Comparative Transcriptome Analysis Reveals bmo-miR-6497-3p Regulate Circadian Clock Genes during the Embryonic Diapause Induction Process in Bivoltine Silkworm

Diapause is one of the survival strategies of insects for confronting adverse environmental conditions. Bombyx mori displays typical embryonic diapause, and offspring diapause depends on the incubation environment of the maternal embryo in the bivoltine strains of the silkworm. However, the molecular mechanisms of the diapause induction process are still poorly understood. In this study, we compared the differentially expressed miRNAs (DEmiRs) in bivoltine silkworm embryos incubated at diapause- (25 °C) and non-diapause (15 °C)-inducing temperatures during the blastokinesis (BK) and head pigmentation (HP) phases using transcriptome sequencing. There were 411 known miRNAs and 71 novel miRNAs identified during the two phases. Among those miRNAs, there were 108 and 74 DEmiRs in the BK and HP groups, respectively. By the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the predicted target genes of the DEmiRs, we found that aside from metabolism, the targets were also enriched in phototransduction-fly and insect hormone biosynthesis in the BK group and the HP group, respectively. Dual luciferase reporter assay illustrated that bmo-miR-6497-3p directly regulated Bmcycle and subsequently regulated the expression of circadian genes. These results imply that microRNAs, as vitally important regulators, respond to different temperatures and participate in the diapause induction process across species.

The urban-adapted underground mosquito, Culex pipiens form molestus, maintains exogenously influenceable circadian rhythms

Genes known to affect circadian rhythms (i.e. ‘clock genes’) also influence the photoperiodic induction of overwintering reproductive diapause in the Northern house mosquito, Culex pipiens f. pipiens. This suggests that molecular changes in one or more clock genes could contribute to the inability to diapause in a second form of this mosquito, Cx. pipiens f. molestus. Temperate populations of Cx. pipiens f. molestus inhabit underground locations generally devoid of predictable photoperiods. For this reason, there could be limited fitness consequences if the hypothesized molecular changes to its clock genes also eliminated this mosquito's ability to regulate circadian rhythms in response to photoperiod variation. Here we demonstrate that in contrast to this prediction, underground derived Cx. pipiens f. molestus retain exogenously-influenceable circadian rhythms. Nonetheless, our genetic analyses indicate that the gene Helicase domino (dom) has a nine-nucleotide, in-frame deletion specific to Cx. pipiens f. molestus. Previous work has shown that splice variants in this gene differentially influence circadian behavior in Drosophila melanogaster. We also find derived, non-synonymous single nucleotide polymorphisms (SNPs) in eight genes that may also affect circadian rhythms and/or diapause induction in Cx. pipiens f. molestus. Finally, four putative circadian genes were found to have no quantifiable expression during any examined life-stage, suggesting potential regulatory effects. Collectively, our findings indicate that the distinct, but molecularly interconnected life-history traits of diapause induction and circadian rhythms are decoupled in Cx. pipiens f. molestus and suggest this taxon may be a valuable tool for exploring exogenously influenced phenotypes in mosquitoes more broadly.

Comparisons in temperature and photoperiodic-dependent diapause induction between domestic and wild mulberry silkworms

The bivoltine strain of the domestic silkworm, Bombyx mori, has two generations per year. It shows a facultative diapause phenotype determined by environmental conditions, including photoperiod and temperature, and nutrient conditions during embryonic and larval development of the mother. However, it remains unclear how the environmental signals received during development are selectively utilized as cues to determine alternative diapause phenotypes. We performed a comparative analysis between the Kosetsu strain of B. mori and a Japanese population of the wild mulberry silkworm B. mandarina concerning the hierarchical molecular mechanisms in diapause induction. Our results showed that for the Kosetsu, temperature signals during the mother’s embryonic development predominantly affected diapause determination through the thermosensitive transient receptor potential ankyrin 1 (TRPA1) and diapause hormone (DH) signaling pathways. However, embryonic diapause in B. mandarina was photoperiod-dependent, although the DH signaling pathway and thermal sensitivity of TRPA1 were conserved within both species. Based on these findings, we hypothesize that TRPA1-activated signals are strongly linked to the signaling pathway participating in diapause induction in Kosetsu to selectively utilize the temperature information as the cue because temperature-dependent induction was replaced by photoperiodic induction in the TRPA1 knockout mutant.

Plasticity of nutrient accumulation patterns in diapausing fall webworm pupae

The accumulation of nutrients during diapause preparation is crucial because any lack of nutrition will reduce the likelihood of insects completing diapause, thereby decreasing their chances of survival and reproduction. The fall webworm, Hyphantria cunea, diapause as overwintering pupae and their diapause incidence and diapause intensity are regulated by the photoperiod. In this study, we test the hypothesis that photoperiod influences energy reserve accumulation during diapause preparation in fall webworm. We found that the body size and mass, lipid and carbohydrate content of pupae with a short photoperiod during the diapause induction phase were significantly greater than those of pupae with a relatively short photoperiod, and the efficiency of converting digested food and ingested food into body matter was greater in the short-photoperiod diapause-destined larvae than the relatively short-photoperiod diapause-destined larvae. We also observed higher lipase and amylase activities in short-photoperiod diapause-destined larvae relative to the counterparts. However, no obvious difference was found in protein and protease in the pupae with a short photoperiod during the diapause induction phase and short-photoperiod diapause-destined larvae compared with the counterparts. Therefore, we conclude that the energy reserve patterns of diapausing fall webworm pupae are plastic and that short-photoperiod diapause-destined larvae increase their energy reserves by improving their feeding efficiency and increase their lipid and carbohydrate stores by increasing the lipase and amylase activities in the midgut.

Comparisons in Temperature and Photoperiodic-dependent Diapause Induction between Domestic and Wild Mulberry Silkworms

The bivoltine strain of the domestic silkworm, Bombyx mori, has two generations per year. It shows a facultative diapause phenotype determined by environmental conditions, including photoperiod and temperature, and nutrient conditions during embryonic and larval development of the mother. However, it remains unclear how the environmental signals received during development are selectively utilized as cues to determine alternative diapause phenotypes. We performed a comparative analysis between the Kosetsu strain of B. mori and a Japanese population of the wild mulberry silkworm B. mandarina concerning the hierarchical molecular mechanisms in diapause induction. Our results showed that for the Kosetsu, temperature signals during the mother’s embryonic development predominantly affected diapause determination through the thermosensitive transient receptor potential ankyrin 1 (TRPA1) and diapause hormone (DH) signaling pathways. However, embryonic diapause in B. mandarina was photoperiod-dependent, although the DH signaling pathway and thermal sensitivity of TRPA1 were conserved within both species. Based on these findings, we hypothesize that TRPA1-activated signals are strongly linked to the signaling pathway participating in diapause induction in Kosetsu to selectively utilize the temperature information as the cue because temperature-dependent induction was replaced by photoperiodic induction in the TRPA1 knockout mutant.