transcriptomic analysis of regulatory pathways involved in female reproductive physiology of Rhodnius prolixus under different nutritional states Jimena Leyria * , ian orchard & Angela B. Lange the triatomine Rhodnius prolixus, a vector of the etiological agent of chagas disease, has long been used as model to understand important aspects of insect physiology. Despite this history, the impact of the nutritional state on regulatory pathways associated with reproductive performance in triatomines has never been studied. the insulin-like peptide/target of rapamycin (iLp/toR) signaling pathway is typically responsible for detecting and interpreting nutrient levels. Here, we analyzed transcriptomes from the central nervous system, fat bodies and ovaries of adult females in unfed and fed conditions, with a focus on the iLp/toR signaling. the results show an up-regulation of transcripts involved in iLp/toR signaling in unfed insects. However, we demonstrate that this signaling is only activated in tissues from fed insects. Moreover, we report that foxo (forkhead box o) factor, which regulates longevity via iLp signaling, is responsible for the up-regulation of transcripts related with iLp/toR signaling in unfed insects. As a consequence, we reveal that unfed females are in a sensitized state to respond to an increase of ILP levels by rapidly activating ILP/ToR signaling. This is the first analysis that correlates gene expression and protein activation of molecules involved with iLp/toR signaling in R. prolixus females in different nutritional states.

In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia.

The blood-sucking hemipteran Rhodnius prolixus is a vector of Chagas disease, one of the most neglected tropical diseases affecting several million people, mostly in Latin America. The blood meal is an event with a high epidemiological impact since adult mated females feed several times, with each meal resulting in a bout of egg laying, and thereby the production of hundreds of offspring. By means of RNA-Sequencing (RNA-Seq) we have examined how a blood meal influences mRNA expression in the central nervous system (CNS), fat body and ovaries in order to promote egg production, focusing on tissue-specific responses under controlled nutritional conditions. We illustrate the cross talk between reproduction and a) lipids, proteins and trehalose metabolism, b) neuropeptide and neurohormonal signaling, and c) the immune system. Overall, our molecular evaluation confirms and supports previous studies and provides an invaluable molecular resource for future investigations on different tissues involved in successful reproductive events. These analyses serve as a starting point for new investigations, increasing the chances of developing novel strategies for vector population control by translational research, with less impact on the environment and more specificity for a particular organism.

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Rhodnius prolixus (the kissing bug and a major vector of Chagas disease) is an obligate blood feeder that in the case of the fifth instar consumes up to 10 times its unfed body weight in a single 20-minute feed. A post-prandial diuresis is initiated, within minutes of the start of gorging, in order to lower the mass and concentrate the nutrients of the meal. Thus, R. prolixus rapidly excretes a fluid that is high in NaCl content and hypo-osmotic to the hemolymph, thereby eliminating 50% of the volume of the blood meal within 3 hours of gorging. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in diuresis. Malpighian tubules are not innervated, and their fine control comes under the influence of the neuroendocrine system that releases amines and neuropeptides as diuretic or antidiuretic hormones. These hormones act upon the Malpighian tubules via a variety of G protein-coupled receptors linked to second messenger systems that influence ion transporters and aquaporins; thereby regulating fluid secretion. Much has been discovered about the control of diuresis in R. prolixus, and other model insects, using classical endocrinological studies. The post-genomic era, however, has brought new insights, identifying novel diuretic and antidiuretic hormone-signaling pathways whilst also validating many of the classical discoveries. This paper will focus on recent discoveries into the neuroendocrine control of the rapid post-prandial diuresis in R. prolixus, in order to emphasize new insights from a transcriptome analysis of Malpighian tubules taken from unfed and fed bugs.

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