Diapause intensity (DI) is a physiological trait represented by the duration of diapause under given conditions of environment. In many species, it is highly variable, probably being controlled by multiple genes and tends to form a cline in response to the latitudinal gradient of selection pressure. DI clines could be established artificially by crossing between lines of a cricket selected for different levels of DI, indicating the importance of genetic factors in the adaptive variation of DI. However, DI may be modified in response to seasonal cues both before and after the onset of diapause. Polymorphism in the intensity of prolonged diapause may split adults of a single population to emerge in different years. A unimodal distribution of DI may also result in polymodal termination of diapause, if DI variation is so large that chilling in one winter is not enough to terminate diapause for all members of a population. Bimodal termination of diapause after overwintering suggests heterogeneity in the final phase of diapause that requires high temperatures in spring. Polymodal termination of diapause subserves a bet-hedging strategy. Variability in DI thus provides insects with an important means of adaptation to their environments changing in space and time.
The cabbage armyworm, Mamestra brassicae, enters diapause in the early pupal stage. Pupal diapause is induced by rearing the larvae under short day lengths. We previously demonstrated that feeding Dopa during last larval instar induces pupal diapause even under long day lengths. In order to elucidate the mechanism by which pupal diapause is induced after experiencing short day lengths or fed Dopa under long day lengths, we analyzed gene expression in the brain of M. brassicae larvae under both of these conditions using a subtractive hybridization technique. After the secondary screen, 49 clones and 28 clones were identified as short day length or Dopa-feeding specific clones, respectively. All of these genes were sequenced and, using the base sequences of these clones, primers were synthesized. To confirm the genes enhanced specifically by these conditions, quantitative real-time PCR was carried out. This quantitative PCR analysis identified 15 and 1 clone whose expression was enhanced by the short day length conditions or Dopa-feeding, respectively. Among these clones, the gene with a high level of identity to receptor for activated protein kinase C (RACK) from Heliothis virescens is the most dramatically up-regulated under both conditions.
A 1962 bp cDNA clone of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (purH) was isolated from diapausing adults of Leptinotarsa decemlineata using RT-PCR and 3' and 5'-RACE. The probe generated from this clone hybridized to a transcript approximately 1960 bp in length on northern blots. The clone encodes for a deduced protein 594 amino acids in length with 73% identity, 83% similarity to purH from Drosophila melanogaster. Northern blot (total RNA) analysis determined that L. decemlineata purH (LdpurH) was downregulated in diapausing beetles stored at 10°C. Developmental studies revealed that LdpurH is expressed at nearly constant high levels in both nondiapausing and prediapause adults. LdpurH expression during the first 20 days of diapause is equivalent to that seen in nondiapausing beetles, after which expression decreases. Exposing 70 day old diapausing beetles to 20°C for 24 h induced an increase in expression of purH indicating that purH is regulated by temperature in diapausing/overwintering beetles.
Many stress-induced genes, including those related to the insect humoral immune response, are upregulated during diapause even in the absence of stress. We further test the relationship between stress genes and diapause in Sarcophaga crassipalpis by cloning sarcotoxin II, a member of the attacin family, and examining its expression pattern in relation to pupal diapause. Unlike several other stress-related genes, sarcotoxin II is not developmentally upregulated during diapause, but it remains fully responsive to immune challenge. Interestingly, the elevation of sarcotoxin II mRNA in response to body wall injury, but not immune challenge, is initiated more slowly and persists longer in diapausing pupae than in nondiapausing individuals.
The activities of three enzymes involved in polyol biosynthesis (aldose reductase, AR; ketose reductase, KR; and polyol dehydrogenase, PDH) were studied in adult females of the linden bug, Pyrrhocoris apterus, collected from the field during 2005/2006. While the activities of three enzymes were low in reproductive females, activities greater by one or two orders were seen in reproductively arrested females. AR and KR showed similar seasonal trends in activity. Activities were low during diapause initation and later increased and stabilized during autumnal diapause development. Further increases of AR and KR activities were seen during low temperature quiescence and finally the activities sharply decreased during vernal resumption of direct development. The activity of PDH was relatively high (but fluctuating) during diapause, then decreased in quiescent insects and almost disapeared in reproductively active females. Insects collected in February were subjected to laboratory de-acclimation (exposure to high temperatures) followed by re-acclimation (exposure to low temperatures) which resulted in loss of activity in all three enzymes and no regain. High activities of AR, KR and PDH in reproductively arrested females thus conform well with their previously observed high capacity to synthesize and accumulate polyol cryoprotectants.