RNA interference directed against ribosomal protein S3a suggests a link between this gene and arrested ovarian development during adult diapause in Culex pipiens

Abstract

Arrested ovarian development is a key characteristic of adult diapause in the mosquito Culex pipiens. In this study we propose that ribosomal protein S3a (rpS3a), a small ribosomal subunit, contributes to this shutdown. RpS3a is consistently expressed in females of C. pipiens that do not enter diapause, but in females programmed for diapause, expression of the rpS3a transcript is dramatically reduced for a brief period in early diapause (7-10 days after adult eclosion). RNA interference directed against rpS3a in nondiapausing females arrested follicle development, mimicking the diapause state. The effect of the dsRNA injection faded within 10 days, allowing the follicles to grow again, thus the suppression of rpS3a caused by RNAi did not permanently block ovarian development, implying that a brief suppression of rpS3a is not the only factor contributing to the diapause response. The arrest in development that we observed in dsRNA-injected females could be reversed with a topical application of juvenile hormone III, an endocrine trigger known to terminate diapause in this species. Though we speculate that many genes contribute to the diapause syndrome in C. pipiens, our results suggest that a shut down in the expression of rpS3a is one of the important components of this developmental response.

Figure 1

Northern blot hybridization showing expression of the mRNA encoding ribosomal protein S3a (rpS3a). (A) RpS3a was continuously expressed in nondiapausing females reared at 18°C long days. (B) In diapause mosquitoes, reared at 18°C short days, rpS3a was down-regulated in early (7–10 days) stages of diapause. (C) Further confirmation of the low expression of rpS3a on day 8 and 10 diapausing females. 28S ribosomal RNA was used as a control.

Figure 2

Northern blot hybridization confirming expression of rpS3a in the ovaries. (A) RpS3a expression was high in ovaries of nondiapausing mosquitoes (ND) 10 days after eclosion, but down-regulated in diapausing females of the same age. RpL19 was used to confirm equal loading. (B) RpS3a expression indicates suppressed expression in the ovaries of nondiapausing females injected with dsrpS3a on day 4 and examined on day 6.

Figure 3

The effect of injection of dsRNA on expression of rpS3a. (A) Mean ± SD length of primary follicles after injection of dsrpS3a (10 follicles measured per mosquito; total of 10 mosquitoes for controls and 25 mosquitoes for others. ND = nondiapause mosquitoes, D = diapause mosquitoes, d = days after adult eclosion, dai = days after injection). Ovarian development was arrested when rpS3a was suppressed by dsRNA injection. dsRNAs of rpL19 and β-gal were injected as internal controls. Bars with the same letters are not significantly different (P > 0.05). (B) Northern blot hybridization confirmed suppression of rpS3a (left) and suppression of rpL19 (right) in nondiapause mosquitoes. 28S ribosomal RNA was used as a control. (C) Primary follicle images: a. Nondiapause mosquito, 6 days, b. Nondiapause mosquito, 8 days, c. Diapause mosquito, 8 days, d. Nondiapause mosquito, 4 days + 2 days after injection of dsrpS3a, e. Nondiapause mosquito, 4 days + 4 days after injection of dsrpS3a. (Scale bar = 50 μm)

Figure 4

The effect of dsrpS3a in nondiapausing mosquitoes at different times after injection. Mean ± SD primary follicle length slightly increased with time after dsrpS3a injection due to the presumed degradation of dsRNA (10 follicles measured per mosquito; total 10 mosquitoes for controls and 25 mosquitoes for others). d = days after adult eclosion, dai = days after injection of dsrpS3a. Bars with the same letters are not significantly different (P > 0.05).

Figure 5

Juvenile hormone (JHIII) rescues the ovarian developmental arrest caused by an injection of dsrpS3a. Mean ± SD primary follicles initiated development when JHIII was applied immediately after a dsrpS3a injection (10 follicles measured per mosquito; total 10 mosquitoes for controls and 25 mosquitoes for others). ND = nondiapause mosquitoes, D = diapause mosquitoes, d = days after adult eclosion, dai = days after injection of dsrpS3a. Bars with the same letters are not significantly different (P > 0.05).