Effect Of Diapause-inducing Temperature And Photoperiod On The Expression Of Diapause-related Genes In The Silkworm, Bombyx Mori

Bombyx mori is the model insect of Lepioloptera with diapausing during its egg stage. Diapause inducements are due to temperature and photoperiod in the incubation process of parental eggs. However, whether or not eggs at progeny enter into diapause depends on temperature after oviposition. This paper investigated the expression of diapause-related genes in Bombyx mori using the classic diapause-inducing incubation conditions, which contained diapause hormone gene, sorbitol dehydrogenase gene, time interval measuring enzyme-esterase A4 gene, superoxide dismutase and catalase genes. The major results are as follows:1 Diapause-related genes had sex specific and tissue specific expression characteristics in Bombyx moriGene microarray analysis displayed that five genes, containing dh, sdh, ea4, sod and cat, had sex specific and tissue specific expression characteristics during postembryonic stage. dh gene expression level in multiple tissues of female is significantly lower than male. sdh gene expression abundance in male is higher than female, but the abundance in testis is obviously lower than ovary. Expression analysis of ea4 showed that it had higher transcription level in head than in posterior silk gland. sod and cat genes also had the tissue specificity, moreover, cat gene significantly had sex specific expression, its expression abundances in male are higher than female except gonads.Express Sequence Tag (EST) profile showed that ea4 shows stage-specific and tissue-specific expression during postembryonic stages, with high ea4 mRNA in pheromone gland, but with low levels in gonads. Expression was relatively high in the adult stage, and the expression was up-regulated in the microbe-infected fat body of fifth-instar larvae. 2 The temperature and photoperiod inducing diapause of offspring eggs up-regulated the expression level of dh geneEggs at progeny are completely diapause when parental eggs are under constant illumination at 25℃(25LL) incubation, and they are completely non-diapause under constant darkness at 15℃(15DD). The dh mRNA of eggs in 25LL stayed at a high level, but it was very low in 15LL. Under 25LL or 20LD, the dh level of parental eggs up-regulated in the late incubation stage, which indicated that B. mori would be sensitive to the external illumination and temperature after embryonic nervous developed. However, the mRNA level of dh was not oscillated with the periodic oscillation of incubation photoperiod and temperature. The dh expression level rose at 25℃during postembryonic developmental stage. In conclusion, the incubation temperature and photoperiod of parental eggs affected the diapause of offspring eggs, but dh was not the direct gene regulated by the zeitgebers.3 The expression level of sdh gene of the whole generation down-regulated when parental eggs were under high temperatureEggs incubated under high temperature and constant illumination (25LL), the expression level of sdh gene would decrease. In 20LD, diapause-inducing temperature and photoperiod directly affected the expression of sdh gene in parental eggs but it did not have the obvious rhythmicity. The expression level of sdh gene in 25LL was lower than 20LD from larva to pupa. And sdh mRNA level was lower in the offspring eggs at 25℃than at 15℃, which indicated that as offspring eggs went into the diapausing stage, the transcription level of sdh subsequently fell.4 The effect of photoperiod on the expression of sdh gene was different between parental eggs and offspring eggsWhen parental eggs were incubated under the low temperature and constant illumination, the expression level of sdh gene in this generation was significantly down-regulated, however, its level of offspring eggs was up-regulated. This indicated that the constant illumination played a reverse role beween parental eggs and offspring eggs.5 The TIME-ATPase activity did not depend on the up-regulated expression level of ea4 gene The expression level of Bmea4 gene was significantly different among eggs incubated in 25LL, 15DD, and 20LD, respectively. However, they were not oscillated with the periodic oscillation of incubation photoperiod and temperature. The expression level of ea4 is different during postembryonic developmental stage, no matter development stages or tissues (organs), and the difference also existed among different diapause-inducing conditions. So it might be not related to diapause inducing conditions but related to free radical metabolism.The expression of ea4 in the processes of diapause incidence and termination showed that eggs from virgin moths (0h eggs) exhibited a high level of ea4 mRNA, but 0.5h-12h after oviposition, the ea4 mRNA level decreased steeply, from 24 to 72 hours after oviposition, and after age-72h, the levels of ea4 mRNA were consistent among DDE, DTE, and NDE during the above two periods. The EA4-ATPase activity in eggs was transiently elevated at 1.5h after common acid-treatment, while the typical peak presented on day-13 at 5℃from age-48h, but ea4 mRNA level did not increase at the same time.6 The research on Haea4 gene from Helicoverpa armigeraThe ORF of Haea4 which codes 199 amino acids is 799 bp. Protein analysis show that the molecular weight of Haea4 is 18768.88(18.8 kD), and the isoelectric point is 5.45. HaEA4 shows the significant signal peptide sequence which indicates that it is a secretory protein. N-glycosylation site, Protein kinase C phosphorylation site, Tyrosine kinase II phosphorylation site, N-myristoylation site, Casein kinase phosphorylation site, Cu/Zn SOD core domain are identified in HaEA4. The protein homology analysis reveals that HaEA4 is of high conservative in different species. Analysis of molecular evolution indicates that the evolution distances between HaEA4/BmEA4/BmaEA4 and Cu/Zn SOD are far. Secondary structure prognosticates results reveal that the characteristics of HaEA4 are much similar with BmEA4. Semi-quantitative RT-PCR showed that Haea4 was highly expressed in midgut, integument and fat body, lowly expressed in head, gonad and silk gonad. It was postulated that this Haea4 gene might be involved in the detoxification of xenobiotics. Genetic distance and homology analysis showed that the TIME-EA4 genetic distances of Helicoverpa armigera and Bombyx mori, Helicoverpa armigera and Bombyx mandarina were 0.536 and 0.548, respectively, in addition, the sequence identities of both them are 44%. 7 The effect of diapause-inducing incubation temperature and photoperiod on the expression of sod and cat genesWhen eggs were incubated in 25LL, their sod mRNA would keep at a high level, but the transcription level was rapidly down-regulated at EAT1800℃.h and 3600℃.h. The expression of sod gene in 20LD was significantly higher than in 15DD, this phenomenon was more obvious when the embryonic nervous had fully grown in the later incubation stage. The expression of cat gene had no obvious differences when eggs were incubated in 25LL, 20LD or 15DD but it was notable that the mRNA level of cat all increased in the late incubation stage, which was in accordance with the fact that the eggs were more sensitive to diapause-inducing incubation temperature and photoperiod of this time.After artificial hatching treatment with hydrochloric acid at 24h after oviposition, the sod mRNA in diapause-terminated eggs (DTE) was much higher than diapause-destined eggs (DDE). From 24 to 192 hours after oviposition, eggs at progeny went into the diapause stage, the transcription level of sod subsequently decreased. After we examined the common effect of illumination and temperature on the sod gene of progeny eggs, it was observed that the sod mRNA increased under constant darkness at 15℃. The cat mRNA level of DTE was lower than DDE from 24 to 48 hours after oviposition, but after age-72h, the mRNA increased, which indicated that the expression of cat might be inhibited by hydrochloric acid at the early activation. We also investigated the common effect of illumination and temperature on the cat gene of progeny eggs and found that the expression level of cat in 20LD was lower than in 25LL and 15DD.