Life-history stage determines the diet of ectoparasitic mites on their honey bee hosts.

Ectoparasitic mites of the genera Varroa and Tropilaelaps have evolved to exclusively exploit honey bees as food sources during alternating dispersal and reproductive life history stages. Here we show that the primary food source utilized by Varroa destructor depends on the host life history stage. While feeding on adult bees, dispersing V. destructor feed on the abdominal membranes to access to the fat body as reported previously. However, when V. destructor feed on honey bee pupae during their reproductive stage, they primarily consume hemolymph, indicated by wound analysis, preferential transfer of biostains, and a proteomic comparison between parasite and host tissues. Biostaining and proteomic results were paralleled by corresponding findings in Tropilaelaps mercedesae, a mite that only feeds on brood and has a strongly reduced dispersal stage. Metabolomic profiling of V. destructor corroborates differences between the diet of the dispersing adults and reproductive foundresses. The proteome and metabolome differences between reproductive and dispersing V. destructor suggest that the hemolymph diet coincides with amino acid metabolism and protein synthesis in the foundresses while the metabolism of non-reproductive adults is tuned to lipid metabolism. Thus, we demonstrate within-host dietary specialization of ectoparasitic mites that coincides with life history of hosts and parasites.

Upper thermal limits of Rhagoletis indifferens (Diptera: Tephritidae) pupae and pteromalid parasitoids (Hymenoptera: Pteromalidae) inside fly puparia.

Determining upper thermal limits of tephritid fly pupae can have practical implications for disinfesting soils and for predicting differential impacts of global warming on flies and their parasites. Here, upper thermal limits of Rhagoletis indifferens Curran (Diptera: Tephritidae) pupae and pteromalid wasps (Hymenoptera: Pteromalidae) inside puparia were determined. Puparia receiving sufficient chill to terminate pupal diapause were exposed to temperatures ramped linearly over 6 h from 21 °C to 47.8, 49.4, 51.1, 55.0, or 60.0 °C for a 0-h hold time. Flies eclosed when pupae were exposed to 47.8 °C but not to 49.4, 51.1, 55.0, or 60.0 °C nor in a separate test to 47.8 °C for 1-3 h hold times. All fly pupae in treatments where no eclosion occurred were dead based on puparial dissections. In contrast, adult wasps eclosed when puparia were exposed to 49.4 and 51.1 °C for 0 h and to 47.8 °C for 1- and 2-h hold times. Despite the greater upper thermal limits of wasps, heat delayed eclosion times of both adult flies and wasps, in 47.8 and 51.1 °C treatments, respectively. In separate tests, longevity of flies exposed as pupae to 47.3-48.6 °C was greater than of control flies, while longevity of control wasps and wasps exposed as immatures to 47.8-51.1 °C did not differ. Flies exposed as pupae to 47.2-48.6 °C produced as many eggs and puparia as control flies. Results suggest heat could be used to disinfest soils of puparia while sparing parasitoids. In addition, extreme heat waves due to global warming might be more detrimental to fly pupae than immature wasps.

Cadmium exposure through the food chain reduces the parasitic fitness of Chouioia cunea to Hyphantria cunea pupae: An ecotoxicological risk to pest control.

The toxicity of heavy metals on various trophic levels along the food chain has been extensively investigated, but no studies have focused on parasitic natural enemy insects. Herein, we constructed a food chain consisting of soil-Fraxinus mandshurica seedlings-Hyphantria cunea pupae-Chouioia cunea to analyze the effects of Cd exposure through food chain on the fitness of parasitic natural enemy insects and its corresponding mechanism. The results showed that the transfer of Cd between F. mandshurica leaves and H. cunea pupae and between H. cunea pupae and C. cunea was a bio-minimization effect. After parasitizing Cd-accumulated pupa, the number of offspring larvae, and the number, individual size (body weight, body length, abdomen length) and life span of offspring adults decreased significantly, while the duration of embryo development extended significantly. The contents of malondialdehyde and H2O2 in Cd-exposed offspring wasps increased significantly, accompanied by a significantly decrease in antioxidant capacity. The cellular immunity parameters significantly decreased in Cd-accumulated pupae, including the number of hemocytes, melanization activity and the expression level of cellular immunity genes (e.g. Hemolin-1 and PPO1). The humoral immunity disorder was found in the Cd-accumulated pupae, as evidenced by that the expression level of immune recognition gene (PGRP-SA), signal transduction genes (IMD, Dorsal, and Tube), as well as all antimicrobial peptide genes (e.g. Lysozym and Attacin) decreased significantly. Cd exposure decreased the content of glucose, trehalose, amino acid, and free fatty acid in H. cunea pupae. The expression of Hk2 in glycolysis pathway and the expression of Idh2, Idh3, Cs, and OGDH in TCA cycle pathway were significantly down-regulated in Cd-accumulated pupae. Taken together, exposure to Cd through the food chain causes oxidative damage on the offspring wasps and disrupts energy metabolism of the host insect, ultimately reducing the parasitic fitness of C. cunea to H. cunea pupae.

On the life cycle of Brachymeria podagrica (Fabricius, 1787) (Hymenoptera: Chalcididae) - a parasitoid of sacrophagid, calliphorid, and muscid flies.

The lifecycle of Brachymeria podagrica, a parasitic wasp with a worldwide distribution, was studied under laboratory conditions using the flesh fly, Sarcophaga dux, as a host. Two hundred parasite-free 3rd instars of S. dux were exposed for 24 h to 20 female B. podagrica. In daily intervals, maggots and later pupae were examined for developmental stages of the parasitoid. The whole pre-imaginal development at a temperature of 26 °C lasted 21 to 26 days. Three morphologically different instars, followed by a prepupal and a pupal stage, were described using light and scanning electron microscopy. In a second experiment with 100 3rd stage Sarcophaga larvae and 10 parasitoids, a total of 70 wasps emerged 20 to 25 days after exposure. Two fly larvae did not pupate and dried out, while 28 pupae contained a dry or caseous content, dead wasp imagos, or their larval stages. No fly imagines emerged from exposed groups, while all 100 unexposed larvae pupated and adults eclosed between day 12 and day 14 after the start of the experiment, while the imagoes of the parasitoids appeared 8 to 12 days later.

Below ground efficiency of a parasitic wasp for Drosophila suzukii biocontrol in different soil types.

The parasitoid wasp Trichopria drosophilae is promising as a biocontrol agent for controlling the ubiquitous pest Drosophila suzukii (Matsumura). Crucial for the successful implementation of any biocontrol agent is a high parasitisation rate by the parasitoid. Most studies investigating the parasitisation rate of D. suzukii pupae have focused on parasitisation in the fruit or in a petri dish. However, the predominant pupation site of D. suzukii in the field is the soil. Unfortunately, little is known on how well parasitoid wasps can detect and parasitise pupae of D. suzukii buried in the soil. Therefore, we conducted soil parasitisation experiments of T. drosophilae on D. suzukii pupae using two pupation depths in three different soil types (loamy sand, loam, and clay). In all three soil types, we found generally low D. suzukii pupae parasitisation rate by T. drosophilae, independent of the pupation depth. The pupation behaviour of D. suzukii and the parasitisation behaviour of T. drosophilae are discussed in detail. For pest control in most soil types, our results mean that the number of D. suzukii larvae pupating in the soil should be reduced, e.g., by adding a layer of sandy soil or covering the soil with plastic mulch. This might increase the probability of success when using T. drosophilae as a biocontrol agent.

First Record of Brachymeria podagrica (Hymenoptera: Chalcididae) in Bisha City, Asir Region, Saudi Arabia.

Chalcid Brachymeria podagrica (Fabricius), formerly known as Brachymeria fonscolombei (Dufour), is a parasitoid wasp that infests dipteran larvae of fly, Sarcophaga dux (Thomson), (Diptera: Sarcophagidae) recovered from the dead body of sheep in Bisha City, Asir region-Saudi Arabia. S. dux larvae were infested by B. podagrica. The first instar larvae and pupae of B. podagrica were found inside the larvae and pupae of S. dux. The adults of the parasitic wasp emerged from the pupae of the host. Adults and larvae of the parasitoid wasp were observed under a dissecting microscope. Identification was made by following the identification keys. It was confirmed that it was B. podagrica, which is herewith reported for the first time in Saudi Arabia.

Non-lethal effects of entomopathogenic nematode infection.

Entomopathogenic nematodes are typically considered lethal parasites of insect hosts. Indeed they are employed as such for biological control of insect pests. The effects of exposure to entomopathogenic nematodes are not strictly limited to mortality, however. Here we explore non-lethal effects of exposure to entomopathogenic nematodes by introducing the relatively non-susceptible pupal stage of Delia antiqua to thirteen different strains. We specifically chose to inoculate the pupal stage because it tends to be more resistant to infection, yet resides in the soil where it could come into contact with EPN biological control agents. We find that there is no significant mortality at the pupal stage, but that there are a host of strain-dependent non-lethal effects during and after the transition to adulthood including altered developmental times and changes in risk of death compared to controls. We also find that exposure to specific strains can reduce risk of mortality. These results emphasize the strain-dependent nature of entomopathogenic nematode infection and highlight the positive and negative ramifications for non-lethal effects for biological control of insect pests. Our work emphasizes the need for strain-specific screening of biological control agents before wide-spread adoption.

Potential of in vivo- and in vitro-cultured entomopathogenic nematodes to infect Lobesia vanillana (Lepidoptera: Tortricidae) under laboratory conditions.

Entomopathogenic nematodes (EPNs) have been successfully applied as biological control agents against above ground and soil stages of insect pests. However, for commercial application, it is crucial to mass culture these nematodes using in vitro liquid culture technology, as it is not attainable when using susceptible insects as hosts. Lobesia vanillana (Lepidoptera: Tortricidae) is regarded a sporadic pest of wine grapes in South Africa. The in vivo- and in vitro-cultured South African EPNs, Steinernema yirgalemense and Steinernema jeffreyense (Rhabditida: Steinernematidae), were evaluated against larvae and pupae of L. vanillana in laboratory bioassays. For larvae, high mortality was observed for all treatments: In vitro-cultured S. yirgalemense (98%) performed better than S. jeffreyense (73%), while within in vivo cultures, there was no difference between nematode species (both 83%). No significant difference was detected between in vivo- and in vitro cultures of the same nematode species. The LD50 of the in vitro-cultured S. yirgalemense, was 7.33 nematodes per larva. Mortality by infection was established by dissecting L. vanillana cadavers and confirming the presence of nematodes, which was > 90% for all treatments. Within in vitro cultures, both S. yirgalemense and S. jeffreyense were able to produce a new cohort of infective juveniles from L. vanillana larvae. Pupae, however, were found to be considerably less susceptible to EPN infection. This is the first study on the use of EPNs to control L. vanillana. The relative success of in vitro-cultured EPN species in laboratory assays, without any loss in pathogenicity, is encouraging for further research and development of this technology.

Effect of Drosophila suzukii on Blueberry VOCs: Chemical Cues for a Pupal Parasitoid, Trichopria anastrephae.

Biocontrol agents such as parasitic wasps use long-range volatiles and host-associated cues from lower trophic levels to find their hosts. However, this chemical landscape may be altered by the invasion of exotic insect species. The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), is a highly polyphagous fruit pest native to eastern Asia and recently arrived in South America. Our study aimed to characterize the effect of SWD attack on the volatile organic compounds (VOCs) of blueberries, a common host fruit, and to correlate these odor changes with the olfactory-mediated behavioral response of resident populations of Trichopria anastrephae parasitoids, here reported for the first time in Uruguay. Using fruit VOC chemical characterization followed by multivariate analyses of the odor blends of blueberries attacked by SWD, we showed that the development of SWD immature stages inside the fruit generates a different odor profile to that from control fruits (physically damaged and free of damage). These differences can be explained by the diversity, frequency, and amounts of fruit VOCs. The behavioral response of T. anastrephae in Y-tube bioassays showed that female wasps were significantly attracted to volatiles from SWD-attacked blueberries when tested against both clean air and undamaged blueberries. Therefore, T. anastrephae females can use chemical cues from SWD-infested fruits, which may lead to a successful location of their insect host. Since resident parasitoids are able to locate this novel potential host, biological control programs using local populations may be plausible as a strategy for control of SWD.

New austral-most records of the genus Heterostylum Macquart (Diptera: Bombyiliidae) in Argentina.

Bee flies of the genus Heterostylum Macquart are medium-sized species (1015 mm) characterized primarily by a robust body covered with long pile and by an indented hind margin of the eye (Cunha et al. 2007). There are several studies on the immatures of some species of Heterostylum (Bohart et al. 1960; Yeates Greathead 1997), which are considered ectoparasites of fossorial solitary bee larvae and pupae (Yeates Greathead 1997).

Evidence for Vertical Transmission of Babesia odocoilei (Piroplasmida: Babesiidae) in Ixodes scapularis (Acari: Ixodidae).

Limited evidence suggests that the cervid parasite, Babesia odocoilei, is transovarially transmitted from adult female Ixodes scapularis Say to offspring. The prevalence of B. odocoilei in unfed larval I. scapularis and whether vertical transmission is crucial to pathogen maintenance are currently unknown. To investigate these questions, 275 unfed larvae from two Wisconsin counties were tested for B. odocoilei genetic material. Sixteen of 29 pools were positive for the parasite. The maximum likelihood estimation for overall larval infection prevalence was 7.8% (95% confidence interval: 4.7-12). This vertically acquired infection appears to be sustained transstadially in nymphal ticks the following year; however, our relatively small sample and replicate size warrants additional evaluation. Our study revealed further evidence of vertical transmission, a low and consistent infection prevalence in larvae, and the potential importance of vertical transmission in B. odocoilei maintenance.

Variation in Parasitoid Virulence of Tetrastichus brontispae during the Targeting of Two Host Beetles.

In host-parasitoid interactions, antagonistic relationship drives parasitoids to vary in virulence in facing different hosts, which makes these systems excellent models for stress-induced evolutionary studies. Venom compositions varied between two strains of Tetrastichus brontispae, Tb-Bl and Tb-On. Tb-Bl targets Brontispa longissima pupae as hosts, and Tb-On is a sub-population of Tb-Bl, which has been experimentally adapted to a new host, Octodonta nipae. Aiming to examine variation in parasitoid virulence of the two strains toward two hosts, we used reciprocal injection experiments to compare effect of venom/ovarian fluids from the two strains on cytotoxicity, inhibition of immunity and fat body lysis of the two hosts. We found that Tb-Onvenom was more virulent towards plasmatocyte spreading, granulocyte function and phenoloxidase activity than Tb-Blvenom. Tb-Blovary was able to suppress encapsulation and phagocytosis in both hosts; however, Tb-Onovary inhibition targeted only B. longissima. Our data suggest that the venom undergoes rapid evolution when facing different hosts, and that the wasp has good evolutionary plasticity.

A salivary chitinase of Varroa destructor influences host immunity and mite's survival.

Varroa destructor is an ectoparasite of honey bees and an active disease vector, which represents one of the most severe threats for the beekeeping industry. This parasitic mite feeds on the host's body fluids through a wound in the cuticle, which allows food uptake by the mother mite and its progeny, offering a potential route of entrance for infecting microorganisms. Mite feeding is associated with saliva injection, whose role is still largely unknown. Here we try to fill this gap by identifying putative host regulation factors present in the saliva of V. destructor and performing a functional analysis for one of them, a chitinase (Vd-CHIsal) phylogenetically related to chitinases present in parasitic and predatory arthropods, which shows a specific and very high level of expression in the mite's salivary glands. Vd-CHIsal is essential for effective mite feeding and survival, since it is apparently involved both in maintaining the feeding wound open and in preventing host infection by opportunistic pathogens. Our results show the important role in the modulation of mite-honey bee interactions exerted by a host regulation factor shared by different evolutionary lineages of parasitic arthropods. We predict that the functional characterization of Varroa sialome will provide new background knowledge on parasitism evolution in arthropods and the opportunity to develop new bioinspired strategies for mite control based on the disruption of their complex interactions with a living food source.

Improved Sentinel Method for Surveillance and Collection of Filth Fly Parasitoids.

Parasitoids are important natural enemies of house flies and other muscoid flies. The two most commonly used methods for collecting fly parasitoids from the field have distinct advantages and disadvantages. Collections of wild puparia depend on the ability to find puparia in sufficient numbers and are prone to localized distortions in relative species abundance because of the overrepresentation of samples from hot spots of fly larval activity. Placement and retrieval of sentinel puparia is convenient and allows consistent sampling over time but is strongly biased in favor of Muscidifurax spp. over Spalangia spp. An improved sentinel method is described that combines some of the advantages of these two methods. Fly medium containing larvae is placed in containers, topped with a screen mesh bag of puparia, and placed in vertebrate-proof wire cages. Cages are placed at sites of actual or potential fly breeding and retrieved 3-7 d later. The modified method collected species profiles that more closely resembled those of collections of wild puparia than those from sentinel pupal bags. A method is also described for isolating puparia individually in 96-well tissue culture plates for parasitoid emergence. Use of the plate method provided a substantial saving of time and labor over the use of individual gelatin capsules for pupal isolation. Puparia from the collections that were housed individually in the wells of tissue culture plates had a higher proportion of emerged Spalangia species than puparia that were held in groups.

A venom protein, Kazal-type serine protease inhibitor, of ectoparasitoid Pachycrepoideus vindemiae inhibits the hemolymph melanization of host Drosophila melanogaster.

Parasitic wasps inject various virulence factors into the host insects while laying eggs, among which the venom proteins, one of the key players in host insect/parasitoid relationships, act in host cellular and humoral immune regulation to ensure successful development of wasp progeny. Although the investigations into actions of venom proteins are relatively ample in larval parasitoids, their regulatory mechanisms have not been thoroughly understood in pupal parasitoids. Here, we identified a venom protein, Kazal-type serine protease inhibitor, in the pupal ectoparasitoid Pachycrepoideus vindemiae (PvKazal). Sequence analysis revealed that PvKazal is packed by a signal peptide and a highly conserved "Kazal" domain. Quantitative polymerase chain reaction analysis recorded a higher transcript level of PvKazal in the venom apparatus relative to that in the carcass, and the PvKazal messenger RNA level appeared to reach a peak on day 5 posteclosion. Recombinant PvKazal strongly inhibited the hemolymph melanization of host Drosophila melanogaster. Additionally, the heterologous expression of PvKazal in transgenic Drosophila reduced the crystal cell numbers and blocked the melanization of host pupal hemolymph. Our present work underlying the roles of PvKazal undoubtedly increases the understanding of venom-mediated host-parasitoid crosstalk.

Biocontrol potential of Steinernema cholashanense (Nguyen) on larval and pupal stages of potato tuber moth, Phthorimaea operculella (Zeller).

The potato tuber moth, Phthorimaea operculella (Zeller), is a serious pest of potato and other commercial crops belonging to the Solanaceae family. In recent years, it has become an emerging problem in potato-growing regions of the Nilgiri hills of southern India. It is responsible for the reduced quality and quantity of marketable potatoes. In this regard, the development of an eco-friendly control method for the management of the potato tuber moth is urgently required. Therefore, in the present study, the virulence of Steinernema cholashanense CPRSUS01 originally isolated from the potato rhizosphere was tested on fourth-instar larvae and pupae of P. operculella. Steinernema cholashanense caused the greatest mortality in the fourth-instar larval stage (100%) than the pupae (30%). In addition to this, penetration and reproduction of this nematode was also studied in fourth-instar larvae of P. operculella and this is the first report of penetration and reproduction of any entomopathogenic nematode species on potato tuber moth larvae. The reproduction capacity of S. cholashanense on P. operculella is higher (702 infective juveniles mg-1 body weight). Our results indicated that S. cholashanense has good potential as an alternative tool for the management of P. operculella. But before including S. cholashanense in the integrated pest management program of P. operculella, its efficacy should be tested under field conditions.

Cold tolerance of the drosophila pupal parasitoid Trichopria drosophilae.

Trichopria drosophilae (Perkins) (Hymenoptera: Diapriidae) is a pupal parasitoid of drosophila flies recorded from several parts of the world. It is currently considered for augmentative biological programs to control the severe agricultural pest Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). Since D. suzukii has invaded regions that experience zero and sub-zero winter temperatures, cold tolerance of the parasitoid is an important aspect to consider. We characterized low temperature tolerance and overwintering capacity of a colony of T. drosophilae collected in Northern Switzerland. We used copper-constantan thermocouples to determine the supercooling point and pre-freeze mortality. Moreover, we subjected honey-fed and unfed adult T. drosophilae as well as developing stages within their drosophila host to short- and long-term acclimation conditions and assessed the duration of their survival at low temperatures. Finally, we exposed adult and sub-adult stages to winter conditions in a semi-field experiment and evaluated their survival. We found that T. drosophilae is chill susceptible like D. suzukii, but adults froze and survived at colder temperatures than those reported for D. suzukii. Adult parasitoids could tolerate several days of exposure to sub-zero temperatures and could reproduce afterwards, whereas sub-adult stages could survive longer periods under these conditions. The provision of honey and water enhanced the survival of adults and long-term acclimation led to longer survival in all stages. The semi-field experiment supported the results of the laboratory tests. Based on these results we suggest that in Central Europe, T. drosophilae survives winters mainly in developing stages but adults are likely able to tolerate short periods of low spring temperatures.

Side effects of chlorantraniliprole, phosalone and spinosad on the egg parasitoid, Trichogramma brassicae.

The combined use of chemicals and biological control is not always a successful strategy owing to the potential side effects on biocontrol agents. Lethal and sublethal effects of three commonly used insecticides were assessed on adult and immature stages of the egg parasitoid Trichogramma brassicae Bezdenko (Hymenoptera: Trichogrammatidae). Recommended field concentrations of chlorantraniliprole, phosalone and spinosad caused mortality on preimaginal stages by 24, 87, and 98%, respectively. Lethal effects on parasitoid adults exposed to the insecticide dry residues were estimated as median lethal concentrations (LC50) that were 13.28, 0.25, and 0.03 µg a.i. ml-1 for chlorantraniliprole, phosalone, and spinosad, respectively. The effect of a low lethal concentration (LC30) of the compounds was evaluated on various adult biological traits, such as longevity, fecundity, emergence rate and other life table parameters. All compounds caused detrimental effects on all the estimated demographical indexes. Chlorantraniliprole affected the net reproductive rate, mean generation time and doubling time in comparison to the control; while, phosalone and spinosad adversely affected all assessed parameters. Phosalone and spinosad significantly reduced gross reproductive rate, net reproductive rate, intrinsic rate of increase, finite rate of increase, mean generation time and doubling time and reduced longevity, fecundity, emergence rate related to other biological parameters in comparison with control. The results suggest that all compounds are not fully compatible with the activity of T. brassicae, and that the inclusion of chlorantraniprole, spinosad and phosalone into Integrated Pest Management (IPM) involving this parasitoid has to be avoided. Nevertheless, further studies in open field conditions and on a multiple generation scale are necessary for providing a more definitive conclusion on the IPM suitability of the three tested insectcides.

Performance of Trichopria drosophilae (Hymenoptera: Diapriidae), a Generalist Parasitoid of Drosophila suzukii (Diptera: Drosophilidae), at Low Temperature.

Survival and parasitism activity of Trichopria drosophilae Perkins adults, a cosmopolitan parasitoid of Drosophila spp., were studied under laboratory conditions using five constant temperatures at the lower range known for this enemy, from 4 to 20°C in 4°C increments. Drosophila suzukii Matsumura, an invasive pest of small fruits, was used as a host. Commercially available adult parasitoids were provided with 1) food and D. suzukii pupae; 2) food and no D. suzukii pupae; 3) no food and no pupae. The results show that adult females of T. drosophilae lived longer than males, and both generally benefitted from food supply. The highest level of survival was observed between 8 and 12°C for fed insects, irrespective of whether they were offered host pupae or not. The absence of food led to the highest mortality, but the parasitoid demonstrated considerably resistance to prolonged starvation. Successful parasitism increased steadily with temperature and reached the highest value at 20°C. Conversely, D. suzukii emergence rate was high after exposure of pupae to parasitoids at 4°C, while pupal mortality increased strongly with temperature until 12°C. The findings indicate that T. drosophilae is well adapted to the relatively cold conditions experienced in early spring and in autumn or at high elevations, when the host pupae could be largely available. The long lifespan of the adults and the ability to parasitize the host at low temperature make T. drosophilae potentially useful for the biocontrol of D. suzukii.

Comparing four methods of rearing Varroa destructor in vitro.

The parasitic mite Varroa destructor Anderson and Trueman continues to devastate western honey bee (Apis mellifera L.) colonies throughout most of the world where they are managed. The development of a method to rear Varroa in vitro would allow for year-round Varroa research, rapidly advancing our progress towards controlling the mite. We created two separate experiments to address this objective. First, we determined which of four in vitro rearing methods yields the greatest number of Varroa offspring. Second, we attempted to improve the rearing rates achieved with that method. The four methods tested included (1) rearing Varroa on honey bee pupae in gelatin capsules, (2) rearing Varroa on in vitro-reared honey bees, (3) group rearing Varroa on honey bee pupae in Petri dishes, and (4) providing Varroa a bee-derived diet. The number of reproducing females and the number of fully mature offspring were significantly higher in the gelatin capsules maintained at 75% RH than in any other method. A 2 × 3 full factorial design was used to test combinations of gelatin capsule size (6 and 7 mm diameter) and relative humidity (65, 75, or 85%) on Varroa rearing success. Varroa reproduction and survival were significantly higher in 7-mm-diameter gelatin capsules maintained at 75% RH than in those maintained in 6-mm capsules and at the other humidities. By identifying factors that influence Varroa reproductive success in vitro, this work provides an important foundation for the development of future rearing protocols.