Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica.

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.

Krüppel homolog 1 regulates photoperiodic reproductive plasticity in the cabbage beetle Colaphellus bowringi.

Many insects exhibit reproductive plasticity where the photoperiod determines whether the insect becomes reproductively active or enters diapause. Adult reproductive diapause is a strategy that allows insects to survive harsh environmental conditions. A deficiency in juvenile hormone (JH) leads to reproductive diapause. However, little is known about the molecular mechanisms by which JH signaling regulates reproductive diapause. In this study, we used the cabbage beetle Colaphellus bowringi, a serious pest, to investigate the role of Krüppel homolog 1 (Kr-h1) in controlling photoperiodic plasticity of female reproduction. We focused on Kr-h1, since it acts as a key mediator of JH signaling. We show here that JH-Methoprene-tolerant signaling upregulated the expression of Kr-h1 in reproductively active C. bowringi females when reared under short day conditions. In the long day-treated diapausing females, Kr-h1 transcripts decreased dramatically. Interfering with Kr-h1 function repressed reproductive development by blocking vitellogenesis and ovarian growth. Further, Kr-h1 depletion induced other diapause-like traits, including elevated lipid accumulation and high expression of diapause-related genes. RNA-Seq showed that Kr-h1 played both activating and repressive roles, depending on whether downstream genes were acting in reproduction- or diapause pathways, respectively. Finally, we identified the DNA replication gene mini-chromosome maintenance 4 and two triacylglycerol lipase genes as critical downstream factors of Kr-h1 that are critical for reproductive plasticity in C. bowringi. These results reveal that Kr-h1 is a key component of the regulatory pathway that coordinates reproduction and diapause in insects in response to photoperiodic input.

Comparative proteomics provides insights into diapause program of Bactrocera minax (Diptera: Tephritidae).

The Chinese citrus fly, Bactrocera minax, is a notorious univoltine pest that causes damage to citrus. B. minax enters obligatory pupal diapause in each generation to resist harsh environmental conditions in winter. Despite the enormous efforts that have been made in the past decade, the understanding of pupal diapause of B. minax is currently still fragmentary. In this study, the 20-hydroxyecdysone solution and ethanol solvent was injected into newly-formed pupae to obtain non-diapause- (ND) and diapause-destined (D) pupae, respectively, and a comparative proteomics analysis between ND and D pupae was performed 1 and 15 d after injection. A total of 3,255 proteins were identified, of which 190 and 463 were found to be differentially abundant proteins (DAPs) in ND1 vs D1 and ND15 vs D15 comparisons, respectively. The reliability and accuracy of LFQ method was validated by qRT-PCR. Functional analyses of DAPs, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and protein-protein interaction (PPI) network construction, were conducted. The results revealed that the diapause program of B. minax is closely associated with several physiological activities, such as phosphorylation, chitin biosynthesis, autophagy, signaling pathways, endocytosis, skeletal muscle formation, protein metabolism, and core metabolic pathways of carbohydrate, amino acid, and lipid conversion. The findings of this study provide insights into diapause program of B. minax and lay a basis for further investigation into its underlying molecular mechanisms.

The Modulation of Trehalose Metabolism by 20-Hydroxyecdysone in Antheraea pernyi (Lepidoptera: Saturniidae) During its Diapause Termination and Post-Termination Period.

Trehalose plays a crucial role in the diapause process of many insects, serving as an energy source and a stress protectant. Trehalose accumulation has been reported in diapause pupae of Antheraea pernyi; however, trehalose metabolic regulatory mechanisms associated with diapause termination remain unclear. Here, we showed that the enhanced trehalose catabolism was associated with an increase in endogenous 20-hydroxyecdysone (20E) in hemolymph of A. pernyi pupae during their diapause termination and posttermination period. Injection of 20E increased the mRNA level of trehalase 1A (ApTre-1A) and trehalase 2 (ApTre-2) of A. pernyi diapause pupae in a dose-dependent manner but did not affect the mRNA level of trehalase 1B (ApTre-1B). Meanwhile, exogenous 20E increased the enzyme activities of soluble and membrane-bound trehalase, leading to a decline in hemolymph trehalose. Conversely, the expression of ApTre-1A and ApTre-2 were down-regulated after the ecdysone receptor gene (ApEcRB1) was silenced by RNA interference or by injection of an ecdysone receptor antagonist cucurbitacin B (CucB), which inhibits the 20E pathway. Moreover, CucB treatment delayed adult emergence, which suggests that ApEcRB1 might be involved in regulating pupal-adult development of A. pernyi by mediating ApTre-1A and ApTre-2 expressions. This study provides an overview of the changes in the expression and activity of different trehalase enzymes in A. pernyi in response to 20E, confirming the important role of 20E in controlling trehalose catabolism during A. pernyi diapause termination and posttermination period.

NMR-based untargeted metabolomic study of hydrogen peroxide-induced development and diapause termination in brine shrimp.

Artemia diapause has been extensively studied in embryonic biology for a long time. It has been demonstrated that hydrogen peroxide (H2O2) can increase the hatching rate in the development and diapause termination of Artemia cysts. This study used an untargeted 1H NMR-based metabolomic approach to explore the physiological regulation of H2O2 in initiating the development and terminating the diapause of Artemia cysts. This experiment was divided into two parts. In the first part, we analyzed three groups with or without H2O2 as control-0h, control-5h and H2O2 (180µM)-5h; in the second part, after 7-d incubation, the non-hatching cysts were treated with different H2O2 concentrations as low as 180µM and as high 1800µM. The results showed that arginine and proline metabolism were up-regulated after 5h, and H2O2 up-regulated valine, leucine and isoleucine biosynthesis in the development of cysts. In the second part, low H2O2 (180µM) showed alanine, aspartate and glutamate metabolism, but high H2O2 (1800µM) also up-regulated arginine and proline metabolism, as in the control group without H2O2 stimulus. These results suggest that enough H2O2 can catalyze cell transcription and translation in Artemia cysts, and it improves the cell growth rate, thus allowing embryo cells to grow again.

HCl Treatment for Preventing Diapause Causes Ca2+ Efflux in Bombyx mori Eggs.

To elucidate the mechanism for preventing entry into embryonic diapause or breakdown of diapause in Bombyx mori by HCl and dimethyl sulfoxide (DMSO) treatment or a combination of cold and HCl treatment, we performed quantitative analysis of Ca2+ and Mg2+ in the chorion and egg content using inductively coupled plasma atomic emission spectrometry (ICP-AES). When diapause eggs that had been incubated at 25°C for 2 days from oviposition and at 4°C for an additional six days were treated with HCl solution, the amount of Ca2+ in the chorion and egg content after HCl treatment was reduced to one-seventh, as compared with the amount before treatment. In contrast, there was no change in the amount of Mg2+ with HCl treatment. The amount of Ca2+ in the HCl solution after the diapause eggs were treated increased 7.5-fold, as compared with that of eggs treated with water. Even when 17-day-old diapausing eggs were treated with HCl, which did not break diapause, the amount of Ca2+ in the chorion and egg content was reduced to one-fifth, as compared with the control. Meanwhile, changes in Ca2+ and Mg2+ contents were not observed in 12-hr-old diapause-destined eggs before or after treatment with DMSO, which effectively prevents diapause. These data may suggest that Ca2+ efflux from diapause eggs by HCl is not directly associated with preventing entry into diapause or breaking of diapause. In addition, we discovered that the amount of Ca2+ in diapause-destined eggs was more than 2.4-fold larger than in non-diapause-destined eggs.

Imidazole derivative KK-42 boosts pupal diapause incidence and delays diapause termination in several insect species.

The imidazole derivative KK-42 is a synthetic insect growth regulator known previously to be capable of averting embryonic diapause in several Lepidoptera, but whether it also affects diapauses occurring in other developmental stages remains unknown. In the present study, we examined the effect of KK-42 on pupal diapause in two species of Lepidoptera, the Chinese oak silkworm Antheraea pernyi and the corn earworm Helicoverpa zea, and in one species of Diptera, the flesh fly Sarcophaga crassipalpis. In A. pernyi, KK-42 delayed pupal diapause termination under the long day conditions that normally break diapause in this species. Likewise, in H. zea, KK-42 delayed termination of pupal diapause, a diapause that, in this species, is normally broken by high temperature. KK-42-treated pupae of these two species eventually terminated diapause and successfully emerged as adults, but the timing of diapause termination was significantly delayed. KK-42 also significantly increased the incidence of pupal diapause in H. zea and S. crassipalpis when administered to larvae that were environmentally programmed for diapause, but it was not capable of inducing pupal diapause in H. zea if larvae were reared under environmental conditions that do not normally evoke the diapause response. Experiments with H. zea showed that the effect of KK-42 on pupal diapause was dose- and stage-dependent, but not temperature-dependent. Results presented here are consistent with a link between KK-42 and the ecdysteroid signaling pathway that regulates pupal diapause.

Diapause prevention effect of Bombyx mori by dimethyl sulfoxide.

HCl treatment has been, for about 80 years, the primary method for the prevention of entry into embryonic diapauses of Bombyx mori. This is because no method is as effective as the HCl treatment. In this study, we discovered that dimethyl sulfoxide (DMSO) prevented entry into the diapause of the silkworm, Bombyx mori. The effect of diapause prevention was 78% as a result of treatment with 100% DMSO concentration, and the effect was comparable to that of the HCl treatment. In contrast, in the case of non-diapause eggs, hatchability was decreased by DMSO in a concentration-dependent manner. The effect of DMSO was restricted within 24 hours after oviposition of diapause eggs, and the critical period was slightly shorter than the effective period of the HCl treatment. DMSO analogs, such as dimethyl formamide (DMF) and dimethyl sulfide (DMS), did little preventive effect against the diapause. Furthermore, we also investigated the permeation effects of chemical compounds by DMSO. When treated with an inhibitor of protein kinase CK2 (CK2) dissolved in DMSO, the prevention rate of the diapause was less than 40%. This means that the inhibition effect by the CK2 inhibitor was the inhibition of embryonic development after diapause prevention by DMSO. These data suggest that DMSO has the effects of preventing from entering into the diapause and permeation of chemicals into diapause eggs.