Circadian clock genes period and cycle regulate photoperiodic diapause in the bean bug Riptortus pedestris males

The photoperiodic response is crucial for many insects to adapt to seasonal changes in temperate regions. It was recently shown that the circadian clock genes period (per) and cycle (cyc) are involved in the photoperiodic regulation of reproductive diapause in the bean bug Riptortus pedestris females. Here, we investigated the involvement of per and cyc both in the circadian rhythm of cuticle deposition and in the photoperiodic diapause of R. pedestris males using RNA interference (RNAi). RNAi of per and cyc disrupted the cuticle deposition rhythm and resulted in distinct cuticle layers. RNAi of per induced development of the male reproductive organs even under diapause-inducing short-day conditions, whereas RNAi of cyc suppressed development of the reproductive organs even under diapause-averting long-day conditions. Thus, the present study suggests that the circadian clock operated by per and cyc governs photoperiodism of males as that of females.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Juvenile hormone induces ovarian development in diapausing cave-dwelling Drosophila species

Drosophila grisea and macroptera were collected in caves overwintering as adults. The females remained in a state of reproductive diapause which extended until May for macroptera and until July for grisea, whereas the males of both species had mature sperm at all times. Termination of the reproductive diapause under laboratory conditions was accomplished in grisea by exposing them to 14 hr of illumination daily and in macroptera by increasing the temperature to 20°C. Topical application of juvenile hormone (JH) on diapausing grisea caused a prompt termination of diapause and maturation of oöcytes within 10 days. Yolk proteins were found in the haemolymph of diapausing flies but not in their ovaries. In the JH-treated flies, yolk proteins were found in both the haemolymph and the ovaries, suggesting that in this species JH regulates the uptake of yolk proteins.     

Diapause-specific gene expression in the northern house mosquito, Culex pipiens L., identified by suppressive subtractive hybridization

In this study we probe the molecular events underpinning diapause observed in overwintering females of Culex pipiens. Using suppressive subtractive hybridization (SSH) we have identified 40 genes that are either upregulated or downregulated during this seasonal period of dormancy. Northern blot hybridizations have confirmed the expression of 32 of our SSH clones, including six genes that are upregulated specifically in early diapause, 17 that are upregulated in late diapause, and two upregulated throughout diapause. In addition, two genes are diapause downregulated and five remain unchanged during diapause. These genes can be categorized into eight functional groups: genes with regulatory functions, metabolically-related genes, those involved in food utilization, stress response genes, cytoskeletal genes, ribosomal genes, transposable elements, and genes with unknown functions.     

Loss of Trx-2 enhances oxidative stress-dependent phenotypes in Drosophila

Overexpression of thioredoxin (TRX) confers oxidative stress resistance and extends lifespan in mammals and insects. However, less is known about phenotypes associated with loss of TRX. We investigated loss-of-function phenotypes of Trx-2 in Drosophila, and found that the mutant flies are hyper-susceptible to paraquat, a free radical generator, but not to hydrogen peroxide. They contain a high amount of protein carbonyl, which dramatically increases with age. Trx-2 mutants express high levels of anti-oxidant genes, such as superoxide dismutase, catalase, and glutathione synthetase. This is the first demonstration of biochemical and physiological consequences caused by loss of Trx-2 in Drosophila.     

Dissecting the Biological Role of Mucin-type O-Glycosylation Using RNA Interference in Drosophila Cell Culture

Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.     

The antioxidant system in diapausing and active red mason bee Osmia bicornis

The red mason bee Osmia bicornis L. (Hymenoptera: Megachilidae) belongs to a group of insects that undergo an obligatory diapause in the imago stage. Red mason bees (O. bicornis) consume oxygen during overwintering diapause, and reactive oxygen species can be produced despite substantial inhibition of metabolism. These are first studies to investigate the antioxidant system in diapausing red mason bees and to compare the antioxidant systems of overwintering and active imagines. The present study analyzes total antioxidant status, glutathione and ascorbic acid levels, and the activity of superoxide dismutase, catalase and peroxidase in overwintering (October to March) and active (April) female and male O. bicornis. Diapause phases (prediapause, diapause and post‐diapause) cannot be distinguished based on the parameters of the antioxidant system of the bees. During overwintering, a significant decrease is noted only in ascorbic acid content. The remaining antioxidants remain fairly stable, which indicates the absence of oxidative stress in diapausing specimens. The analyzed parameters distinguish diapausing bees from active insects. Excluding total antioxidant status, the evaluated parameters are significantly higher in active individuals than in overwintering specimens. Sex‐related differences are found only for catalase, the level of which is consistently higher in males than in females.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

The environmental regulation of adult diapause in Drosophila littoralis

Drosophila littoralis overwinters in the adult stage in a reproductive diapause. During the warm season there are one or two generations in Finland. The diapause appears to be a prolongation of the post-eclosion immaturity of young females. The termination of diapause is controlled by a combination of adequate temperature and sufficiently long photophases. The diapausing status of females is ascertained by inspecting the developmental stage of their ovaries. In laboratory experiments the maturity of ovaries is not closely correlated with the receptivity of females.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.