Characterizing the relative abundance of circadian transcription factors in diapausing and nondiapausing Northern house mosquitoes.

The Northern house mosquito (Culex pipiens) is a major vector of West Nile virus. To survive harsh conditions in winter adult females of Cx. pipiens enter a state of arrested reproductive development called diapause. Diapause is triggered by the short daylengths of late summer and early fall. The methods by which Cx. pipiens measures daylength are still unknown. However, it is suspected that clock genes, which provide information on daylength, may also regulate diapause. The proteins produced by these genes often cycle in abundance throughout the day in diapausing and nondiapausing insects. Two clock genes suspected to control diapause are cycle (cyc) and Par domain protein1 (Pdp1) as they encode circadian transcription factors that may regulate genes that are involved in diapause. Using Western blotting we measured the relative protein abundance of CYC and PDP1 throughout the day in the whole bodies and the heads of Cx. pipiens reared under either long-day, diapause-averting conditions or short-day, diapause-inducing conditions. We found that in whole bodies there was no significant oscillation of CYC or PDP1 abundance in both long day and short day-reared mosquitoes. In the heads of long day-reared mosquitoes both CYC and PDP1 cycled. In contrast, only PDP1 abundance showed diel differences in abundance in the heads of short day-reared mosquitoes. These data bring us one step closer to understanding the role that CYC and PDP1 may play in regulating diapause and other biological processes.

Circadian transcription factors differentially regulate features of the adult overwintering diapause in the Northern house mosquito, Culex pipiens.

The short days of late summer and early fall are the environmental cues that most temperate insects and other animals use to predict winter's arrival. Although it is still unclear precisely how insects measure daylength, there is mounting evidence that the circadian clock regulates seasonal responses including photoperiodic diapause. Females of the Northern house mosquito, Culex pipiens, enter an adult reproductive diapause in response to short daylengths. While in this state, females divert their resources from reproduction to survival, arresting egg follicle development and increasing fat content. Here, we characterized the expression profile of two circadian transcription factors, vrille (vri) and Par domain protein 1 (Pdp1), as well as genes downstream of the clock, takeout (to) and Nocturnin (Noc) and under different seasonal conditions. We saw that while vri mRNA oscillated under both long day and short day conditions, Pdp1 expression oscillated only under long day conditions and was constitutively upregulated in diapausing females. We saw similar expression profiles for to and Noc, suggesting that PDP1 might regulate their expression or that Pdp1, to and Noc might be regulated by the same transcription factor. We suppressed vri and Pdp1 using RNA interference. dsRNA against vri provided inconsistent results, sometimes stimulating autogenous egg follicle development in both long and short day-reared females, and other times had no effect. In contrast, knocking down Pdp1 prevented short day-reared females from accumulating fat reserves, but increased expression of to and Noc. Taken together, these data suggest that the circadian transcription factors Vri and Pdp1 may independently regulate signaling pathways underlying arrested egg follicle development and fat accumulation in diapausing females of Cx. pipiens.

Endocrine regulation of non-circadian behavior of circadian genes in insect gut.

The linden bug Pyrrhocoris apterus exhibits a robust diapause response to photoperiod. Photoperiod strongly affected basal levels of circadian gene transcripts in the gut, via the neuroendocrine system. Cryptochrome 2 (cry2) mRNA level was much higher in diapause promoting short days (SD) than in reproduction promoting long days (LD), while Par Domain Protein 1 (Pdp1) mRNA level was higher in LD than in SD. The effect of photoperiod on gene expression was mediated by the neurosecretory cells of the pars intercerebralis (PI) and the juvenile hormone (JH) producing corpus allatum (CA). In LD-females, CA ablation resulted in SD-like levels of gene transcripts, while PI ablation had little effect. Conversely, in SD-females, CA ablation had only a little effect, while PI ablation resulted in LD-like levels of gene transcripts. Thus, the CA is responsible for LD-like characteristics of gene expression in reproducing females and the PI is responsible for SD-like characteristics of gene expression in diapausing females. A simultaneous ablation of both PI and CA revealed two roles of PI in SD-females: (1) inhibition of CA, and (2) weak CA-independent stimulation of cry2 mRNA. Overall, our results indicate that peripheral circadian gene expression in the gut reflects the physiological state of females (with respect to diapause or reproduction) rather than the external light-dark cycle.