Molecular structure of the prothoracicotropic hormone gene in the northern house mosquito, Culex pipiens, and its expression analysis in association with diapause and blood feeding

Abstract

We cloned the gene that encodes prothoracicotropic hormone (PTTH) in the northern house mosquito, Culex pipiens, and investigated its expression profile in short-day (diapause-destined) and long-day (nondiapause-destined) individuals from the fourth-instar larval stage to 2 months of adulthood, as well as after a blood meal. The deduced C. pipiens PTTH (Cupip-PTTH) amino acid sequence contains seven cysteines with a specific spacing pattern. Sequence alignment suggests that Cupip-PTTH is 23% identical to Drosophila melanogaster PTTH, but is ≥59% identical to the PTTHs of other mosquitoes. Cupip-PTTH has structural characteristics similar to those of Bombyx mori PTTH and some vertebrate nerve growth factors with cysteine-knot motifs. PTTH transcripts exhibit a daily cycling profile during the final (fourth) larval instar, with peak abundance occurring late in the scotophase. The fourth-larval instar stage is one day longer in short-day larvae than in long-day larvae, resulting in larger larvae and adults. This additional day of larval development is associated with one extra PTTH cycle. No cycling was observed in pupae, but PTTH transcripts were slightly higher in short-day pupae than in long-day pupae throughout much of the pupal stage. PTTH expression persisted at a nearly constant level in diapausing adult females for the first month but then dropped by ∼50%, while expression decreased at the beginning of adulthood in nondiapausing females and then remained at a low level as long as the females were denied a blood meal. However, when nondiapausing females were offered a blood meal, PTTH transcripts rose approximately 7 fold in 2 h and remained elevated for 24 h. A few diapausing females (∼10%) will take a blood meal when placed in close proximity to a host, but much of the blood is ejected and such meals do not result in mature eggs. Yet, elevated PTTH mRNA expression was also observed in diapausing females that were force fed. Our results thus point to several distinctions in PTTH expression between short-day and long-day mosquitoes, but both types of females responded to a blood meal by elevating levels of PTTH mRNA.