Catalase and superoxide dismutase-2 enhance survival and protect ovaries during overwintering diapause in the mosquito Culex pipiens

Abstract

Lifespan extension and stress resistance are two important features of diapause that are essential for successful overwintering. We present several lines of evidence suggesting that genes encoding two antioxidant enzymes, catalase and superoxide dismutase-2, are critical in generating these characteristics during diapause in overwintering adults of the mosquito Culex pipiens. Expression of both catalase and sod-2 was dramatically higher in young diapausing females than in their nondiapausing counterparts at the same age. Suppression of catalase, but not sod-2, resulted in increased damage to the ovaries, as evidenced by signs of apoptosis in ovarian follicle cells. Adult survival time was shortened when levels of either catalase or sod-2 were suppressed using RNAi. Together these results imply that these two antioxidants are particularly important in promoting survival in diapausing females, while elevation of catalase also contributes to protection of the ovaries. In addition, RNAi directed against forkhead transcription factor (foxo), a gene thought to be upstream of the genes encoding these antioxidants, resulted in suppression of both catalase and sod-2. The linkage with FOXO suggests that the genes encoding these two antioxidants are components of an important gene network regulated by this transcription factor.

Fig. 1

Quantitative real-time PCR showing expression of catalase, sod-1, -2, -3 and gpx in C. pipiens females, 3 days and 1 week after adult eclosion. D (black) = programmed by short daylength for diapause, ND (white) = programmed by long daylength for nondiapause. Both groups were maintained at 18°C. Bars (mean ± s.e., n=3) with asterisks (*) indicate significant differences at P<0.05, t-test.

Fig. 2

RNAi targeted against foxo also suppresses expression of catalase and sod-2 genes in C. pipiens females, 1 and 3 days after adult eclosion. Catalase and sod-2 transcript levels in females injected with dsFOXO (gray bars) were compared with the double-strand RNA of β-galactosidase (dsβ-gal) controls (white bars). Bars (mean ± s.e., n=3) with asterisks (*) indicate significant differences at P < 0.05, t-test

Fig. 3

RNA interference efficiency in catalase and sod-2 knocked-down mosquitoes that were programmed by short daylength for diapause. Catalase and sod-2 transcript levels in females injected with dsCatalase (gray bars) or dsSOD-2 (black bars) were compared with the dsβ-gal controls (white bars). Expression levels were measured by qRT-PCR 1 week after RNAi injection. Ribosomal protein large subunit 19 (rpl19) was used to confirm equal loading. Bars (mean ± s.e., n=3) with asterisks (*) indicate significant differences at P < 0.05, t-test.

Fig. 4

Primary follicles from diapausing females, prepared with neutral red and acridine orange dyes 7 days after eclosion under white light (A) and GFP filter (B). Primary follicles from double-strand RNA of catalase (dsCatalase) under white light (C) and GFP filter (D). These follicles were in the process of apoptosis. Scale bar = 50 μm.

Fig. 5

The relative percentages (mean ± s.e., n=4) of surviving mosquitoes after injection of dsRNA targeting against β-gal (control, ds-β-gal), catalase (dsCatalase) and sod-2 (dsSOD-2). (*) indicate significant differences at P = 0.05, t-test. N = 4 groups of 20 individuals for each data point. White boxes = 1 days after adult dsRNA injection (1 d.p.i), Gray boxes = 5 days (5 d.p.i), and Black boxes = 10 days (10 d.p.i.).