A.
      • Foster L.J.
      Proteogenomics: Recycling public data to Improve genome annotations.
      ,
      • Nesvizhskii A.I.
      Proteogenomics: Concepts, applications and computational strategies.
      ) can help overcome this problem by sequencing the expressed protein regions in a relatively unbiased survey of the genomic landscape. Since protein expression is dynamic throughout an organism's life cycle, high-resolution omics data that cross developmental stages and sexes are very well-suited for this purpose.
      Investigating global protein expression profiles throughout development of both sexes simultaneously provides a foundational understanding of Varroa biology and creates an opportunity to improve upon existing gene annotations. We present here the first Varroa proteome crossing all major developmental stages (egg, protonymph, deutonymph, adult) of both males and females, where distinguishable (Fig. 1). Through a proteogenomics effort, we identified 519 new protein-coding regions—301 of which are supported by two or more peptides. We also analyze the chemical properties of these sequences and their sequence similarity to other organisms to investigate reasons why underannotation continues to be a problem. We identified 3,102 proteins overall, nearly half (1,433) of which were significantly differentially expressed through development and 101 of which were differentially expressed between sexes. Functional enrichment suggested that carbohydrate and amino acid metabolism underpin developmental transitions, so we investigated proteins involved in glycolysis and the Krebs cycle in detail. Cuticle formation is clearly a process associated with mite aging, and closer analysis suggests the mites utilize different chitin structural proteins as they mature. In addition, chromatin remodeling and positive regulation of transcription may be key factors in sexual differentiation. Building on our previous honey bee protein atlas (
      • Chan Q.W.
      • Chan M.Y.
      • Logan M.
      • Fang Y.
      • Higo H.
      • Foster L.J.
      Honey bee protein atlas at organ-level resolution.
      ), we provide a web-based interactive platform (http://foster.nce.ubc.ca/varroa/index.html) where researchers can query proteins for visual displays of expression patterns, enabling further hypothesis generation and maximizing the utility of this information for the scientific community.

      DISCUSSION

      The work presented here provides a foundation to begin to unravel the fundamentals of Varroa biology, including developmental transitions, sexual differentiation, diet, and host–virus interactions, as well as assisting with improving the genome annotation. Prior to this, there has been one published Varroa RNA-seq transcriptomics (
      • Campbell E.M.
      • Budge G.E.
      • Watkins M.
      • Bowman A.S.
      Transcriptome analysis of the synganglion from the honey bee mite, Varroa destructor and RNAi knockdown of neural peptide targets.
      ) and two proteomics studies, one of which—as far as we can tell—identified only virus and honey bee proteins and none from Varroa (
      • Erban T.
      • Harant K.
      • Hubalek M.
      • Vitamvas P.
      • Kamler M.
      • Poltronieri P.
      • Tyl J.
      • Markovic M.
      • Titera D.
      In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.
      ), and the other only analyzed foundresses (
      • Surlis C.
      • Carolan J.C.
      • Coffey M.F.
      • Kavanagh K.
      Proteomic analysis of Bayvarol® resistance mechanisms in the honey bee parasite Varroa destructor.
      ). With almost 20,000 unique peptides identified, our study represents the deepest Varroa proteome to date. Overall, we identified 3,102 proteins, 2,626 of which were quantified by LFQ and incorporated into an interactive web-based Varroa proteome to serve as a community resource.
      Genome sequencing is becoming relatively easy, but accurately annotating the genome is an arduous and imperfect process. The most common model organisms (e.g. M. musculus, D. melanogaster, C. elegans, etc.) have benefitted from decades of genetic research that has refined their genome annotations over time, resulting in highly reliable and accurate gene sets on which most tools for analyzing global gene and protein expression rely. Our data clearly show that the new Varroa gene annotation is far better than the provisional draft (Fig. 2), but our proteogenomics initiative, which identified 1,464 unique unannotated peptides, suggests that there is still room for improvement. While some of these novel peptides simply harbor nonsynonymous sequence polymorphisms, that itself is worth reporting, and this information can be used to augment the protein databases used for mass spectrometry searches (
      • Schandorff S.
      • Olsen J.V.
      • Bunkenborg J.
      • Blagoev B.
      • Zhang Y.
      • Andersen J.S.
      • Mann M.
      A mass spectrometry-friendly database for cSNP identification.
      ). Other peptides, however, clearly corresponded to exons of unannotated genes (Figs. 5C and 5D) that showed significant homology to vitellogenin. This observation, along with finding nothing unusual about the sequence properties of the newly identified coding regions (Fig. 4B, supplemental Fig. 1) led us to question why they were not already annotated.
      The annotation process is not only influenced by the genome itself (chemical and physical properties, completeness, etc.) but also by the quality of guiding transcript assemblies and a number of human-determined parameters (e.g. the annotation software employed, hard or soft repeat masking, splice site awareness, etc.), and availability of prior gene models (
      • Yandell M.
      • Ence D.
      A beginner's guide to eukaryotic genome annotation.
      ,
      • Ekblom R.
      • Wolf J.B.
      A field guide to whole-genome sequencing, assembly and annotation.
      ). Furthermore, some parameters may need to be altered on a species-by-species basis, but there is no inherent pathway for finding the optimal settings. Proteomics and RNA-seq data could serve as tools to not only confirm expression of predicted genes but also to help define these parameters in the first place since the resulting protein and gene IDs are sensitive to database accuracy. The data we present here are all publicly available (PXD006072), and we urge future iterations of annotation refinement to take full advantage of this peptide evidence when developing new Varroa gene models.
      In mass-spectrometry-based proteomics, it is important that the protein database reflects the proteins that could be present in the sample. Since Varroa feeds on honey bee tissues and others have detected honey bee proteins in Varroa (
      • Erban T.
      • Harant K.
      • Hubalek M.
      • Vitamvas P.
      • Kamler M.
      • Poltronieri P.
      • Tyl J.
      • Markovic M.
      • Titera D.
      In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.
      ), we included honey bee proteins in the search database and found that 167 of them were significantly differentially abundant (supplemental Fig. 3). The eggs were largely lacking in honey bee proteins, which is in keeping with the developing embryos not yet being able to feed on wounded honey bee pupae. The presence of some honey bee proteins in the egg suggests these are contamination; however, the deutonymph stage of both sexes, which are actively feeding on hemolymph, showed the highest abundance of honey bee proteins. This suggests that the deutonymphs require large amounts of food, possibly to support energetically expensive developmental processes such as metamorphosis.
      Our analysis of developmentally regulated proteins revealed some intriguing trends regarding the energetic demands throughout development (Fig. 6A). The foundress had consistently high abundances of enzymes that participate in glycolysis and the citric acid cycle, which may be required to meet the energetic demands of producing and laying eggs. We speculate that many of the differences in metabolic processes are also driven by the unique energetic requirements of metamorphosis, when energetically expensive morphological rearrangements must occur while the mite does not eat.
      During maturation, protonymph and deutonymph mites transition from having a soft, translucent cuticle to acquiring a harder and more durable exoskeleton. The phoretic and foundress mites have rigid armor to protect against injury by grooming honey bees and other environmental hazards. To investigate the possible mechanisms behind these transitions, we compared the expression profiles of significantly differentially expressed proteins that are related to cuticle development (chitin structural protein, chitinases, and chitin-binding proteins; Fig. 6B). The egg contains large amounts of one chitinase and one chitin structural protein, which could be related to the breakdown of the egg case or the developing mite larva as it becomes a protonymph. Deutonymphs display a specific profile of highly abundant structural proteins and chitin-binding proteins, and from this point on, there is a clear separation between male and female expression profiles. The male mite appears not to invest energy in forming a tough exoskeleton like the female does, which is consistent with the lack of environmental exposure during the male life cycle.
      In our analysis of sexually regulated proteins, we found that chromatin remodeling and transcription activation were significantly enriched processes. Chromatin remodeling could be required to decondense chromosomal regions that are highly expressed in males or females and vice versa. Indeed, histone lysine N-methyltransferase was one of the most significant differentially expressed proteins, with ∼30-fold higher levels in males compared with females (Fig. 7B), and peptidyl-amino acid modification was the most significantly enriched biological process (Table III). This kind of on–off regulation could thus be very important for sex determination. We also found that HSP83, which is critically important for spermatogenesis in Drosophila (
      • Yue L.
      • Karr T.L.
      • Nathan D.F.
      • Swift H.
      • Srinivasan S.
      • Lindquist S.
      Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis.
      ), displayed the greatest fold change (∼50-fold) out of those with known functions. Broadening our analysis to all identified HSPs, we found that there is a core group of HSPs that are specific to the foundress and another group that is specific to males (Fig. 7C), suggesting that these HSPs are involved in regulating the transcription of sex-specific genes.
      The work we present here represents a first-of-its-kind, high-resolution analysis of the Varroa proteome. With some 1,433 proteins that are differentially expressed, these data provide a first glimpse into the changes that take place during Varroa development. In addition, 101 strongly sexually regulated proteins provide clues for discovering the mechanisms behind sex determination and general dimorphism. We hope that the interactive web tool will maximize the utility of this information for the research community and will help generate further hypotheses for future experiments on this major honey bee pest.

      DATA AVAILABILITY

      Raw mass spectrometry data can be downloaded from the PRIDE Archive (www.ebi.ac.uk/pride/archive/), accession PXD006072. Annotated spectra are available through MS-viewer (http://msviewer.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msviewer) with search keys wuh30b9smr and msmx6z444s.

      Acknowledgments

      We would like to acknowledge Alexandra Sebastien, Walter Wasserman, Greg Stacey, and Jason Rogalski for their assistance during manuscript preparation.

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