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.     

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.     

Three Heat Shock Protein Genes from Bactrocera (Tetradacus) minax Enderlein: Gene Cloning,Characterization, and Association with Diapause

Bactrocera (Tetradacus) minax Enderlein is a major pest to wild and cultivated species of citrus. Bactrocera minax produces one generation per year with a long pupal diapause period of over 6 months, which hinders efforts to obtain vast numbers of insects under standard room conditions. Determining the mechanisms of diapause is significantly important for obtaining large quantities of these insects. To characterize the heat shock protein (Hsp) genes of B. minax and to unravel their potential contribution to diapause, we performed 3′ and 5′ RACE to isolate the complementary DNA (cDNA) sequences, bioinformatics to examine the phylogenetic relationships, and real-time quantitative PCR to detect the expression patterns of three Hsp genes during various developmental stages. These results represent the first characterization of the three Hsp genes of B. minax; the open reading frames of Bmhsp23, Bmhsp70, and Bmhsp90 were 510, 1,911, and 1,089 bp, encoding 170, 636, and 363 amino acids, respectively. BmHsp70 and BmHsp90 displayed high identity to previously identified Hsp70 and Hsp90 genes, respectively. BmHsp23 displayed varying similarity, from 28 to 83%, to previously identified small Hsps. Bmhsp23 messenger RNA (mRNA) expression was found to be upregulated during diapause initiation, maintenance, and termination. Bmhsp70 mRNA expression peaked during diapause initiation. Bmhsp90 mRNA expression remained at a relatively low level during deep diapause. Our present results suggest that Bmhsp70 might play an important role in diapause initiation, while Bmhsp23 in diapause initiation and maintenance and Bmhsp90 in diapause regulation. These results improve our understanding of the mechanism of diapause in B. minax at the molecular level.     

Short-day and long-day expression patterns of genes involved in the flesh fly clock mechanism: period,timeless, cycle and cryptochrome

Though our knowledge of the molecular details of the circadian clock has advanced rapidly, the functional elements of the photoperiodic clock remain largely unknown. As a first step to approach this issue, we report here the sequences and expression patterns of period (per), timeless (tim), cycle (cyc) and cryptochrome (cry) mRNAs in the flesh fly Sarcophaga crassipalpis. Nucleotide and deduced amino acid sequences of the genes in S. crassipalpis show high similarity to homologous genes in other insects that have been investigated. S. crassipalpis TIM has a unique C-terminus that contains a poly Q region. A diel rhythmicity of per and tim mRNA abundance was detected in the adult heads (peak during scotophase), while cry and cyc mRNA abundance remained fairly constant throughout. The abundance of cyc mRNA was quite low when compared to per, tim and cry mRNA. Rearing temperature affected the amount of per and tim mRNAs: abundance of per mRNA increased at 20 °C when compared to 25 °C, but that of tim mRNA decreased. Photoperiod influenced the expression patterns of per and tim mRNA: the peak of per mRNA expression shifted in concert with onset of the scotophase, while a shift in tim mRNA expression was less pronounced. The amplitude of tim mRNA was severely dampened under long daylength, but that of per mRNA was not affected. These distinct patterns of expression suggest that this information could be used to determine photoperiodic responses such as diapause.