Uninfected Eggs Research Articles

Cardinium symbionts are pervasive in Iranian populations of the spider mite Panonychus ulmi despite inducing an infection cost and no demonstrable reproductive phenotypes when Wolbachia is a symbiotic partner.

Maternally transmitted symbionts such as Cardinium and Wolbachia are widespread in arthropods. Both Cardinium and Wolbachia can cause cytoplasmic incompatibility, a reproductive phenotype that interferes with the development of uninfected eggs that are fertilized by infected sperm. In haplodiploid hosts, these symbionts can also distort sex allocation to facilitate their spread through host populations. Without other fitness effects, symbionts that induce strong reproductive phenotypes tend to spread to high and stable infection frequencies, whereas variants that induce weak reproductive phenotypes are typically associated with intermediate and variable frequencies. To study the spread of Cardinium in a haplodiploid host, we sampled Iranian populations of the economically important spider mite Panonychus ulmi in apple orchards. Within several field populations, we also studied the Wolbachia infection frequencies. All P. ulmi field populations carried a Cardinium infection and exhibited high infection frequencies. In contrast, Wolbachia frequency ranged between ca. 10% and ca. 70% and was only found in co-infected mites. To test whether Cardinium induce reproductive phenotypes in P. ulmi, a Cardinium-cured derived line was generated by antibiotic treatment from a co-infected field population. Genetic crosses indicated that Cardinium do not induce demonstrable levels of cytoplasmic incompatibility and sex allocation distortion in co-infected P. ulmi. However, Cardinium infection was associated with a longer developmental time and reduced total fecundity for co-infected females. We hypothesize that Cardinium spread through P. ulmi populations via uncharacterized fitness effects and that co-infection with Wolbachia might impact these drive mechanisms.

Interacting host modifier systems control Wolbachia-induced cytoplasmic incompatibility in a haplodiploid mite.

Reproductive parasites such as Wolbachia spread within host populations by inducing cytoplasmic incompatibility (CI). CI occurs when parasite‐modified sperm fertilizes uninfected eggs and is typified by great variation in strength across biological systems. In haplodiploid hosts, CI has different phenotypic outcomes depending on whether the fertilized eggs die or develop into males. Genetic conflict theories predict the evolution of host modulation of CI, which in turn influences the stability of reproductive parasitism. However, despite the ubiquity of CI‐inducing parasites in nature, there is scarce evidence for intraspecific host modulation of CI strength and phenotype. Here, we tested for intraspecific host modulation of Wolbachia‐induced CI in haplodiploid Tetranychus urticae mites. Using a single CI‐inducing Wolbachia variant and mitochondrion, a nuclear panel was created that consisted of infected and cured near‐isogenic lines. We performed a highly replicated age‐synchronized full diallel cross composed of incompatible and compatible control crosses. We uncovered host modifier systems that cause striking variation in CI strength when carried by infected T. urticae males. We observed a continuum of CI phenotypes in our crosses and identified strong intraspecific female modulation of the CI phenotype. Crosses established a recessive genetic basis for the maternal effect and were consistent with polygenic Mendelian inheritance. Both male and female modulation interacted with the genotype of the mating partner. Our findings identify spermatogenesis as an important target of selection for host modulation of CI strength and underscore the importance of maternal genetic effects for the CI phenotype. Our findings reveal that intraspecific host modulation of CI is underpinned by complex genetic architectures and confirm that the evolution of reproductive parasitism is contingent on host genetics.

Cause of increased size of acipenseriform eggs infected with Polypodium hydriforme.

Eggs of acipenseriform fish infected with the parasite Polypodium hydriforme become enlarged during later stages of development. This study examined if the increase in size is due to the increase in nutrients or water in the infected eggs and if the polypodium eggs affect the nutrient levels of the neighbouring eggs in the ovary. Infected and uninfected eggs were collected from parasitized Paddlefish, Polyodon spathula, hosts and unparasitized individuals. Levels of water, protein, carbohydrates, lipids and amino acids were determined for each egg. Although there were nutritional differences between eggs infected with P. hydriforme and uninfected eggs there was no indication that there was an increase in resource allocation to the infected eggs. The amount of water was much higher in infected eggs, suggesting the size increase was due to a greater influx of water. Levels of free amino acids were much higher in infected eggs and we hypothesize they could be used to increase the solute concentration to increase the influx of water, a mechanism that is also used by marine teleosts.

Paternal transmission of the Wolbachia CidB toxin underlies cytoplasmic incompatibility

Wolbachia are widespread endosymbiotic bacteria that manipulate the reproduction of arthropods through a diversity of cellular mechanisms. In cytoplasmic incompatibility (CI), a sterility syndrome originally discovered in the mosquito Culex pipiens, uninfected eggs fertilized by sperm from infected males are selectively killed during embryo development following the abortive segregation of paternal chromosomes in the zygote. Despite the recent discovery of Wolbachia CI factor (cif) genes, the mechanism by which they control the fate of paternal chromosomes at fertilization remains unknown. Here, we have analyzed the cytological distribution and cellular impact of CidA and CidB, a pair of Cif proteins from the Culex-infecting Wolbachia strainwPip. We show that expression of CidB in Drosophila S2R+ cells induces apoptosis unless CidA isco-expressed and associated with its partner. In transgenic Drosophila testes, both effectors colocalize in germ cells until the histone-to-protamine transition in which only CidB is retained in maturing spermatid nuclei. We further show that CidB is similarly targeted to maturing sperm of naturally infected Culex mosquitoes. At fertilization, CidB associates with paternal DNA regions exhibiting DNA replication stress, as a likely cause of incomplete replication of paternal chromosomes at the onset of the first mitosis. Importantly, we demonstrate that inactivation of the deubiquitylase activity of CidB does not abolish its cell toxicity or its ability to induce CI in Drosophila. Our study thus demonstrates that CI functions as a transgenerational toxin-antidote system and suggests that CidB acts by poisoning paternal DNA replication in incompatible crosses.

Metabolomics provide new insights into mechanisms of Wolbachia-induced paternal defects in Drosophila melanogaster.

Wolbachia is a group of intracellular symbiotic bacteria that widely infect arthropods and nematodes. Wolbachia infection can regulate host reproduction with the most common phenotype in insects being cytoplasmic incompatibility (CI), which results in embryonic lethality when uninfected eggs fertilized with sperms from infected males. This suggests that CI-induced defects are mainly in paternal side. However, whether Wolbachia-induced metabolic changes play a role in the mechanism of paternal-linked defects in embryonic development is not known. In the current study, we first use untargeted metabolomics method with LC-MS to explore how Wolbachia infection influences the metabolite profiling of the insect hosts. The untargeted metabolomics revealed 414 potential differential metabolites between Wolbachia-infected and uninfected 1-day-old (1d) male flies. Most of the differential metabolites were significantly up-regulated due to Wolbachia infection. Thirty-four metabolic pathways such as carbohydrate, lipid and amino acid, and vitamin and cofactor metabolism were affected by Wolbachia infection. Then, we applied targeted metabolomics analysis with GC-MS and showed that Wolbachia infection resulted in an increased energy expenditure of the host by regulating glycometabolism and fatty acid catabolism, which was compensated by increased food uptake. Furthermore, overexpressing two acyl-CoA catabolism related genes, Dbi (coding for diazepam-binding inhibitor) or Mcad (coding for medium-chain acyl-CoA dehydrogenase), ubiquitously or specially in testes caused significantly decreased paternal-effect egg hatch rate. Oxidative stress and abnormal mitochondria induced by Wolbachia infection disrupted the formation of sperm nebenkern. These findings provide new insights into mechanisms of Wolbachia-induced paternal defects from metabolic phenotypes.

Infertility and fecundity loss of Wolbachia-infected Aedes aegypti hatched from quiescent eggs is expected to alter invasion dynamics.

The endosymbiotic bacterium Wolbachia shows viral blocking in its mosquito host, leading to its use in arboviral disease control. Releases with Wolbachia strains wMel and wAlbB infecting Aedes aegypti have taken place in several countries. Mosquito egg survival is a key factor influencing population persistence and this trait is also important when eggs are stored prior to releases. We therefore tested the viability of mosquitoes derived from Wolbachia wMel and wAlbB-infected as well as uninfected eggs after long-term storage under diurnal temperature cycles of 11–19°C and 22–30°C. Eggs stored at 11–19°C had higher hatch proportions than those stored at 22–30°C. Adult Wolbachia density declined when they emerged from eggs stored for longer, which was associated with incomplete cytoplasmic incompatibility (CI) when wMel-infected males were crossed with uninfected females. Females from stored eggs at both temperatures continued to show perfect maternal transmission of Wolbachia, but storage reduced the fecundity of both wMel and wAlbB-infected females relative to uninfected mosquitoes. Furthermore, we found a very strong negative impact of the wAlbB infection on the fertility of females stored at 22–30°C, with almost 80% of females hatching after 11 weeks of storage being infertile. Our findings provide guidance for storing Wolbachia-infected A. aegypti eggs to ensure high fitness adult mosquitoes for release. Importantly, they also highlight the likely impact of egg quiescence on the population dynamics of Wolbachia-infected populations in the field, and the potential for Wolbachia to suppress mosquito populations through cumulative fitness costs across warm and dry periods, with expected effects on dengue transmission.

Identification of Serratia marcescens isolated from Antheraea pernyi eggs and determination of bacterial pathogenicity and transmission pathway

The Chinese oak silkworm, Antheraea pernyi, is an important alternate host for mass production of Trichogramma parasitoids, which play a vital role in the biological control of lepidopterous pests in China. The quality of A. pernyi eggs is particularly important for mass production of these parasitoids. In this study, a pathogenic bacterium, isolated and purified from A. pernyi eggs that had turned gray in color, was identified as Serratia marcescens. We used morphology, biochemistry and 16S rDNA analysis to characterize the strain, which was named “APE strain”. Serratia marcescens APE strain was determined to be the causal bacterium associated with the disease in the eggs, verified by a test based on Koch’s Postulates. We tested the pathogenicity of S. marcescens APE strain on A. pernyi eggs; the percentage of diseased (gray) eggs reached 57.78% when uninfected eggs were exposed to a concentration of 1 × 109 cfu/mL bacterial suspension for 7 h. S. marcescens was transmitted mechanically by Trichogramma parasitoids. The transmission rate was 25.56%. In a horizontal transmission test, the highest percentage of uninfected eggs that developed infections was 51.43% after being treated with contents of diseased eggs for 12 h. In a vertical transmission test, the number of infected eggs per treated adult female was 63.8–92.3 after treatment with different S. marcescens concentrations, significantly lower than the 304.3 eggs per female in the control group. Furthermore, the percentage of infected (gray) eggs produced by adult moths exposed to bacteria in the larval stage was 80.5–85.3%.

Paternal Grandmother Age Affects the Strength of Wolbachia-Induced Cytoplasmic Incompatibility in Drosophila melanogaster.

Wolbachia are obligate intracellular bacteria that are globally distributed in half of all arthropod species. As the most abundant maternally inherited microbe in animals, Wolbachia manipulate host reproduction via reproductive parasitism strategies, including cytoplasmic incompatibility (CI). CI manifests as embryonic death when Wolbachia-modified sperm fertilize uninfected eggs but not maternally infected eggs. Thus, CI can provide a relative fitness advantage to Wolbachia-infected females and drive the infection through a population. In the genetic model Drosophila melanogaster, the Wolbachia strain wMel induces variable CI, making mechanistic studies in D. melanogaster cumbersome. Here, we demonstrate that sons of older paternal D. melanogaster grandmothers induce stronger CI than sons of younger paternal grandmothers, and we term this relationship the "paternal grandmother age effect" (PGAE). Moreover, the embryos and adult sons of older D. melanogaster grandmothers have higher Wolbachia densities, correlating with their ability to induce stronger CI. In addition, we report that Wolbachia density positively correlates with female age and decreases after mating, suggesting that females transmit Wolbachia loads that are proportional to their own titers. These findings reveal a transgenerational impact of age on wMel-induced CI, elucidate Wolbachia density dynamics in D. melanogaster, and provide a methodological advance to studies aimed at understanding wMel-induced CI in the D. melanogaster model.IMPORTANCE Unidirectional cytoplasmic incompatibility (CI) results in a postfertilization incompatibility between Wolbachia-infected males and uninfected females. CI contributes to reproductive isolation between closely related species and is used in worldwide vector control programs to drastically lower arboviral vector population sizes or to replace populations that transmit arboviruses with those resistant to transmission. Despite decades of research on the factors that influence CI, penetrance is often variable under controlled laboratory conditions in various arthropods, suggesting that additional variables influence CI strength. Here, we demonstrate that paternal D. melanogaster grandmother age influences the strength of CI induced by their sons. Older D. melanogaster females have higher Wolbachia densities and produce offspring with higher Wolbachia densities that associate with stronger CI. This work reveals a multigenerational impact of age on CI and expands our understanding of host-Wolbachia interactions and the biology of CI induced by the Wolbachia strain infecting the most widely used arthropod model, D. melanogaster.

Effect of infectious bursal disease virus infection on energy metabolism in embryonic chicken livers

ABSTRACT 1. The purpose of this study was to investigate ATP levels and the activities of important enzymes involved in glycolysis and TCA cycle in livers of embryonated chicken eggs infected by infectious bursal disease virus (IBDV). 2. Embryonated chicken eggs (9 days) were randomly divided into two groups (50 eggs per group). The first group was inoculated with a very virulent IBDV (vvIBDV) isolate into the chorioallantoic membrane. The second group was maintained as uninfected control eggs and inoculated with physiological saline. Embryo survival was assessed daily, and six embryos were sacrificed at 24, 48, 72, 96, and 120 hpi for examining livers. Viral loads in the livers were evaluated by qRT-PCR. A comparative analysis of markers associated with the regulation of energy metabolism across several functional classes (ATP, pyruvic and lactic acids, mitochondrial protein, NAD+/NADH ratios, and enolase, lactic acid dehydrogenase and the respiratory chain complex I activities) were examined in the context of IBDV infection. 3. The results indicated that increases in the enzymatic activities associated with glycolytic metabolism in turn affected the synthesis and cytoplasmic concentrations of ATP at early timepoints in infected chicken embryos. Subsequently, energy metabolism was inhibited through the pathological perturbations of metabolic enzymes and mitochondrial damage, as inferred from reduced ATP generation. 4. These results suggested impaired bioenergetics, which may lead to liver dysfunction consequent to IBDV infection, contributing to the disease pathogenesis.

The influence of microsporidian pathogens from commercially available lady beetles on larval development of the green lacewing, Chrysoperla carnea, in the absence of infection.

In North America, more than 70 species of natural enemies are available for pest control, including the aphid predators, Adalia bipunctata L. (two-spotted lady beetle) and Hippodamia convergens Guérin-Méneville (convergent lady beetle), and the generalist predator Chrysoperla carnea Stephens (green lacewing). The two lady beetle species are known to host microsporidian pathogens: Nosema adaliae was originally described from Adalia bipunctata and Tubulinosema hippodamiae from H. convergens. Microsporidia are spore-forming pathogens that typically produce chronic, debilitating disease. Because the spores of both pathogens are transovarially transmitted through beetle eggs, the predation behavior of lacewing larvae provides an opportunity for the transmission of these pathogens when infected lady beetles and lacewings share the same local environment. In this study, uninfected and microsporidia-infected eggs from A. bipunctata and H. convergens were offered to C. carnea larvae. The development of larvae that consumed N. adaliae-infected eggs was not affected, but larval development was prolonged by almost 3 days for those that consumed two or more T. hippodamiae-infected eggs. Prolonged larval development is considered to be costly because larvae remain vulnerable to cannibalization by sibling larvae or other predators. Longevity did not differ significantly between sexes of C. carnea, and the sex ratio of newly eclosed adults did not differ from the previously reported sex ratio of 1♂: 1♀. Upon examination by light microscopy at the end of the trial, two C. carnea larvae were infected with N. adaliae and none were infected with T. hippodamiae, suggesting that T. hippodamiae influenced lacewing larval development without establishing an infection.

Microfungi Associated with Pteroptyx bearni (Coleoptera: Lampyridae) Eggs and Larvae from Kawang River, Sabah (Northern Borneo)

Overlooking the importance of insect disease can have disastrous effects on insect conservation. This study reported the microfungi that infect Pteroptyx bearni eggs and larvae during ex-situ rearing project. Two different species of microfungi that infected the firefly’s immature life stages were isolated and identified. Penicillium citrinum infected the firefly’s eggs while Trichoderma harzianum infected the firefly during the larval stage. Both microfungi species caused absolute mortality once infection was observed; out of 244 individual eggs collected, 75 eggs (32.5%) were infected by Penicillium citrinum. All 13 larvae that hatched from the uninfected eggs were infected by Trichoderma harzianum. This study was the first to document the infection of Pteroptyx bearni’s eggs and larvae by Penicillium citrinum and Trichoderma harzianum.

Water mold infection but not paternity induces selective filial cannibalism in a goby.

Many animals heavily invest in parental care but still reject at least some of their offspring. Although seemingly paradoxical, selection can favor parents to neglect offspring of particularly low reproductive value, for example, because of small survival chances. We here assess whether filial cannibalism (FC), where parents routinely eat some of their own young, is selective in response to individual offspring reproductive value. We performed two independent laboratory experiments in the common goby (Pomatoschistus microps) to test whether caring fathers preferentially cannibalize eggs of a given infection history and paternity. While males did not discriminate kin from nonkin eggs, they consumed significantly more eggs previously exposed to water mold compared to uninfected eggs. Our findings clearly show that parents differentiate between eggs based on differences in egg condition, and thus complement the prevailing view that FC arises for energetic reasons. By preventing the spread of microbial infections, the removal of molded eggs can constitute an important component of parental care and may represent a key driver of selective FC in a wide array of parental fish.

<strong>Effects of <em>Wolbachia</em> infection in <em>Tetranychus urticae</em> (Acari: Tetranychidae) on predation by <em>Neoseiulus cucumeris</em> (Acari: Phytoseiidae)</strong>

Abstract Wolbachia are endosymbiotic bacteria that are widely distributed in arthropods. The bacteria cause reproductive disorders and affect fitness-related traits of their hosts. The generalist predatory mite Neoseiulus cucumeris(Oudemans) (Acari: Phytoseiidae) is a cosmopolitan species commonly used as a biological control agent for mites. Here we investigated the effect of Wolbachia infection in the prey, Tetranychus urticae, on predation by N. cucumeris by using both functional response tests (effect of prey egg density on predation) and choice and no-choice tests. The rate of egg consumption decreased with increasing egg density for both infected and uninfected eggs (Type II responses). In a no-choice test, N. cucumeris consumed approximately equal numbers of infected and uninfected eggs, nymphs and adults but consumed fewer infected larvae than uninfected larvae. In the first choice test, different developmental stages of T. urticae were provided, N. cucumeris preferred eggs and larvae over nymphs ...

A Wolbachia-Sensitive Communication between Male and Female Pupae Controls Gamete Compatibility in Drosophila

Gamete compatibility is fundamental to sexual reproduction. Wolbachia are maternally inherited endosymbiotic bacteria that manipulate gamete compatibility in many arthropod species. In Drosophila, the fertilization of uninfected eggs by sperm from Wolbachia-infected males often results in early developmental arrest. This gamete incompatibility is called cytoplasmic incompatibility (CI). CI is highest in young males, suggesting that Wolbachia affect sperm properties during male development. Here, we show that Wolbachia modulate testis development. Unexpectedly, this effect was associated with Wolbachia infection in females, not males. This raised the possibility that females influenced testis development by communicating with males prior to adulthood. Using a combinatorial rearing protocol, we provide evidence for such a female-to-male communication during metamorphosis. This communication involves the perception of female pheromones by male olfactory receptors. We found that this communication determines the compatibility range of sperm. Wolbachia interfere with this female-to-male communication through changes in female pheromone production. Strikingly, restoring this communication partially suppressed CI in Wolbachia-infected males. We further identified a reciprocal male-to-female communication at metamorphosis that restricts the compatibility range of female gametes. Wolbachia also perturb this communication by feminizing male pheromone production. Thus, Wolbachia broaden the compatibility range of eggs, promoting thereby the reproductive success of Wolbachia-infected females. We conclude that pheromone communication between pupae regulates gamete compatibility and is sensitive to Wolbachia in Drosophila.

Application of wMelPop Wolbachia Strain to Crash Local Populations of Aedes aegypti.

The endosymbiotic bacteria Wolbachia pipientis (wMel strain) has been successfully established in several populations of Aedes aegypti, the primary dengue vector. The virulent Wolbachia strain wMelPop is known to cause several pathological impacts (increased egg mortality, life shortening, etc.) reducing overall fitness in the mosquito Ae. aegypti. Increased egg mortality could substantially reduce egg banks in areas with a lengthy monsoonal dry season, and be employed to eliminate local populations. We tested this application under semi-field cage conditions. First, we determined that wMelPop infection significantly reduced the survival of desiccation-resistant eggs of the dengue vector Ae. aegypti, with shade and temperature having a significant impact; nearly all wMelPop-infected eggs failed to hatch after 6 and 10 weeks in summer and winter conditions, respectively. In laboratory selection experiments we found that egg desiccation resistance can be increased by selection, and that this effect of wMelPop infection is due to the nuclear background of the host rather than Wolbachia. We then conducted an invasion of wMelPop within a semi-field cage using sustained weekly releases of wMelPop infected mosquitoes, with fixation achieved after 9 weeks. The egg populations wMelPop infected and an uninfected control were then subjected to a simulated prolonged monsoonal dry season (2.5 months) before flooding to induce hatching. The wMelPop infected eggs suffered significantly greater mortality than the controls, with only 0.67% and 4.35% of respective infected and uninfected eggs held in 99% shade hatching after 80 days. These studies suggest that wMelPop could be used to locally eliminate populations of Ae. aegypti that are exposed to prolonged dry conditions, particularly if combined with vector control.

Sex ratio distortion in the Nesolynx thymus (Hymenoptera: Eulophidae), an ecto-pupal parasitoid of uzifly, Exorista sorbillans (Diptera: Tachinidae)

The reproductive alterations induced by maternally inherited α-proteo-bacteria Wolbachia to their hosts is a well-document- ed phenomenon. In Nesolynx thymus, a gregarious hymenopterous ecto-pupal parasitoid of the uzifly, Exorista sorbillans, diagnostic PCR assay using specific primers revealed the presence of Wolbachia. Following genetic crossing experiments, we observed a female biased sex ratio of 1 : 9.5 at 25°C and 1 : 3 male to female ratio when the populations were exposed to heat shock 33°C for six hours. Furthermore, we found infection polymorphism, where female parasitoids are infected by Wolbachia but males are not infected. In- fected eggs develop into females, whereas uninfected eggs develop parthenogenetically into males. The results are discussed in the context of the possible mechanism of sex-ratio bias caused by Wolbachia.

Improvement of sampling plans for Salmonella detection in pooled table eggs by use of real-time PCR

Eggs and egg products have been described as the most critical food vehicles of salmonellosis. The prevalence and level of contamination of Salmonella on table eggs are low, which severely affects the sensitivity of sampling plans applied voluntarily in some European countries, where one to five pools of 10 eggs are tested by the culture based reference method ISO 6579:2004. In the current study we have compared the testing-sensitivity of the reference culture method ISO 6579:2004 and an alternative real-time PCR method on Salmonella contaminated egg-pool of different sizes (4–9 uninfected eggs mixed with one contaminated egg) and contamination levels (10°–101, 101–102, 102–103CFU/eggshell). Two hundred and seventy samples corresponding to 15 replicates per pool size and inoculum level were tested. At the lowest contamination level real-time PCR detected Salmonella in 40% of contaminated pools vs 12% using ISO 6579. The results were used to estimate the lowest number of sample units needed to be tested in order to have a 95% certainty not falsely to accept a contaminated lot by Monte Carlo simulation. According to this simulation, at least 16 pools of 10 eggs each are needed to be tested by ISO 6579 in order to obtain this confidence level, while the minimum number of pools to be tested was reduced to 8 pools of 9 eggs each, when real-time PCR was applied as analytical method. This result underlines the importance of including analytical methods with higher sensitivity in order to improve the efficiency of sampling and reduce the number of samples to be tested.

Wolbachiainfection lowers fertile sperm transfer in a moth

The endosymbiotic bacterium Wolbachia pipientis manipulates host reproduction by rendering infected males reproductively incompatible with uninfected females (cytoplasmic incompatibility; CI). CI is believed to occur as a result of Wolbachia-induced modifications to sperm during maturation, which prevent infected sperm from initiating successful zygote development when fertilizing uninfected females' eggs. However, the mechanism by which CI occurs has been little studied outside the genus Drosophila. Here, we show that in the sperm heteromorphic Mediterranean flour moth, Ephestia kuehniella, infected males transfer fewer fertile sperm at mating than uninfected males. In contrast, non-fertile apyrene sperm are not affected. This indicates that Wolbachia may only affect fertile sperm production and highlights the potential of the Lepidoptera as a model for examining the mechanism by which Wolbachia induces CI in insects.

Survival and Replication of Dengue-2 Virus in Diapausing Eggs of Aedes albopictus (Diptera: Culicidae)

Survival and replication of dengue-2 virus (family Flaviviridae, genus Flavivirus, DENV-2) was determined in diapausing eggs of Aedes albopictus (Skuse) (Diptera: Culicidae) to evaluate the importance of this mechanism for viral maintenance during adverse climatic conditions. Infected and uninfected eggs of Ae. albopictus were induced to diapause under low temperature and short photoperiod. Virus was detected by reverse transcription-polymerase chain reaction and isolated in C6/36 cells from both diapausing eggs and nondiapausing eggs. Nucleic acid hybridization was used to monitor viral replication in diapausing eggs. DENV-2 might survive in infected diapausing eggs in a relatively quiescent status based on the absence of a replicative intermediate RNA. In contrast, the viral replicative intermediate RNA along with the replicative form RNA was detected in nondiapausing eggs. It seemed that the virus replicated more actively in nondiapausing eggs than in diapausing eggs. Infected diapausing eggs may play an important role in the maintenance of DENV during adverse climatic conditions in nature.

Survival and Replication of Dengue-2 Virus in Diapausing Eggs of <I>Aedes albopictus</I> (Diptera: Culicidae)

Survival and replication of dengue-2 virus (family Flaviviridae, genus Flavivirus, DENV-2) was determined in diapausing eggs of Aedes albopictus (Skuse) (Diptera: Culicidae) to evaluate the importance of this mechanism for viral maintenance during adverse climatic conditions. Infected and uninfected eggs of Ae. albopictus were induced to diapause under low temperature and short photoperiod. Virus was detected by reverse transcription-polymerase chain reaction and isolated in C6/36 cells from both diapausing eggs and nondiapausing eggs. Nucleic acid hybridization was used to monitor viral replication in diapausing eggs. DENV-2 might survive in infected diapausing eggs in a relatively quiescent status based on the absence of areplicative intermediate RNA. In contrast, the viral replicative intermediate RNA along with the replicative form RNA was detected in nondiapausing eggs. It seemed that the virus replicated more actively in nondiapausing eggs than in diapausing eggs. Infected diapausing eggs may play an important role in the maintenance of DENV during adverse climatic conditions in nature.