Adult Molt Research Articles

Age-dependent body mass changes in adult blackfield crickets Teleogryllus commodus (Walker) (Orthoptera: Gryllidae)

Body mass should not be regarded as constant during an adult insect's life span. To investigate this hypothesis in blackfield crickets Teleogryllus commodus (Walker, 1869), the mean body mass (mg) of a cohort of adult male (n=20) and single-mated female (n=20) crickets was measured daily for 60 days from adult moult to late senescence. Both male and female crickets increased in body mass during the first 20 days and then showed a continuous decrease in body mass. Whilst any body mass gained between prematurity (P) (first 5 days) and sexual maturity (SM) (day 6 to 40) phases was not statistically significant in either sex, body mass loss between sexual maturity and senescence (S) (day 41 till death) was statistically significant.

Exposure of female lobsters to sediments spiked with emamectin benzoate: Effects on eggs and larvae early development

The salmon aquaculture industry is an important economic activity established on both the west and east coast of Canada. To control sea lice infestations, in-feed products like emamectin benzoate (EMB) are widely used. Due to its low solubility and persistence EMB can accumulate in marine sediments and be potentially bioavailable to non-target organisms from months to years. The American lobster (Homarus americanus) is a key species in the Northwest Atlantic with high economic and ecological value. It may be exposed to therapeutants considering lobster habitats overlap with aquaculture locations requiring a better understanding of the potential impact of these therapeutants through varied pathways of exposure. In this study, we investigated the exposure of gravid female lobsters to EMB spiked sediment to mimic the likely presence of these females at aquaculture sites for a 10-day period. We completed testing by assessing EMB effects on adult molting and quality, embryo hatching rates, and larval offspring quality and larval molting. Our results show that a single, 10-day exposure of ovigerous females to EMB concentrations higher than environmentally relevant values did not affect females or their offspring.

Sex- and age-dependent breeding plumage acquisition in monomorphic species, the Black-headed Gull Chroicocephalus ridibundus

In monomorphic species, like in the Black-headed Gull, both sexes look alike in breeding plumage. With large sets of data on captured and photographed birds and using the Underhill–Zucchini moult model, we provided a detailed pattern of breeding plumage development in this species by age and sex. This study, similar to other studies, documented first adults with the initial stage of head moult at the beginning of January, yet the mean start date of nuptial moult in adults was the end of February. Half of adults acquired full breeding plumage about mid-March and almost all of them before mid-April. The start date of nuptial moult was more variable in immatures. The mean start of head moult in immatures was 19th April, which is 52 days later than in adults, and the majority, i.e., 70%, did not complete moult until the end of May. We showed for the first-time sex-dependent breeding plumage acquisition in monomorphic species. According to the Underhill–Zucchini moult model, males started to moult on average 7 days earlier than females and their moult lasted 7 days longer. Hence, the final date of completed head moult was the same in both sexes. A fully developed hood is an important part of the status signalling during pairing; therefore, completing the moult before mating is important for both sexes.

Juvenile hormone suppresses sensory organ precursor determination to block Drosophila adult abdomen morphogenesis

Juvenile hormone (JH) has a classic "status quo" action at both the pupal and adult molts when administrated exogenously. In Drosophila, treatment with JH at pupariation inhibits the formation of abdominal bristles, which are derived from the histoblasts. However, the mechanism via which JH exerts this effect remains poorly understood. In this study, we analyzed the effect of JH on histoblast proliferation, migration, and differentiation. Our results indicated that whereas the proliferation and migration of histoblasts remained unaffected following treatment with a JH mimic (JHM), their differentiation, particularly the specification of sensor organ precursor (SOP) cells, was inhibited. This effect was attributable to downregulated proneural genes achaete (ac) and Scute (sc) expression levels, which prevented the specification of SOP cells in proneural clusters. Moreover, Kr-h1 was found to mediate this effect of JHM. Histoblast-specific overexpression or knockdown of Kr-h1, respectively mimicked or attenuated the effects exerted by JHM on abdominal bristle formation, SOP determination, and transcriptional regulation of ac and sc. These results indicated that the defective SOP determination was responsible for the inhibition of abdominal bristle formation by JHM, which, in turn, was mainly mediated via the transducing action of Kr-h1.

Sores of boreal moose reveal a previously unknown genetic lineage of parasitic nematode within the genus Onchocerca.

Long-standing reports of open sores on the hind legs of moose (Alces alces) have been recorded in Alaska (as well as Canada, Europe, and Michigan), eliciting concerns about causes and infection. We used histological and genomic methods to investigate the sores from 20 adult moose on the Kenai Peninsula, Alaska. We paired this with thermal imagery and molt scoring of adult moose to further describe sore formation and understand its timing. Severe, ulcerative and eosinophilic dermatitis was found in all moose with sores present, and microfilariae within intraepidermal pustules were additionally found in four samples. Genetic analysis of sores from moose revealed a previously unknown genetic lineage of Onchocerca. Adult moose molt and lose their barrier of protection against flies in June and July during peak fly activity, leaving them vulnerable and allowing the development of sores. In summary, our results indicate that the cause for the sores on the hindleg of moose is a previously unknown genetic lineage of Onchocerca, probably transmitted by black flies, in timing with the molt cycle of adult moose. These sores leave moose exposed to pathogens, making them vulnerable, and challenging their health and fitness.

Strategic pheromone signalling by mate searching females of the sexually cannibalistic spider Argiope bruennichi.

Reproduction often requires finding a mating partner. To this end, females of many arthropods advertise their presence to searching males via volatile chemical signals. Such pheromones are considered low-cost signals, although this notion is based on little evidence and has recently been challenged. Even when using comparatively low-cost signals, females should signal as little as possible to minimize costs while still ensuring mate attraction. Here, we test the strategic-signalling hypothesis using Argiope bruennichi. In this orb-weaving spider, egg maturation commences with adult moult, and females that do not attract a male in time will lay a large batch of unfertilized eggs approximately three weeks after maturation. Using GC-MS analyses, we show that virgin females enhance their signalling effort, i.e. pheromone quantity per unit body mass, with increasing age and approaching oviposition. We further demonstrate that pheromone release is condition dependent, suggesting the occurrence of physiological costs. Mate choice assays revealed that pheromone quantity is the only predictor of female attractiveness for males. In support of the strategic-signalling hypothesis, pheromone signals by female A. bruennichi become stronger with increased need as well as body condition, and might thus qualify as an honest signal of female quality.

Juvenile hormone analog enhances Zika virus infection in Aedes aegypti

In recent years, there has been a rise in the emergence of arboviruses of public health importance, including Zika, chikungunya, dengue, and yellow fever viruses. Insecticide-based mosquito control has been the primary method for mitigating transmission of arboviruses. The consequences for the application of insecticides include both lethal and sublethal effects, and associated development of insecticide resistance. However, little is known about the influence on arboviral transmission. Mosquitoes with phenotypes that exhibit insecticide resistance or experience sublethal effects may be associated with altered susceptibility to arbovirus infection and transmission. Juvenile hormone analogs (JHAs) are insecticides that prevent pupa to adult molting of mosquitoes by mimicking the action of their natural juvenile hormone. Here, we examined whether the JHA pyriproxyfen interacts with ambient temperature (20 °C and 30 °C) during juvenile stages to influence life-history traits, population growth (λ'), and Zika virus (ZIKV) infection in Aedes aegypti. Development time of females was lengthened at 20 °C and in the presence of JHA. Prevention of pupa to adult molting by JHA was differentially higher at elevated temperature than low temperature. Size of females was larger at 20 °C and smaller at 30 °C. Infection, disseminated infection, and transmission of ZIKV in females were enhanced by JHA at both 20 °C and 30 °C relative to the controls. These results demonstrate that mosquito life-history and vector competence parameters are strongly influenced by interactive effects of JHA and temperature. The JHA-induced enhancement of ZIKV infection in females should be a consideration when implementing JHA in vector control strategies.

Identification and RNAi-Based Functional Analysis of Four Chitin Deacetylase Genes in Sogatella furcifera (Hemiptera: Delphacidae).

Chitin deacetylases (CDAs) are chitin-degrading enzymes that play a key role in insect molting. In this study, we identified and characterized four full-length cDNAs of CDAs from Sogatella furcifera (Horváth). Developmental expression showed that SfCDA1 and SfCDA2 were expressed at all nymph developmental stages, SfCDA3 and SfCDA4 were mainly expressed in the third-instar to fifth-instar nymph stages, whereas tissue-specific analyses indicated that four CDA genes were mainly high expressed in the integument and head during the fifth-instar nymph. RNA interference (RNAi) results revealed that SfCDA1, SfCDA2, and SfCDA4 are associated with molting defect and high mortality with nymph–adult molting. Furthermore, transcripts of chitin synthase 1 variants (SfCHS1, SfCHS1a, and SfCHS1b) were significantly downregulated and causing significant changes in the expression levels of trehalases (TRE1 and TRE2) in the SfCDA1, SfCDA2, and SfCDA4 dsRNA treatment groups. By contrast, no significant phenotypic characteristics were observed after dsSfCDA3 injection. Taken together, our results suggest that SfCDA1, SfCDA2, and SfCDA4 play a vital role in nymph-adult transition, and these genes could regulate chitin biosynthesis expression levels.

Functional analysis of ABCG and ABCH transporters from the red flour beetle, Tribolium castaneum.

ATP-binding cassette transporter (ABC transporter) subfamilies ABCA-C and ABCG-H have been implicated in insecticide detoxification, mostly based on findings of elevated gene expression in response to insecticide treatment. We previously characterized TcABCA-C genes from the model beetle and pest Tribolium castaneum and demonstrated that TcABCA and TcABCC genes are involved in the elimination of diflubenzuron, because RNA interference (RNAi)-mediated gene silencing increased susceptibility. In this study, we focused on the potential functions of TcABCG and TcABCH genes in insecticide detoxification. When we silenced the expression of TcABCG-H genes using RNAi, we noticed a previously unreported developmental RNAi phenotype for TcABCG-4F, which is characterized by 50% mortality and ecdysial arrest during adult moult. When we knocked down the Drosophila brown orthologue TcABCG-XC, we did not obtain apparent eye colour phenotypes but did observe a loss of riboflavin uptake by Malpighian tubules. Next, we determined the expression profiles of all TcABCG-H genes in different tissues and developmental stages and analysed transcript levels in response to treatment with four chemically unrelated insecticides. We found that some genes were specifically upregulated after insecticide treatment. However, when we determined insecticide-induced mortalities in larvae that were treated by double-stranded RNA injection to silence those TcABCG-H genes that were upregulated, we did not observe a significant increase in susceptibility to insecticides. Our findings suggest that the observed insecticide-dependent induction of TcABCG-H gene expression reflects an unspecific stress response, and hence underlines the significance of functional studies on insecticide detoxification. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Functional Analysis of Nuclear Factor Y in the Wing-Dimorphic Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae).

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor with the ability to bind to a CCAAT box in nearly all eukaryotes. However, the function of NF-Y in the life-history traits of insects is unclear. Here, we identified three NF-Y subunits, NlNF-YA, NlNF-YB, and NlNF-YC, in the wing-dimorphic brown planthopper (BPH), Nilaparvata lugens. Spatio-temporal analysis indicated that NlNF-YA, NlNF-YB, and NlNF-YC distributed extensively in various body parts of fourth-instar nymphs, and were highly expressed at the egg stage. RNA interference (RNAi)-mediated silencing showed that knockdown of NlNF-YA, NlNF-YB, or NlNF-YC in third-instar nymphs significantly extended the fifth-instar duration, and decreased nymph-adult molting rate. The addition of 20-hydroxyecdysone could specifically rescue the defect in adult molting caused by NlNF-YARNAi, indicating that NlNF-Y might modulate the ecdysone signaling pathway in the BPH. In addition, NlNF-YARNAi, NlNF-YBRNAi, or NlNF-YCRNAi led to small and moderately malformed forewings and hindwings, and impaired the normal assembly of indirect flight muscles. Adult BPHs treated with NlNF-YARNAi, NlNF-YBRNAi, or NlNF-YCRNAi produced fewer eggs, and eggs laid by these BPHs had arrested embryogenesis. These findings deepen our understanding of NF-Y function in hemipteran insects.

Adult moulting and dimorphism in a new species of Sphenoderes (Kinorhyncha: Kentrorhagata)—Is the exception becoming the rule?

AbstractAdult dimorphism (i.e. non‐sexual dimorphism observed among sexually mature specimens) and the existence of two morphologically distinct adult stages are for the first time reported from a species of the kentrorhagid kinorhynch genus Sphenoderes. The dimorphism was found in a new species, Sphenoderes neptunus sp. nov., from the Gulf of Naples, Italy, and a formal description is provided. While dimorphism between adult life stages of kinorhynchs so far have been restricted to differences in the two posteriormost segments, the dimorphism in the new species is expressed in spine lengths throughout the entire trunk, as well as in presence or absence of cuspidate spines. The implications for comparative kinorhynch morphology and taxonomy are discussed, and it is concluded that adult dimorphism and the occurrence of more than one adult life stage always should be considered as a possibility when exploring kinorhynchs of the orders Kentrorhagata and Xenosomata. Despite the comparative challenges prompted by the adult dimorphism, species of Sphenoderes can still be distinguished by differences in shape of midventral placid and distribution of sensory spots. Conversely, spine patterns as differential characters should be used with caution, and only after ensuring that specimens belonging to the same adult stage are compared.

What determines the occurrence of fertilized females in hibernating populations of Pyrrhocoris apterus (Heteroptera: Pyrrhocoridae) in Central Europe?

The current climate warming is associated with the development of a second generation in populations of species such as Pyrrhocoris apterus (L.) (Heteroptera: Pyrrhocoridae), which, in Central Europe, were univoltine in the 1980s. Females of the first generation that develop in spring either enter diapause immediately following the adult moult or after a period of oviposition that gives rise to a second generation. In 2005-2010, in overwintering populations, 3 ± 0.6% of females were fertilized. We explored the possibility that the females that overwintered in a fertilized state were those that had oviposited in the previous season. To test this hypothesis, we reared females of the first generation that moulted to the adult stage in June-August under natural conditions. For these females, we recorded oviposition, mortality and overwintering in the fertilized state. In 2004-2007, the majority (78%) of the first-generation females that oviposited in summer also died before winter and the rest entered dormancy and overwintered. Most of the overwintering females that had laid eggs before overwintering were fertilized. However, the majority (63%) of females that overwintered in the fertilized state had not laid eggs in the previous season; they only copulated and stored the sperm until the next spring. The presence of fertilized females in a hibernating population thus indicates pre-winter sexual activity but not the percentage of first-generation females that oviposit before overwintering.

A Retrospective Assessment of Temperature Trends in Northern Europe Reveals a Deep Impact on the Life Cycle of Ixodes ricinus (Acari: Ixodidae).

This study modelled the changes in the development processes of the health-threatening tick Ixodes ricinus in Northern Europe as driven by the trends of temperature (1950–2018). We used the ECA&D dataset to calculate the annual accumulated temperature to obtain the development rates of the oviposition, incubation, larva–nymph, and nymph–adult molts. Annual values were used to ascertain the trend in development rates of each stage. The ecological classification of Northern Europe (LANMAP2) was used to summarize results. The temperature in 1950–2018 clearly increased in the target territory. The development rates of every tested life cycle process were faster along the time series. Faster oviposition and incubation rates resulted in central Sweden, Baltic countries, and parts of Finland. Faster molting rates were observed in the same territories and in large areas of Western Norway. The trend of temperature in the period 1950–2018 shows a consistent inflection point around 1990, demonstrating that the increased annual accumulated temperature has a deeper impact on the life cycle of I. ricinus since approximately 1990. Faster development rates could be part of the processes driving the reported spread of the tick in the target area and should be considered as a serious threat to human health.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval–pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval–pupal and pupal–adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.

Regulatory mechanisms underlying the specification of the pupal-homologous stage in a hemimetabolous insect.

Juvenile hormones and the genetic interaction between the transcription factors Krüppel homologue 1 (Kr-h1) and Broad (Br) regulate the transformation of insects from immature to adult forms in both types of metamorphosis (holometaboly with a pupal stage versus hemimetaboly with no pupal stage); however, knowledge about the exact instar in which this occurs is limited. Using the hemimetabolous cricket Gryllus bimaculatus (Gb), we demonstrate that a genetic interaction occurs among Gb'Kr-h1, Gb'Br and the adult-specifier transcription factor Gb'E93 from the sixth to final (eighth) nymphal instar. Gb'Kr-h1 and Gb'Br mRNAs were strongly expressed in the abdominal tissues of sixth instar nymphs, with precocious adult moults being induced by Gb'Kr-h1 or Gb'Br knockdown in the sixth instar. The depletion of Gb'Kr-h1 or Gb'Br upregulates Gb'E93 in the sixth instar. By contrast, Gb'E93 knockdown at the sixth instar prevents nymphs transitioning to adults, instead producing supernumerary nymphs. Gb'E93 also represses Gb'Kr-h1 and Gb'Br expression in the penultimate nymphal instar, demonstrating its important role in adult differentiation. Our results suggest that the regulatory mechanisms underlying the pupal transition in holometabolous insects are evolutionarily conserved in hemimetabolous G. bimaculatus, with the penultimate and final nymphal periods being equivalent to the pupal stage. This article is part of the theme issue 'The evolution of complete metamorphosis'.

Resting metabolic rate in migratory and non‐migratory geese following range expansion: go south, go low

While many species suffer from human activities, some like geese benefit and may show range expansions. In some cases geese (partially) gave up migration and started breeding at wintering and stopover grounds. Range expansion may be facilitated and accompanied by physiological changes, especially when associated with changes in migratory behaviour. Interspecific comparisons found that migratory tendency is associated with a higher basal or resting metabolic rate (RMR). We compared RMR of individuals belonging to a migratory and a sedentary colony of barnacle geeseBranta leucopsis. The migratory colony is situated in the traditional arctic breeding grounds (Russia), whereas the sedentary colony has recently been established in the now shared wintering area (the Netherlands). We measured RMR by oxygen consumption () during two ontogenetic phases (juvenile growth and adult wing moult). We also investigated juvenile growth rates and adult body mass dynamics.Mass‐independentwas 13.6% lower in goslings from the sedentary colony than in goslings from the migratory colony. Similarly, in adult geese, mass‐independentwas 15.5% lower in sedentary than in migratory conspecifics. Goslings in the Netherlands grew 36.2% slower than goslings in Russia, while we found no differences in body dimensions in adults. Adult geese from both colonies commenced wing moult with similar body stores, but whereas Russian barnacle geese maintained this level throughout moult, body stores in geese from the Netherlands fell, being 8.5% lower half‐way through the moult.We propose that the colony differences in resting metabolic rate, growth rate and body mass dynamics during moult can be explained by environmental and behavioural differences. The less stringent time constraints combined with poorer foraging opportunities allow for a smaller ‘metabolic machinery’ in non‐migratory geese. Our analysis suggests that range expansion may be associated with changes in physiology, especially when paired with changes in migratory tendency.

North American Condyloderes (Kinorhyncha: Cyclorhagida: Kentrorhagata): Female dimorphism suggests moulting among adult Condyloderes

Two new species of Condyloderes are described, C. rohalorum sp. nov. from the deep-sea off California, and Condyloderes flosfimbriatus sp. nov. from the continental shelf in the northern Gulf of Mexico. Additional species are furthermore reported from the two regions, including Condyloderes kurilensis from the Californian deep-sea, one potentially new species from each of the two regions, and the presence of specimens similar to, and potentially conspecific with, the two new species, i.e., Condyloderes cf. C. rohalorum sp. nov. in the Gulf of Mexico, and Condyloderes cf. C. flosfimbriatus sp. nov. from the Californian deep-sea. Examinations of C. rohalorum sp. nov. reveal that the species has two different adult female stages, hence representing the first example of adult female dimorphism known from this genus, and indicating that adult moulting might happen amongst species of Condyloderes.

Imaginal disc growth factor 4 regulates development and temperature adaptation in Bactrocera dorsalis

The oriental fruit fly Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) is an important invasive pest with high reproductive capacity and invasiveness; it has shown remarkable range expansion and brings higher risk to the environment and agriculture. The insect cuticle serves as skin and skeleton, protecting insects against numerous harmful stresses. One gene named imaginal disc growth factor 4 (idgf4) which is involved in cuticle formation, plays an important role in organizing proteins in the chitin-matrix, as well as in adult molting. This gene in the poorly-described glycoside hydrolase 18 (GH 18) family was chosen to study the function of chitinases in insect defense barrier against heat and molting using quantitative real-time PCR (qRT-PCR) and RNA interference (RNAi). qRT- PCR showed that idgf4 was expressed in all nine developmental stages and was mainly expressed in the early and late pupal, as well as adult stages. Knocking down the idgf4 gene via RNAi in 3rd instar larvae led to the decreased survival of larvae under high temperatures and malformed individuals as adults. The results indicated the function of the idgf4 gene in the fruit fly’s defense barrier and development. It can provide new insights into understanding the function of one member in the GH 18 family, and may reveal a new potential gene for pest control.

Aimed limb movements in a hemimetabolous insect are intrinsically compensated for allometric wing growth by developmental mechanisms.

ABSTRACTFor aimed limb movements to remain functional, they must be adapted to developmental changes in body morphology and sensory-motor systems. Insects use their limbs to groom the body surface or to dislodge external stimuli, but they face the particular problem of adapting these movements to step-like changes in body morphology during metamorphosis or moulting. Locusts are hemimetabolous insects in which the imaginal moult to adulthood results in a sudden and dramatic allometric growth of the wings relative to the body and the legs. We show that, despite this, hind limb scratches aimed at mechanosensory stimuli on the wings remain targeted to appropriate locations after moulting. In juveniles, the tips of the wings extend less than halfway along the abdomen, but in adults they extend well beyond the posterior end. Kinematic analyses were used to examine the scratching responses of juveniles (fifth instars) and adults to touch of anterior (wing base) and posterior (distal abdomen) targets that develop isometrically, and to wing tip targets that are anterior in juveniles but posterior in adults. Juveniles reach the (anterior) wing tip with the distal tibia of the hind leg using anterior rotation of the thoraco-coxal and coxo-trochanteral (‘hip’) joints and flexion of the femoro-tibial (‘knee’) joint. Adults, however, reach the corresponding (but now posterior) wing tip using posterior rotation of the hip and extension of the knee, reflecting a different underlying motor pattern. This change in kinematics occurs immediately after the adult moult without learning, indicating that the switch is developmentally programmed.

Spermatozoa production in male Varroa destructor and its impact on reproduction in worker brood of Apis mellifera.

Reproduction in Varroa destructor exclusively takes place within the sealed honey bee brood cell and is, therefore, limited by the duration of the postcapping period. Oogenesis, ontogenetic development and mating must be optimized to ensure the production of as many mated daughter mites as possible. One adult male mite has to mate with up to five sister mites and transfer 30-40 spermatozoa to each female. We analyzed the production and transfer of male spermatozoa during a reproductive cycle by counting all spermatozoa in the genital tracts of the male and daughter mites in 80 worker brood cells at defined times after cell capping. We could show that spermatozoa production in male mites is an ongoing process throughout their adult lifetime starting after the adult molt. The spermatozoa are transferred to the females in an early non-capacitated stage and require further maturation within the female's genital tract. Our study points out that a Varroa male has at any time in the brood cell enough spermatozoa to inseminate all daughter mites but does not waste energy in producing a big surplus. In total one male produced, on average, 125 spermatozoa during a reproductive cycle in worker brood which is sufficient for successful matings with at least three daughter mites. Spermiogenesis in Varroa males represents therefore a further adaptation to the limited time available for reproduction.