Silencing of ATP synthase β induces female sterility in a leafhopper phytoplasma vector

Abstract

The leafhopper Euscelidius variegatus Kirschbaum (Hemiptera: Cicadellidae) is a natural vector of the 'Candidatus Phytoplasma asteris' and a laboratory vector of the phytoplasma associated with grapevine Flavescence dorée. Previous studies indicated that RNA interference (RNAi) by dsRNA injection efficiently works in E. variegatus and that silencing of ATP synthase β increases mortality and impairs phytoplasma multiplication. Here, a nearly complete female sterility was observed when ATP synthase β was silenced. The sterility was associated with the absence of mature eggs in the ovaries. On the contrary, male genitalia morphology and sperm motility were similar to those observed in E. variegatus control specimens, treated with dsRNAs targeting green fluorescent protein (dsGFP). An over-expression of hexamerin (amino acid storage protein) and cathepsin L (lysosome proteinase) was observed at transcript and protein level in dsATPinjected females in comparison with dsGFP-injected insects, in both whole body and dissected ovary samples. Conversely, the expression of other genes known to be involved in oocyte development, namely vitellogenin, perilipin and digestive cystein protein, was not altered in dsATP-injected E. variegatus insects in comparison with dsGFP ones. Possible roles of ATP synthase β, hexamerin and cathepsin L in oocyte and egg development are discussed. Insecticide treatments against vectors are the main strategies to counteract phytoplasma diseases, with negative impact on environment and public health. RNAi is a promising sustainable approach against insect vectors and ATP synthase β could be a valid target gene, as its silencing, besides increasing mortality and reducing phytoplasma multiplication, induces female sterility.

Silencing of ATP Synthase β Impairs Egg Development in the Leafhopper Scaphoideus titanus, Vector of the Phytoplasma Associated with Grapevine Flavescence Dorée

“…As previously observed in E. variegatus [ 31 ], upon silencing of the ATP synthase β gene, the reproduction of the phytoplasma vector S. titanus might be impaired due to the inability to develop mature eggs in ovaries. Silenced females also showed high levels of hexamerin together with low levels of ATP synthase β gene product.…”

“…Previous results showed that in E. variegatus , silencing of ATP synthase β induces female sterility by inhibiting oocyte development concurrently with an overexpression of hexamerin and cathepsin L together with a reduction in vitellogenin in silenced females [ 31 ]. Here, the same effects were analyzed in S. titanus .…”

“…Silenced S. titanus females showed a high level of cathepsin L protein in ovaries and a low level of corresponding transcript in the remaining parts of the body after ovary dissection. Previous results from E. variegatus showed an over-expression of this gene in the ovaries of silenced females both at the transcript and protein levels [ 31 ]. Cathepsins are lysosomal proteases involved in several processes such as development, apoptosis, and immunity of arthropods [ 53 , 54 ].…”

“…Previous results demonstrated that silencing of ATP synthase β induces female sterility in E. variegatus [ 31 ]. To evaluate the phenotypic effects on egg development before deposition, ovaries were collected from injected females of S. titanus and E. variegatus , caged soon after the injection with untreated males of corresponding species.…”

“…The present work describes the RNAi machinery of S. titanus , sets up optimal parameters to deliver dsRNAs to this species by injection, determines its efficiency at decreasing both transcript and protein levels and evaluates its specificity using different constructs and E. variegatus as closely phylogenetic related non-target species. ATP synthase β was selected as the target gene as its silencing in E. variegatus decreases survival rate [ 26 ], impairs phytoplasma multiplication [ 30 ], and induces female sterility by inhibiting oocyte development [ 31 ]. Here, we investigated whether a similar effect on egg development also occurs in S. titanus females.…”