TOR as a Regulatory Target in Rhipicephalus microplus Embryogenesis.

Camila Waltero,Carlos Logullo,Rodrigo Nunes-Da-Fonseca,Itabajara Da Silva Vaz,Leonardo Araujo De Abreu,Thayná Alonso

doi:10.3389/fphys.2019.00965

Abstract

Embryogenesis is a metabolically intensive process carried out under tightly controlled conditions. The insulin signaling pathway regulates glucose homeostasis and is essential for reproduction in metazoan model species. Three key targets are part of this signaling pathway: protein kinase B (PKB, or AKT), glycogen synthase kinase 3 (GSK-3), and target of rapamycin (TOR). While the role of AKT and GSK-3 has been investigated during tick embryonic development, the role of TOR remains unknown. In this study, TOR and two other downstream effectors, namely S6 kinase (S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), were investigated in in vitro studies using the tick embryonic cell line BME26. First, we show that exogenous insulin can stimulate TOR transcription. Second, TOR chemical inhibition led to a decrease in BME26 cell viability, loss of membrane integrity, and downregulation of S6K and 4E-BP1 transcription. Conversely, treating BME26 cells with chemical inhibitors of AKT or GSK-3 did not affect S6K and 4E-BP1 transcription, showing that TOR is specifically required to activate its downstream targets. To address the role of TOR in tick reproduction, in vivo studies were performed. Analysis of relative transcription during different stages of tick embryonic development showed different levels of transcription for TOR, and a maternal deposition of S6K and 4E-BP1 transcripts. Injection of TOR double-stranded RNA (dsRNA) into partially fed females led to a slight delay in oviposition, an atypical egg external morphology, decreased vitellin content in eggs, and decreased larval hatching. Taken together, our data show that the TOR signaling pathway is important for tick reproduction, that TOR acts as a regulatory target in Rhipicephalus microplus embryogenesis and represents a promising target for the development of compounds for tick control.

Highlights

The cattle tick, Rhipicephalus microplus, is an obligate hematophagous ectoparasite of veterinary and economical relevance in tropical and subtropical regions, due to its role as animal disease vector for Babesia spp. and Anaplasma spp., and its impact on livestock production (Grisi et al, 2002; Guerrero et al, 2012; Parizi et al, 2012)
This paper demonstrates that the role of target of rapamycin (TOR) is conserved in ticks and fundamental in the embryogenesis of the cattle tick R. microplus
In order to investigate components of TSP in the cattle tick R. microplus, we sequenced and analyzed either partial or complete nucleotide sequences coding for RmTOR, RmS6K and Rm4E-BP1

Summary

Introduction

The cattle tick, Rhipicephalus microplus, is an obligate hematophagous ectoparasite of veterinary and economical relevance in tropical and subtropical regions, due to its role as animal disease vector for Babesia spp. and Anaplasma spp., and its impact on livestock production (Grisi et al, 2002; Guerrero et al, 2012; Parizi et al, 2012). Insulin signaling pathway (ISP) and its components, phosphatidylinositol 3-OH kinase (PI3K), protein kinase B (PKB or AKT) and glycogen synthase kinase 3 (GSK-3), play important roles in metabolic control (Supplementary Figure S1) (de Abreu et al, 2009, 2013; Fabres et al, 2010). We demonstrated that R. microplus embryonic cell line, BME26, harbors an insulin-responsive machinery, and that AKT/GSK3 axis integrates glycogen metabolism and cell survival (de Abreu et al, 2009, 2013). We observed that GSK-3, a key enzyme in glycogen metabolism, is required for cell viability, oviposition, and larval hatching in the cattle tick (Fabres et al, 2010; de Abreu et al, 2013) (Supplementary Figure S1). In order to improve the current knowledge of tick physiology, new targets must be identified and studied

Methods

Results

Conclusion