Varroa destructor rearing in laboratory conditions: importance of foundress survival in doubly infested cells and reproduction of laboratory-born females

International audienceA considerable part of the knowledge about the honey bee parasite Varroa destructor emerged from rearing protocols in semi-natural or laboratory conditions, yet a durable protocol over several generations of mites is still lacking. The development of such multigenerational rearing relies on the emergence of a sufficient number of new fertile females in the first generation of V. destructor. The optimization of the parasite’s reproductive success in laboratory conditions thus represents an important prerequisite. The number of foundress mites in a cell is known to impact the probability of male survival and thus the number of mated daughters. We therefore investigated the effect of the degree of bee larvae infestation under laboratory conditions. The results showed that the probability of finding at least one foundress alive at the end of the rearing was significantly higher in doubly infested cells. This leads to the improvement of the reproductive parameters and more specifically of the number of daughters per mite. In doubly infested cells with one dead foundress, the presence of a surviving female would in fact allow both its descendants and those of the dead mite to complete their development. The mated daughters from this system were used in a subsequent experiment to test their ability to complete their reproductive cycle in laboratory conditions, from the perspective of developing a multigenerational rearing. The reproduction and development of the offspring measured were similar to those of the first generation. However, many of the females from the second generation died before the completion of their first reproductive cycle. We suggest that these females are fertile but might lack the energy necessary to survive throughout reproduction. The results from our bioassay could constitute a basis for the development of a durable V. destructor laboratory rearing and for the improvement of our understanding of the parasite’s reproductive cycle

Fourmis : une chimiothèque de nouveaux anticancéreux

La composition des venins est très compliquée à déterminer à cause de leur complexité et de leur diversité moléculaire, cependant l’étude transcriptomique des glandes à venin de la fourmi Tetramorium bicarinatum révèle une grande diversité fonctionnelle des peptides/protéines qui pourraient être produits par ces glandes. Ces molécules peptidiques correspondent pour partie à des toxines connues mais de nombreuses séquences ne sont répertoriées dans aucune base de données et pourraient correspondre à des peptides de venins originaux non décrits auparavant. Nos travaux étaient les premiers à rapporter le séquençage du transcriptome de la glande à venin chez une fourmi, d’abord par la technique de la SSH (Suppression Subtractive Hybridization), puis par une technique de séquençage profond permettant de progresser dans le degré d’informations que renferme le transcriptome (Illumina). Nos expériences montrent clairement que l’extrait de venin brut induit l’apoptose de certaines lignées de cellules tumorales résistantes à tous les traitements actuels et l’analyse en spectrométrie de masse nous a orientées vers une molécule dont la structure nous est encore inconnue. L’identification et la caractérisation du mécanisme d’action de la molécule responsable de cette cytotoxicité constituent donc un espoir de découvrir de nouvelles stratégies thérapeutiques dans la lutte contre cette pathologie encore aujourd’hui incurable. D’une manière générale, l’extraordinaire diversité taxonomique des fourmis laisse présager d’une grande diversité de leurs toxines. La probabilité de découvrir de nouvelles molécules d’intérêt pour l’homme n’en est que renforcée

Transmission of deformed wing virus between Varroa destructor foundresses, mite offspring and infested honey bees

International audienceBackground: Varroa destructor is the major ectoparasite of the western honey bee (Apis mellifera). Through both its parasitic life-cycle and its role as a vector of viral pathogens, it can cause major damage to honey bee colonies. The deformed wing virus (DWV) is the most common virus transmitted by this ectoparasite, and the mite is correlated to increased viral prevalence and viral loads in infested colonies. DWV variants A and B (DWV-A and DWV-B, respectively) are the two major DWV variants, and they differ both in their virulence and transmission dynamics. Methods: We studied the transmission of DWV between bees, parasitic mites and their offspring by quantifying DWV loads in bees and mites collected in in vitro and in situ environments. In vitro, we artificially transmitted DWV-A to mites and quantified both DWV-A and DWV-B in mites and bees. In situ, we measured the natural presence of DWV-B in bees, mites and mites' offspring. Results: Bee and mite viral loads were correlated, and mites carrying both variants were associated with higher mortality of the infected host. Mite infestation increased the DWV-B loads and decreased the DWV-A loads in our laboratory conditions. In situ, viral quantification in the mite offspring showed that, after an initially non-infected egg stage, the DWV-B loads were more closely correlated with the foundress (mother) mites than with the bee hosts. Conclusions: The association between mites and DWV-B was highlighted in this study. The parasitic history of a mite directly impacts its DWV infection potential during the rest of its life-cycle (in terms of variant and viral loads). Regarding the mite's progeny, we hypothesize that the route of contamination is likely through the feeding site rather than by vertical transmission, although further studies are needed to confirm this hypothesis

Varroa destructor from the Laboratory to the Field: Control, Biocontrol and IPM Perspectives—A Review

Varroa destructor is a real challenger for beekeepers and scientists: fragile out of the hive, tenacious inside a bee colony. From all the research done on the topic, we have learned that a better understanding of this organism in its relationship with the bee but also for itself is necessary. Its biology relies mostly on semiochemicals for reproduction, nutrition, or orientation. Many treatments have been developed over the years based on hard or soft acaricides or even on biocontrol techniques. To date, no real sustainable solution exists to reduce the pressure of the mite without creating resistances or harming honeybees. Consequently, the development of alternative disruptive tools against the parasitic life cycle remains open. It requires the combination of both laboratory and field results through a holistic approach based on health biomarkers. Here, we advocate for a more integrative vision of V. destructor research, where in vitro and field studies are more systematically compared and compiled. Therefore, after a brief state-of-the-art about the mite’s life cycle, we discuss what has been done and what can be done from the laboratory to the field against V. destructor through an integrative approach

Lose Your Grip: Challenging <i>Varroa destructor</i> Host Attachment with Tartaric, Lactic, Formic, and Citric Acids

Beekeepers can use a variety of treatments against Varroa destructor, the parasitic mite of Apis mellifera. However, sustainable and easy-to-use solutions are still scarce, considering the complexity of reaching the parasite alone. Current treatments involve soft acaricides, although their mechanism of action is not well understood. We investigated the effects of organic acids such as tartaric, lactic, formic, and citric acids on the attachment abilities of V. destructor under laboratory conditions. Preventing parasites from gripping or holding on to their hosts is a crucial target for mite control strategies. We challenged grip skills through the Rotavar setup after the direct application of acids to mites’ arolia. We also tested the potential for mites to fall off honeybees after bee treatment. We found that tartaric, citric, and lactic acids were good candidates to impair the attachment of V. destructor twenty-four hours post-treatment. However, lactic acid remained the only candidate at a reasonable concentration to destabilise mites after the honey bee’s treatment without reducing their lifespan. While we conducted these experiments under artificial conditions, our results improved our comprehension of the organic acids’ potential impact on V. destructor. They can also help with the development of new methods for hive application for beekeepers worldwide

Standard Methods for Dissection of Varroa destructor Females

Varroa destructor (Anderson and Trueman) is known as a major pest of Apis mellifera L, especially in the Northern Hemisphere where its effects can be deleterious. As an obligate parasite, this mite relies entirely on its host to reproduce and complete its cycle. Studies focusing on isolated organs are needed to better comprehend this organism. To conduct such targeted molecular or physiological studies, the dissection of V. destructor mites is crucial as it allows the extraction of specific organs. Here, we propose a technical article showing detailed steps of females V. destructor dissection, illustrated with pictures and videos. These illustrated guidelines will represent a helpful tool to go further in V. destructor research

Varroa destructor s’appuie sur des signaux physiques pour se nourrir dans des conditions artificielles

Olfaction is a major sense in Varroa destructor. In natural conditions, it is known that this honey bee parasite relies on kairomones to detect its host or to reproduce. Yet, in artificial conditions, the parasite is able to feed and survive for a few days even though most honey bee pheromones are lacking. Other key cues are thus probably involved in V. destructor perception of its close environment. Here, we used several artificial feeding designs to explore the feeding behaviour of the parasite when it is deprived of olfactory cues. We found that V. destructor is still able to feed only guided by physical cues. The detection of the food source seems to be shape-related as a 3D membrane triggers arrestment and exploration more than a 2D membrane. The tactile sense of V. destructor could thus be essential to detect a feeding site, although further studies are needed to assess the importance of this sense combined with olfaction in natural conditions.L’olfaction est un sens prépondérant chez Varroa destructor. En conditions naturelles, ce parasite de l’abeille domestique dépend en effet de kairomones qui lui permettent de détecter son hôte ou de se reproduire. Pourtant, lorsqu’il se retrouve en conditions artificielles, le parasite se nourrit et survit plusieurs jours malgré l’absence de la majorité des phéromones émises par l’abeille. Des indices clés autres qu’olfactifs sont donc très probablement impliqués dans la perception de l’environnement de V. destructor. Dans cette étude, plusieurs dispositifs d’alimentation artificielle ont été testés afin d’explorer le comportement de nourrissage du parasite lorsqu’il est privé d’indices olfactifs. Les résultats montrent que V. destructor est tout à fait capable de se nourrir en étant uniquement guidé par des indices physiques. En l’occurrence, la détection de la source nutritive semble être liée à sa forme puisqu’une membrane 3D provoque des comportements exploratoires plus prononcés qu’une membrane plane (2D). Le sens du toucher serait donc essentiel à V. destructor pour trouver son site de nourrissage. Des études complémentaires permettraient néanmoins d’évaluer les importances relatives des sens olfactif et tactile en conditions naturelles

Vitellogenin gene expression from mites in relation with the three treatments.

<p>Within stage log-transformed normalized absolute expression of two vitellogenin transcripts of mites that experienced distinct phoretic conditions: mites that spent 3 days on forager bees, mites that stayed 3 days on nurse bees, mites that spent 7 days on nurse bees. The stages of the parasite cycle are: <b>pre</b> = prephoretic, mites randomly extracted from brood cells containing different stages of honey bee development and ready to be transferred onto adults; <b>postphoretic</b>, mites sampled right after their stay on adult bees in experimental cages; <b>PP3</b>, Prepupal stage mites; <b>pbm</b>, parasites sampled on brown eyed, medium pigmented thorax honey bee pupae; <b>emerging</b>, parasites on the day of honeybee emergence; <b>daughter</b>, newly fully molted <i>Varroa</i> distinguishable from the mothers. Only the statistical significance for phoretic condition is showed NS = non significant (ANOVA model and post hoc comparisons).</p

Expression of two vitellogenin genes from mites at different times of the development.

<p>A) Normalized absolute expression of the two vitellogenin transcripts at different stages of the parasite cycle, the labels are similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153482#pone.0153482.g003" target="_blank">Fig 3</a>: <b>prephoretic</b>, before the transfer onto adults; <b>postphoretic</b>, after the stay on adult bees; <b>PP3</b>, on prepupal bees; <b>pbm</b>, on brown eyed, medium pigmented thorax honey bee pupae; <b>emerging</b> mites; <b>daughter</b>. Barplot of the mean ± standard error: capital letters indicate the significant differences in the VdVg1 expression, small letters the significance of statistical tests for the VdVg2 expression (ANOVA model and post hoc compaisons p_Bonferroni<0.003). B) Focus on the normalized absolute expression of the two VdVg transcripts of newly born <i>Varroa</i> females after division into two groups according to their age: older daughters (= dpd: darkly pigmented daughters) and younger daughters (= lpd: lightly pigmented daughters). Letters showed the significance of the Mann-Whitney-Wilcoxon test conducted on VdVg1 (capital letter) and VdVg2 (small letter) p_Bonferroni<0.0083.</p

Honey Bee Larval Hemolymph as a Source of Key Nutrients and Proteins Offers a Promising Medium for Varroa destructor Artificial Rearing

International audienceVarroa destructor, a major ectoparasite of the Western honey bee Apis mellifera, is a widespread pest that damages colonies in the Northern Hemisphere. Throughout their lifecycle, V. destructor females feed on almost every developmental stage of their host, from the last larval instar to the adult. The parasite is thought to feed on hemolymph and fat body, although its exact diet and nutritional requirements are poorly known. Using artificial Parafilm™ dummies, we explored the nutrition of V. destructor females and assessed their survival when fed on hemolymph from bee larvae, pupae, or adults. We compared the results with mites fed on synthetic solutions or filtered larval hemolymph. The results showed that the parasites could survive for several days or weeks on different diets. Bee larval hemolymph yielded the highest survival rates, and filtered larval plasma was sufficient to maintain the mites for 14 days or more. This cell-free solution therefore theoretically contains all the necessary nutrients for mite survival. Because some bee proteins are known to be hijacked without being digested by the parasite, we decided to run a proteomic analysis of larval honey bee plasma to highlight the most common proteins in our samples. A list of 54 proteins was compiled, including several energy metabolism proteins such as Vitellogenin, Hexamerin, or Transferrins. These molecules represent key nutrient candidates that could be crucial for V. destructor survival