Biology subjects

Posada-Florez, F.

Publications and source records attributed to Posada-Florez, F..

8 recordsLinked to original sources

bioRxiv2022

Kleptocytosis: A Novel Parasitic Strategy for Accelerated Reproduction via Host Protein Stealing in Varroa destructor

Varroa destructor must produce mature offspring within the inflexible nine-day window framing the pupal development of their honey bee host. Missing this deadline renders the foundress mites fitness zero, establishing evolutionary pressure to accelerate reproduction and development. Through micro-computed tomography and modeling Varroas energy budget, we found each egg grows to constitute [~]18% of the foundresss body volume in 30 hours, yet accounts for less than 10% of her energy usage. We hypothesized that this small energy investment is a successful strategy because of Varroas long-ignored ability to traffic intact host proteins. Through gel electrophoresis, concomitant immunodetection, and MS/MS, we detected several intact, bee-derived proteins in mite eggs, including metamorphic proteins (hexamerins) and egg-yolk precursors (vitellogenin and apolipophorin) which likely reduce the parasites direct reproductive investment. We then expressed recombinant Halotag labelled Apis vitellogenin to determine the route conveying exogenous proteins into the mites oocyte. We detected fluorescent vitellogenin in the lyrate organ and observed a tube-like extension of the lyrate organ connecting to the ovum, likely providing an avenue for intact host proteins. Finally, we tested the hypothesis that exogenous proteins maintain functionality in the parasite. Varroa metamorphose despite their inability to produce hexamerins, critical metamorphic proteins. Through label-free quantification of proteins in metamorphosing mites, we observed a hexamerin depletion pattern consistent with usage as a metamorphic amino acid reservoir. We describe this process as "kleptocytosis," denoting movement of stolen intact macromolecules from host to a parasite cell. Given their fixed developmental timeline, this pathway presents a promising target for novel Varroa management strategies. Significance StatementGlobal honey bee health is threatened by the parasitic mite Varroa destructor; its success due in part to its rapid reproduction and accelerated development. By combining biological modeling, micro-computed tomography, fluorescence imaging, and quantitative proteomics, we show that Varroa siphon intact, ostensibly functional host proteins conveying them directly to their oocyte. This avoids the energetically inefficient process of digesting and reconstructing ingested proteins and affords the capacity to utilize proteins that it does not produce de novo facilitating rapid reproduction and accelerated development. We call this process "kleptocytosis," and identify anatomical adaptations which apparently facilitate protein movement. This work exposes a new target in Varroas physiology, providing a promising direction for more effective management strategies.

zoology↗

bioRxiv2022

The vectoring competence of the mite Varroa destructor for Deformed wing virus of honey bees is highly dynamic and affects survival of the mite.

The ectoparasitic mite, Varroa destructor and the viruses it vectors, including types A and B of Deformed wing virus (DWV), pose a major threat to honey bees, Apis mellifera. Analysis of 256 mites collected from the same set of colonies on five occasions from May to October 2021 showed that less than a half of them, 39.8% (95% confidence interval (CI): 34.0 - 46.0%), were able to induce an overt-level DWV infection with more than 109 viral genomes per bee in the pupa after 6 days of feeding, with both DWV-A and DWV-B being vectored at similar rates. To investigate the effect of the phoretic stage on the mites ability to vector DWV, the mites from two field collection events were divided into two groups, one of which was tested immediately for their infectiveness, and the other was kept with adult worker bees in cages for 12 days prior to testing their infectiveness. We found that while 39.2 % (95% CI: 30.0 - 49.1%) of the immediately tested mites induced overt-level DWV infections, 12-day phoretic passage significantly increased the infectiousness of mites to 89.8% (95% CI: 79.2 - 95.6%). It is likely that Varroa mites that survive brood interruptions in field colonies are increasingly infectious. We found that mite lifespan was significantly affected by the type of DWV it transmitted to pupae. The mites, which induced overt DWV-B but not DWV-A infection had an average lifespan of 15.5 days (95% CI: 11.8 - 19.2 days), which was significantly shorter than those of the mites which induced overt DWV-A but not DWV-B infection, with an average lifespan of 24.3 days (95% CI: 20.2 - 28.5), or the mites which did not induce high levels of DWV-A or DWV-B, with an average survival of 21.2 days (95% CI: 19.0 - 23.5 days). The mites which transmitted high levels of both DWV-A and DWV-B had an intermediate average survival of 20.5 days (95% CI: 15.1 - 25.9 days). The negative impact of DWV-B on mite survival could be a consequence of the ability of DWV-B, but not DWV-A to replicate in Varroa mites.

microbiology↗

bioRxiv2020

Pupal cannibalism by worker honey bees contributes to the spread of Deformed wing virus.

Transmission routes impact pathogen virulence and genetics, therefore comprehensive knowledge of these routes and their contribution to pathogen circulation is essential for understanding host-pathogen interactions and designing control strategies. Deformed wing virus (DWV), a principal viral pathogen of honey bees associated with increased honey bee mortality and colony losses, became highly virulent with the spread of its vector, the ectoparasitic mite Varroa destructor. Reproduction of Varroa mites occurs in capped brood cells and mite-infested pupae from these cells usually have high levels of DWV. The removal of mite-infested pupae by worker bees, Varroa Sensitive Hygiene (VSH), leads to cannibalization of pupae with high DWV loads, thereby offering an alternative route for virus transmission. We used genetically tagged DWV to investigate virus transmission to and between worker bees following pupal cannibalisation under experimental conditions. We demonstrated that cannibalization of DWV-infected pupae resulted in high levels of this virus in worker bees and that the acquired virus was then transmitted between bees via trophallaxis, allowing circulation of Varroa-vectored DWV variants without the mites. Despite the known benefits of hygienic behaviour, it is possible that higher levels of VSH activity may result in increased transmission of DWV via cannibalism and trophallaxis.

microbiology↗

bioRxiv2020

Development of a honey bee RNA virus vector based on the genome of Deformed wing virus.

We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee (Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV LP and/or VP2 proteins. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.

microbiology↗

bioRxiv2019

Aberrant cocoons found on honey bee comb cells are found to be Osmia cornifrons (Radoszkowski) (Hymenoptera Megachilidae)

Potential biological threats to honey bees must be addressed and validated quickly, before making disruptive and costly decisions. Here we describe numerous Osmia cornifrons (Hymenoptera: Megachilidae) cocoons in honey bee cells from one bee hive in Ohio. The developing Osmia cells presented themselves as a mystery at first, catching the attention of regulatory agencies. Along with identifying this species as a presumably benign resident in honey bee colonies, our observations suggest Osmia may use stored honey bee resources to provision offspring. Conceivably, resident honey bees might even act as surrogates, by provisioning Osmia offspring with pollen. Since the cocoons were attached to one another with honey bee wax, it seems likely that honey bee hosts were present during Osmia development. Osmia females have some plasticity when selecting nesting resources, and, upon discovering honey bee comb can use this resource for raising offspring. Along with resolving a potentially new biotic threat to honey bees, this diagnosis suggests a method for mass production of Osmia pollinators using an array of single cell foundation.

zoology↗

bioRxiv2019

A novel system for maintaining Varroa destructor mites on artificial diets and its application for studying mites as a vector for honey bee viruses.

The mite Varroa destructor is one of the most destructive parasites of the honey bee (Apis mellifera) and the primary cause of colony collapse in most regions of the world. These mites cause serious injury to their hosts, especially during the larval and pupal stages, and serve as the vector for several viruses, which affect honey bee health causing colony death. Attempts by beekeepers to control these mites have yielded limited success. The inability to rear populations of mites in vitro that excludes contact with their honey bee hosts has stymied research of Varroa biology. Previous attempts to rear and/or maintain Varroa mites in vitro by feeding them on artificial diets have had limited success. Several methods were plagued by mechanical failures including leaking membranes and, thus far, none have been widely adopted. Here we report a robust system for maintaining Varroa mites that includes an artificial diet, which does not contain honey bee tissue-derived components, thus making it particularly valuable in studying mite vectoring of honey bee viruses. With our system we demonstrated for the first time that Varroa mites maintained on an artificial diet supplemented with the particles of honey bee viruses, cDNA clone-derived genetically tagged Varroa destructor virus-1 and wild-type Deformed wing virus, can acquire and later transmit these viruses to recipient honey bee pupae. Along with providing an opportunity to study parasites and pathogens in the absence of honey bee hosts, this in vitro system for Varroa mite maintenance is both scalable and consistent. These features can be used to better understand mite nutritional needs, metabolic activity, responses to chemicals and other biological functions.

zoology↗

bioRxiv2019

Deformed wing virus type A, a major honey bee pathogen, is vectored by the mite Varroa destructor in a non-propagative manner.

Honey bees, the primary managed insect pollinator, suffer considerable losses due to Deformed wing virus (DWV), an RNA virus vectored by the mite Varroa destructor. Mite vectoring has resulted in the emergence of virulent DWV variants. The basis for such changes in DWV is poorly understood. Most importantly, it remains unclear whether replication of DWV occurs in the mite. In this study, we exposed Varroa mites to DWV type A via feeding on artificially infected honey bees. A significant, 357-fold increase in DWV load was observed in these mites after 2 days. However, after 8 additional days of passage on honey bee pupae with low viral loads, the DWV load dropped by 29-fold. This decrease significantly reduced the mites ability to transmit DWV to honey bees. Notably, negative-strand DWV RNA, which could indicate viral replication, was detected only in mites collected from pupae with high DWV levels but not in the passaged mites. We also found that Varroa mites contain honey bee mRNAs, consistent with the acquisition of honey bee cells which would additionally contain DWV replication complexes with negative-strand DWV RNA. We propose that transmission of DWV type A by Varroa mites occurs in a non-propagative manner.

microbiology↗

bioRxiv2019

Dynamic evolution in the key honey bee pathogen Deformed wing virus

The impacts of invertebrate RNA virus population dynamics on virulence and infection outcomes are poorly understood. Deformed wing virus (DWV), the main viral pathogen of honey bees, negatively impacts bee health which can lead to colony death. Despite previous reports on the reduction of DWV diversity following the arrival of the parasitic mite Varroa destructor, the key DWV vector, we found high genetic diversity of DWV in infested United States (US) honey bee colonies. Phylogenetic analysis showed that the divergent US DWV genotypes are of monophyletic origin, which were likely generated as a result of diversification after a genetic bottleneck. To investigate the population dynamics of this divergent DWV, we designed a series of novel infectious cDNA clones corresponding to co-existing DWV genotypes, thereby devising a reverse genetic system for an invertebrate RNA virus quasispecies. Equal replication rates were observed for all clone-derived DWV variants in single infections. Surprisingly, individual clones replicated to the same high levels as their mixtures and even the parental highly diverse natural DWV population, suggesting that complementation between genotypes was not required to replicate to high levels. Mixed clone-derived infections showed a lack of strong competitive exclusion, suggesting that the DWV genotypes were adapted to co-exist. Mutational and recombination events were observed across clone progeny providing new insights into the forces that drive and constrain virus diversification. Accordingly, herein we propose a new model of Varroa-induced DWV dynamics whereby an initial selective sweep is followed by virus diversification fueled by negative frequency-dependent selection for new genotypes. This selection likely reflects the ability of rare lineages to evade host defenses, specifically antiviral RNA interference (RNAi). In support of this, we show that RNAi induced against one DWV strain is less effective against an alternate strain from the same population.\n\nAuthor SummaryVirulence of Deformed wing virus (DWV), a major pathogen of honey bees, showed a sharp and significant increase following the introduction of its vector, the mite Varroa destructor. Varroa vectoring resulted in genetic changes of DWV, including reduction of DWV diversity to nearly clonal levels in the UK and Hawaii. Contrary to the previous reports, we discovered that virulent DWV populations circulating across the Varroa-infested United States included many divergent genotypes generated following a strong bottleneck event. We designed a series of the full-length infectious cDNA clones that captured the diversity of a typical virulent DWV population from a declining Varroa-infested colony, effectively establishing first reverse genetic system for an invertebrate RNA virus quasispecies, in order to investigate interactions between the virus genotypes. We demonstrated that individual co-existing DWV genotypes and diverse natural DWV populations replicated equally well indicating that complementation between isolates was not required to enable DWV replication to high levels. Also, no obvious competitive exclusion was detected between genotypes in mixed infections suggesting DWV genotypes are adapted to co-exist to maintain overall population diversity. We suggest that introduction of Varroa resulted in an initial selective sweep of DWV diversity which was followed by DWV diversification driven by selection for new genotypes capable of evading host defenses, specifically antiviral RNA interference.

microbiology↗