Suppression of allatotropin simulates reproductive diapause in the mosquito Culex pipiens

Abstract

The cessation of juvenile hormone (JH) production is a key endocrine event that halts ovarian development and hence initiates diapause in females of the mosquito, Culex pipiens. The shutdown in endocrine activity of the corpora allata (CA), the source of JH, was manifested in the smaller size of CA in females reared under short daylengths (diapause) compared to those reared under long daylengths (nondiapause), as well as in low expression of the mRNA encoding allatotropin, the neuropeptide that promotes JH biosynthesis in the CA. Genes encoding both allatotropin and allatostatin were identified in C. pipiens, but only expression levels of allatotropin differed in the two types of females. Knockdown of allatotropin mRNA using RNA interference in females programmed for nondiapause resulted in a cessation of ovarian development akin to diapause. This arrest in development could be reversed with an application of JH. Our results thus suggest that suppression of allatotropin is a critical link in regulating the shutdown of the CA during diapause.

Keywords: Allatostatin; Allatotropin; Culex pipiens; Diapause; Juvenile hormone.

Fig. 1

Morphological distinctions in ovarian follicles and corpora allata between nondiapausing (ND) and diapausing (D) females of Culex pipiens. Ovarian follicles from (A) ND females and (B) D females one week after adult eclosion show the cessation of development in D females. Corpora allata from (C) ND females and (D) D females dissected one week after adult eclosion, showing that the CA are larger and exhibit increased cell counts in ND females (scale bar, 50 Hm). (E) Mean ± s.d. length of corpora allata in ND and D females dissected one week after adult eclosion, n = 11–12 females.

Fig. 2

Quantitative real-time PCR showing expression of AT and AS-C in C pipiens females at 1, 3, 6 and 10 days after adult eclosion. ND (■) = programmed by long daylength for nondiapause, D (◊) = programmed by short daylength for diapause. Both groups were maintained at 18°C. Bars (mean ± s.d.) with asterisk(s) indicate significant differences at *P=0.05 and **P=0.001, T-test. Ribosomal protein large subunit (RpL19) was used to confirm equal loading. Data are expressed as mean transcript level ± s.d.; n=3 groups of 10 individuals each.

Fig. 3

RNA interference efficiency of dsAT in C pipiens. (A) AT transcript levels in females injected with dsAT (white bars) were compared with the dsβ-gal controls (black bars). Expression levels were measured by qRT-PCR at 3 and 6 days after RNAi injection and normalized using a Ribosomal protein large subunit 19 (RpL19) as a loading control. (B) RpL19 transcript levels in females injected with dsAT (white bars) were compared with the dsβ-gal controls (black bars). Expression levels were not significantly different between treated and control groups at P=0.05, T-test.

Fig. 4

Juvenile hormone rescue of ovarian development in AT knocked-down mosquitoes that were programmed by long daylength for nondiapause. Mean ± s.d. length of ovarian follicles in dsAT injected ND females that subsequently received graded doses of JH III. n = 15–29 females for each treatment. Bars with the same letters were not significantly different at P=0.05, ANOVA.