Vitellogenin RNAi Halts Ovarian Growth and Diverts Reproductive Proteins and Lipids in Young Grasshoppers

Abstract

Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph.

Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers

“…Ovariectomized females still produce vitellogenin; however, there is no ovary to sequester the vitellogenin, so instead there is a 5- to 10-fold increase of vitellogenin in the hemolymph (Hatle et al 2003, 2008). This represents a substantial investment of resources to reproduction by females despite failing to produce mature eggs (Tokar et al 2014). …”

“…As a second, separate means of directly reducing reproduction, we can use RNA interference to decrease vitellogenin mRNA (Tokar et al 2014). Vitellogenin is important because it may hold a key role in life extension, in part due to its role in lipid metabolism (Brandt et al 2005).…”

“…Indeed, overexpression of a vitellogenin from another species decreased lifespan of Drosophila (Ren and Hughes 2014). The vitellogenin RNAi (VgRNAi) treatment we used was characterized in young lubber grasshoppers, and it reduced vitellogenin mRNA levels 35-fold, doubled fat body mass, and prevented ovarian development (Tokar et al 2014). Upon VgRNAi treatment vitellogenin protein was still present in the hemolymph, but it was not taken up into the ovary, and females did not produce mature eggs.…”

“…The 515 bp sequence of dsRNA used for knockdown had three 11 bp regions identical to the vitellogenin receptor from Leucophaea maderae (cockroach). As a result, this treatment may partly block transport of vitellogenin from the hemolymph into the ovary (Tokar et al 2014). Nonetheless, this VgRNAi treatment is a genetic means of reducing reproduction with all organs remaining intact, in contrast to the surgical means of reducing reproduction via ovariectomy.…”

“…Ovariectomized females accumulate high levels of non-vitellogenic protein in the hemolymph in old age (Hatle et al 2008). Knockdown of the most abundant storage protein (Hex-90) does not affect reproduction through the first clutch (Tokar et al 2014). Because of this, we chose to use knockdown of Hex-90 as a control for general, non-specific RNAi effects.…”