Lipids in Insect Reproduction: Where, How, and Why.

Modern insects have inhabited the earth for hundreds of millions of years, and part of their successful adaptation lies in their many reproductive strategies. Insect reproduction is linked to a high metabolic rate that provides viable eggs in a relatively short time. In this context, an accurate interplay between the endocrine system and the nutrients synthetized and metabolized is essential to produce healthy offspring. Lipids guarantee the metabolic energy needed for egg formation and represent the main energy source consumed during embryogenesis. Lipids availability is tightly regulated by a complex network of endocrine signals primarily controlled by the central nervous system (CNS) and associated endocrine glands, the corpora allata (CA) and corpora cardiaca (CC). This endocrine axis provides hormones and neuropeptides that significatively affect tissues closely involved in successful reproduction: the fat body, which is the metabolic center supplying the lipid resources and energy demanded in egg formation, and the ovaries, where the developing oocytes recruit lipids that will be used for optimal embryogenesis. The post-genomic era and the availability of modern experimental approaches have advanced our understanding of many processes involved in lipid homeostasis; therefore, it is crucial to integrate the findings of recent years into the knowledge already acquired in the last decades. The present chapter is devoted to reviewing major recent contributions made in elucidating the impact of the CNS/CA/CC-fat body-ovary axis on lipid metabolism in the context of insect reproduction, highlighting areas of fruitful research.

Topical delivery of dsRNA in two hemipteran species: Evaluation of RNAi specificity and non-target effects.

Double-stranded (ds) RNA-based technologies could provide novel and potential tool for pest management with efficiency and specificity of action. However, before applying this technique in the field, it is necessary to identify effective delivery methods and evaluate the non-target effects that may occur. In this article, we evaluated the effectiveness of dsRNA by topical delivery on a species of great agricultural interest, Halyomorpha halys. The specificity of action of the dsRNA was also investigated in Rhodnius prolixus, an insect phylogenetically close to H. halys. Of the three investigated genes (putative ATPase N2B, ATPase, serine/threonine-protein phosphatase PP1-ß catalytic subunit, PP1, and IAP repeat-containing protein 7-B-like, IAP), IAP and ATPase were able to induce higher mortality in H. halys nymphs compared to the control, with specific concentrations for each gene targeted. However, when the same RNAs were topically delivered to both R. prolixus 2nd and 3rd instar nymphs, no gene silencing and mortality were observed. For this reason, to assess dsRNA application-mediated non-target effects, we injected both H. halys and R. prolixus specific dsRNA in R. prolixus 5th instar nymphs. When the dsRNA targeting H. halys IAP was microinjected into R. prolixus 5th instar nymphs, no mortality was observed, suggesting a strong RNAi specificity. Together, these data suggest that the topical delivery could be suitable for the dsRNA to control H. halys population. Furthermore, its specificity of action would allow treatments towards single harmful species with limited non-target effects.

The hormonal and neural control of egg production in the historically important model insect, Rhodnius prolixus: A review, with new insights in this post-genomic era.

Rhodnius prolixus, the blood gorging kissing bug, is a model insect, extensively used by Sir Vincent Wigglesworth and others, upon which the foundations of insect physiology, endocrinology, and development are built. It is also medically important, being a principal vector of Trypanosoma cruzi, the causative agent of Chagas disease in humans. The blood meal stimulates and enables egg production, and since an adult mated female can take several blood meals, each female can produce hundreds of offspring. Understanding the reproductive biology of R. prolixus is therefore of some critical importance for controlling the transmission of Chagas disease. The R. prolixus genome is available and so the post-genomic era has arrived for this historic model insect. This review focuses on the female reproductive system and coordination over the production of eggs, emphasizing the classical (neuro)endocrinological studies that led to a model describing inputs from feeding and mating, and the neural control of egg-laying. We then review recent insights brought about by molecular analyses, including transcriptomics, that confirm, support, and considerably extends this model. We conclude this review with an updated model describing the events leading to full expression of egg production, and also provide a consideration of questions for future exploration and experimentation.

Impact of JH Signaling on Reproductive Physiology of the Classical Insect Model, Rhodnius prolixus.

In adult females of several insect species, juvenile hormones (JHs) act as gonadotrophic hormones, regulating egg production. JH binds to its nuclear receptor, Methoprene tolerant (Met), triggering its dimerization with the protein Taiman (Tai). The resulting active complex induces transcription of JH response genes, such as Krüppel homolog 1 (Kr-h1). In this study we report for the first time the participation of the isoform JH III skipped bisepoxide (JHSB3) and its signaling pathway in the reproductive fitness of the classical insect model Rhodnius prolixus. The topical application of synthetic JHSB3 increases transcript and protein expression of yolk protein precursors (YPPs), mainly by the fat body but also by the ovaries, the second source of YPPs. These results are also confirmed by ex vivo assays. In contrast, when the JH signaling cascade is impaired via RNA interference by downregulating RhoprMet and RhoprTai mRNA, egg production is inhibited. Although RhoprKr-h1 transcript expression is highly dependent on JHSB3 signaling, it is not involved in egg production but rather in successful hatching. This research contributes missing pieces of JH action in the insect model in which JH was first postulated almost 100 years ago.

Crosstalk between Nutrition, Insulin, Juvenile Hormone, and Ecdysteroid Signaling in the Classical Insect Model, Rhodnius prolixus.

The rigorous balance of endocrine signals that control insect reproductive physiology is crucial for the success of egg production. Rhodnius prolixus, a blood-feeding insect and main vector of Chagas disease, has been used over the last century as a model to unravel aspects of insect metabolism and physiology. Our recent work has shown that nutrition, insulin signaling, and two main types of insect lipophilic hormones, juvenile hormone (JH) and ecdysteroids, are essential for successful reproduction in R. prolixus; however, the interplay behind these endocrine signals has not been established. We used a combination of hormone treatments, gene expression analyses, hormone measurements, and ex vivo experiments using the corpus allatum or the ovary, to investigate how the interaction of these endocrine signals might define the hormone environment for egg production. The results show that after a blood meal, circulating JH levels increase, a process mainly driven through insulin and allatoregulatory neuropeptides. In turn, JH feeds back to provide some control over its own biosynthesis by regulating the expression of critical biosynthetic enzymes in the corpus allatum. Interestingly, insulin also stimulates the synthesis and release of ecdysteroids from the ovary. This study highlights the complex network of endocrine signals that, together, coordinate a successful reproductive cycle.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

Ovarian nutritional resources during the reproductive cycle of the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae): focus on lipid metabolism.

In this study, we have analyzed the changes of the ovarian nutritional resources in Dipetalogaster maxima at representative days of the reproductive cycle: previtellogenesis, vitellogenesis, as well as fasting-induced early and late atresia. As expected, the amounts of ovarian lipids, proteins, and glycogen increased significantly from previtellogenesis to vitellogenesis and then, diminished during atresia. However, lipids and protein stores found at the atretic stages were higher in comparison to those registered at previtellogenesis. Specific lipid staining of ovarian tissue sections evidenced remarkable changes in the shape, size, and distribution of lipid droplets throughout the reproductive cycle. The role of lipophorin (Lp) as a yolk protein precursor was analyzed by co-injecting Lp-OG (where OG is Oregon Green) and Lp-DiI (where DiI is 1,10-dioctadecyl-3,3,30,30-tetramethylindocarbocyanine) to follow the entire particle, demonstrating that both probes colocalized mainly in the yolk bodies of vitellogenic oocytes. Immunofluorescence assays also showed that Lp was associated to yolk bodies, supporting its endocytic pathway during vitellogenesis. The involvement of Lp in lipid delivery to oocytes was investigated in vivo by co-injecting fluorescent probes to follow the fate of the entire particle (Lp-DiI) and its lipid cargo (Lp-Bodipy-FA). Lp-DiI was readily incorporated by vitellogenic oocytes and no lipoprotein uptake was observed in terminal follicles of ovaries at atretic stages. Bodipy-FA was promptly transferred to vitellogenic oocytes and, to a much lesser extent, to previtellogenic follicles and to oocytes of ovarian tissue at atretic stages. Colocalization of Lp-DiI and Lp-Bodipy-FA inside yolk bodies indicated the relevance of Lp in the buildup of lipid and protein oocyte stores during vitellogenesis.

Endocrine factors modulating vitellogenesis and oogenesis in insects: An update.

The endocrine system plays a pivotal role in shaping the mechanisms that ensure successful reproduction. With over a million known insect species, understanding the endocrine control of reproduction has become increasingly complex. Some of the key players include the classic insect lipid hormones juvenile hormone (JH) and ecdysteroids, and neuropeptides such as insulin-like peptides (ILPs). Individual endocrine factors not only modulate their own target tissue but also play crucial roles in crosstalk among themselves, ensuring successful vitellogenesis and oogenesis. Recent advances in omics, gene silencing, and genome editing approaches have accelerated research, offering both fundamental insights and practical applications for studying in-depth endocrine signaling pathways. This review provides an updated and integrated view of endocrine factors modulating vitellogenesis and oogenesis in insect females.

Octopamine is required for successful reproduction in the classical insect model, Rhodnius prolixus.

In insects, biogenic amines function as neurotransmitters, neuromodulators, and neurohormones, influencing various behaviors, including those related to reproduction such as response to sex pheromones, oogenesis, oviposition, courtship, and mating. Octopamine (OA), an analog of the vertebrate norepinephrine, is synthesized from the biogenic amine tyramine by the enzyme tyramine ß-hydroxylase (TßH). Here, we investigate the mechanisms and target genes underlying the role of OA in successful reproduction in females of Rhodnius prolixus, a vector of Chagas disease, by downregulating TßH mRNA expression (thereby reducing OA content) using RNA interference (RNAi), and in vivo and ex vivo application of OA. Injection of females with dsTßH impairs successful reproduction at least in part, by decreasing the transcript expression of enzymes involved in juvenile hormone biosynthesis, the primary hormone for oogenesis in R. prolixus, thereby interfering with oogenesis, ovulation and oviposition. This study offers valuable insights into the involvement of OA for successful reproduction in R. prolixus females. Understanding the reproductive biology of R. prolixus is crucial in a medical context for controlling the spread of the disease.

Functional characterization of the kinin receptor in the Chagas disease vector Rhodnius prolixus; activity of native kinins and potent biostable Aib-containing insect kinin analogs.

The causative agent for Chagas disease, Trypanosoma cruzi, is transmitted to a human host in the urine/feces of the kissing bug, Rhodnius prolixus, following blood feeding. Kinins are important chemical messengers in the overall control of blood feeding physiology in R. prolixus, including hindgut contractions and excretion. Thus, disruption in kinin signaling would have damaging consequences to the insect but also interfere with the transmission of Chagas Disease. Here, a heterologous functional receptor assay was used to confirm the validity of the previously cloned putative kinin G-protein-coupled receptor, RhoprKR, in Rhodnius prolixus. Three native R. prolixus kinins were chosen for analysis; two possessing the typical kinin WGamide C-terminal motif and one that possesses an atypical C-terminal WAamide. All three are potent (EC50 values in the nM range), with high efficacy, on CHO-K1-aeq cells expressing the RhoprKR, thereby confirming ligand binding. Members of three other R. prolixus peptide families, which are also myotropins (tachykinins, pyrokinins and sulfakinins) elicited little or no response. In addition, this heterologous receptor assay was used to test characteristics of kinin mimetics previously tested on tick and mosquito kinin receptors. Five α-aminoisobutyric acid (Aib) containing analogs were tested, and four found to have considerably higher potencies than the native kinins, with EC50 values in the pM range. Interestingly, adding Aib to the atypical WAamide kinin improves its EC50 value from 2 nM to 39 pM. Biostable kinin analogs may prove useful leads for novel pest control strategies. Since T. cruzi is transmitted to a human host in the urine/feces after blood feeding, disruption in kinin signaling would also interfere with the transmission of Chagas Disease.

The Male Reproductive System of the Kissing Bug, Rhodnius prolixus Stål, 1859 (Hemiptera: Reduviidae: Triatominae): Arrangements of the Muscles and the Myoactivity of the Selected Neuropeptides.

The gross anatomy of the male reproductive structures and their associated musculature are described in the blood-gorging vector of Chagas disease, Rhodnius prolixus. The male reproductive system is composed of muscular tissues each performing contractions that aid in the movement of sperm out of the testis into the vas deferens, seminal vesicle and then into the ejaculatory duct, along with proteins and lipids from the transparent and opaque accessory glands. Phalloidin staining shows the various patterns of muscle fiber layers, from thin circular to more complex crisscross patterns, implying subtle differences in the form of the contractions and movement of each of the structures, allowing for waves of contractions or twisting patterns. The transcripts for the receptors for proctolin, myosuppressin (RhoprMS) and for the extended FMRFamides are expressed in the various regions of the reproductive system, and the nerve processes extending over the reproductive structures are positive for FMRFamide-like immunoreactivity, as are neurosecretory cells lying on the nerves. Proctolin and AKDNFIRFamide are strong stimulators for the frequency of the contractions, and RhoprMS can inhibit the proctolin-induced contractions. Taken together, this work implicates these two families of peptides in coordinating the male reproductive structures for the successful transfer of sperm and the associated accessory gland fluid to the female during copulation.

Tyraminergic control of vitellogenin production and release in the blood-feeding insect, Rhodnius prolixus.

In insects, the biogenic amine tyramine (TA) has been shown to control several physiological processes. Recently, the involvement of the type 1 tyramine receptor (TAR1) in reproductive processes has been demonstrated in different insects. Here, we investigate the putative role of Rhodnius prolixus TAR1 (RpTAR1) in reproduction in female R. prolixus. RpTAR1 transcript was highly expressed in tissues associated with egg development. Moreover, after a blood meal, which is the stimulus for full egg development, RpTAR1 transcript was upregulated in the ovaries and in the fat body. After RNAi-mediated RpTAR1 knockdown, an ovarian phenotype characterized by the absence or reduction of egg production was observed. Furthermore, protein and Vg accumulation in the fat body was observed, suggesting an impairment in protein release from the fat body into the hemolymph. However, even though fewer eggs were produced and laid, there was no difference in hatching ratio of those laid, in comparison to the controls, indicating that the overall low protein uptake by the ovaries did not influence the viability of individual eggs produced. Interestingly, the eggs from dsTAR1-treated insects appeared more red, indicating a higher content of RHBP compared to the control. A higher colocalization between Vg and Rab11, a marker for the recycling endosome pathway, was observed after dsTAR1 injection, suggesting that a more active lysosome degradation pathway in response to the Vg accumulation may occur. In addition to the Vg accumulation in the fat body, dsTAR1 treatment altered JH pathway. However, it remains to be elucidated whether this event is either directly related to the RpTAR1 downregulation or for a consequence to the Vg accumulation. Lastly, the RpTAR1 action on Vg synthesis and release in the fat body was monitored in the presence or absence of yohimbine, the antagonist of TAR1, in an ex-vivo experiment. Yohimbine antagonises the TAR1 stimulated release of Vg. These results provide critical information concerning the role of TAR1 in Vg synthesis and release in R. prolixus. Furthermore, this work opens the way for further investigation into innovative methods for controlling R. prolixus.

Malpighian tubules of Rhodnius prolixus: More than post-prandial diuresis.

Rhodnius prolixus, a major vector of Chagas disease, may be considered the model upon which the foundations of insect physiology and biochemistry were built. It is an obligate blood feeder in which the blood meal triggers growth, development and reproduction. The blood meal also triggers a post-prandial diuresis to maintain osmotic homeostasis. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in this diuresis, and much has been learned about diuresis in R. prolixus, and in other model insects. But the post-genomic era has brought new insights, identifying functions quite apart from diuresis for Malpighian tubules. Indeed, microarrays, transcriptomes, and proteomics have revealed the major roles that Malpighian tubules play in immunity, detoxification, pesticide resistance, and in tolerance to overall stress. This is particularly relevant to R. prolixus since gorging on blood creates several challenges in addition to osmotic balance. Xenobiotics may be present in the blood or toxins may be produced by metabolism of blood; and these must be neutralized and excreted. These processes have not been well described at the molecular level for Malpighian tubules of R. prolixus. This paper will review the involvement of Malpighian tubules in immunity and detoxification, identifying new aspects for Malpighian tubule physiology of R. prolixus by virtue of a transcriptome analysis. The transcriptome analysis indicates the potential of Malpighian tubules of R. prolixus to mount a robust innate immune response, and to contribute to antioxidant production and heme detoxification.

Effects of mating on female reproductive physiology in the insect model, Rhodnius prolixus, a vector of the causative parasite of Chagas disease.

The blood-sucking hemipteran Rhodnius prolixus is one of the main vectors of Chagas disease, a neglected tropical disease that affects several million people worldwide. Consuming a blood meal and mating are events with a high epidemiological impact since after each meal, mated females can lay fertile eggs that result in hundreds of offspring. Thus, a better knowledge of the control of R. prolixus reproductive capacity may provide targets for developing novel strategies to control vector populations, thereby reducing vector-host contacts and disease transmission. Here, we have used a combination of gene transcript expression analysis, biochemical assays, hormone measurements and studies of locomotory activity to investigate how mating influences egg development and egg laying rates in R. prolixus females. The results demonstrate that a blood meal increases egg production capacity and leads to earlier egg laying in mated females compared to virgins. Virgin females, however, have increased survival rate over mated females. Circulating juvenile hormone (JH) and ecdysteroid titers are increased in mated females, a process mainly driven through an upregulation of the transcripts for their biosynthetic enzymes in the corpus allatum and ovaries, respectively. Mated females display weaker locomotory activity compared to virgin females, mainly during the photophase. In essence, this study shows how reproductive output and behaviour are profoundly influenced by mating, highlighting molecular, biochemical, endocrine and behavioral features differentially expressed in mated and virgin R. prolixus females.

Gonadulin: A newly discovered insulin-like peptide involved in ovulation and oviposition in Rhodnius prolixus, a vector of Chagas disease.

Insulin-like peptides (ILPs) are vital hormones involved in a wide range of physiological processes in all organisms. In insects, insulin signaling has a key role in detecting and interpreting nutrient levels for egg production. Based on publicly available transcriptomes, a new ILP named gonadulin has been reported and suggested to be expressed by the gonads (hence its name). Although the identification of gonadulin establishes its existence, its physiological relevance remains poorly understood. Rhodnius prolixus is an obligate hematophagous insect and a primary vector of Trypanosoma cruzi, the etiological agent of Chagas disease. In this study, we report for the first time the participation of gonadulin in reproductive performance of an hemipteran. By quantitative PCR and fluorescence in situ hybridization (FISH), we find that the R. prolixus gonadulin transcript is highly expressed in the reproductive system, particularly in the calyx, a structure through which eggs move into the lumen of the lateral oviducts during ovulation. The putative gonadulin receptor, a member of the leucine-rich repeat-containing G protein-coupled receptor subfamily (LGR3), is most highly expressed in the central nervous system with lower levels in the reproductive tissue and other tissues. Interestingly, when the gonadulin signaling cascade is impaired using RNA interference (RNAi), eggs are retained primarily in the ovarioles and calyx, indicating that ovulation and oviposition are inhibited. Understanding the physiological processes involved in reproduction in R. prolixus will shed light on potential targets for effective production of biopesticides by translational research, thereby controlling insect populations and transmission of the disease.

Exploring the role of glycoprotein hormone GPA2/GPB5 in the medically important insect, Rhodnius prolixus.

Glycoprotein hormones are formed by the heterodimerization of alpha and beta subunits. In vertebrates, there are five glycoprotein hormones, four of which have a common alpha subunit (GPA1) bound to a specific beta subunit (GPB1, GPB2, GPB3, or GPB4), and the fifth, thyrostimulin, is formed by the dimerization of GPA2 and GPB5 subunits. These hormones mediate physiological events such as development, metabolism, and reproduction, although the functional role of thyrostimulin in vertebrates has not been fully elucidated. Recent reports in invertebrates, specifically in holometabolous insects, suggest that GPA2/GPB5 plays a critical role in development, diuresis, and reproduction. In this study, we clone and characterize the transcripts for the glycoprotein hormone GPA2/GPB5 and its receptor (LGR1) in fifth instar Rhodnius prolixus, a hemimetabolous insect vector of Chagas disease. Sequence analyses reveals considerable identity and similarity between GPA2/GPB5 and LGR1 and those reported in other arthropod species. Quantitative PCR (qPCR) shows that both subunit transcripts, GPA2 and GPB5, and LGR1 transcripts are present in a variety of tissues, with greatest expression of the subunits in the central nervous system (CNS) and highest LGR1 expression in the Malpighian tubules (MT). Results from temporal qPCR analyses reveal a decrease in transcript expression 24 h after feeding, followed by an increase as the days post-feeding advance. Using immunohistochemistry, we show that GPB5 is expressed throughout the CNS, and importantly is present in neurosecretory cells in the brain and abdominal neuromeres and their neurohemal organs, indicating a neurohormonal role for this signaling pathway. A reduction in LGR1 transcript expression (via RNA interference) led to a greater weight loss and mortality rate in unfed insects. In addition, when a blood meal is offered, the insects with reduced LGR1 consume a significantly smaller blood meal and have higher mortality rates as the days post-feeding advance. Overall, the results suggest that the GPA2/GPB5 signaling pathway may play roles during a prolonged unfed state and in feeding-related events.

Role of Methoprene-tolerant in the regulation of oogenesis in Dipetalogaster maxima.

Juvenile hormone (JH) signalling, via its receptor Methoprene-tolerant (Met), controls metamorphosis and reproduction in insects. Met belongs to a superfamily of transcription factors containing the basic Helix Loop Helix (bHLH) and Per Arnt Sim (PAS) domains. Since its discovery in 1986, Met has been characterized in several insect species. However, in spite of the importance as vectors of Chagas disease, our knowledge on the role of Met in JH signalling in Triatominae is limited. In this study, we cloned and sequenced the Dipetalogaster maxima Met transcript (DmaxMet). Molecular modelling was used to build the structure of Met and identify the JH binding site. To further understand the role of the JH receptor during oogenesis, transcript levels were evaluated in two main target organs of JH, fat body and ovary. Functional studies using Met RNAi revealed significant decreases of transcripts for vitellogenin (Vg) and lipophorin (Lp), as well as their receptors. Lp and Vg protein amounts in fat body, as well as Vg in hemolymph were also decreased, and ovarian development was impaired. Overall, these studies provide additional molecular insights on the roles of JH signalling in oogenesis in Triatominae; and therefore are relevant for the epidemiology of Chagas´ disease.

The involvement of insulin/ToR signaling pathway in reproductive performance of Rhodnius prolixus.

Insulins are peptide hormones widely studied for their important regulatory roles in metabolism, growth and development. In insects, insulin signaling along with the target of rapamycin (ToR) are involved in detecting and interpreting nutrient levels. Recently, by transcriptome analysis we reported an up-regulation of transcripts involved in insulin/ToR signaling in unfed Rhodnius prolixus; however, this signaling pathway is only activated in fed insects. Here, continuing with the blood-gorging triatomine R. prolixus as a model, we report the direct effect of insulin/ToR signaling on reproductive performance. By immunofluorescence we identified cells in the brain with positive signal to the R. prolixus ILP (Rhopr-ILP1) and show that the insulin receptor and protein effectors downstream of insulin/ToR signaling activation, are differentially expressed in ovarian follicles dependent on their developmental stage. Using qPCR we find that the expression of transcripts involved in insulin signaling in the central nervous system (CNS), fat body and ovaries increase as the state of starvation progresses, promoting a more highly sensitized state to respond rapidly to ILP/IGF levels. In addition, using dsRNA injection and in vivo and ex vivo assays to promote signaling activation we demonstrate a direct participation of insulin/ToR signaling in coordinating the synthesis of the main yolk protein precursor, vitellogenin, thereby influencing the numbers of eggs laid per female. We thereby show a mechanism by which nutritional signaling regulates reproductive performance in a vector of Chagas disease. As reproduction is responsible for propagation of insect populations, this work is important for the development of innovative biocontrol methods.

Regulation of a Trehalose-Specific Facilitated Transporter (TRET) by Insulin and Adipokinetic Hormone in Rhodnius prolixus, a Vector of Chagas Disease.

Using the blood-sucking kissing bug, Rhodnius prolixus as an experimental model, we have studied the involvement of insulin-like peptides (ILPs) and adipokinetic hormone (AKH) signaling in carbohydrate metabolism, focusing on the regulation of the trehalose-specific facilitated transporter (Rhopr-TRET), particularly in the ovaries. We find that trehalose stores in ovaries increase after feeding, synchronously with the beginning of vitellogenesis, but that the transcript expression of enzymes involved in trehalose synthesis show no changes between unfed and blood-fed animals. However, an eightfold increase in Rhopr-TRET transcript expression is observed in the ovaries post-blood meal. In vivo and ex vivo assays using exogenous insulins and Rhopr-AKH, reveal that Rhopr-TRET is up-regulated in ovaries by both peptide families. In accordance with these results, when ILP and AKH signaling cascades are impaired using RNA interference, Rhopr-TRET transcript is down-regulated. In addition, trehalose injection induces an up-regulation of Rhopr-TRET transcript expression and suggests an activation of insulin signaling. Overall, the results support the hypothesis of a direct trehalose uptake by ovaries from the hemolymph through Rhopr-TRET, regulated by ILP and/or AKH. We also show that Rhopr-TRET may work cooperatively with AKH signaling to support the release of trehalose from the ovaries into the hemolymph during the unfed (starved) condition. In conclusion, the results indicate that in females of R. prolixus, trehalose metabolism and its hormonal regulation by ILP and AKH play critical roles in adapting to different nutritional conditions and physiological states.

Identification of Gonadulin and Insulin-Like Growth Factor From Migratory Locusts and Their Importance in Reproduction in Locusta migratoria.

Many insect species have several genes coding for insulin-related peptides (IRPs), but so far only a single IRP gene has been identified in migratory locusts. Here, we report and characterize two other genes coding for peptides that are related to insulin, namely gonadulin and arthropod insulin-like growth factor (aIGF); peptides postulated to be orthologs of Drosophila melanogaster insulin-like peptides 8 and 6 respectively. In Locusta migratoria the aIGF transcript is expressed in multiple tissues as was previously reported for IRP in both L. migratoria and Schistocerca gregaria, but there are significant differences in expression patterns between the two species. The gonadulin transcript, however, seems specific to the ovary, whereas its putative receptor transcript is expressed most abundantly in the ovary, fat body and the central nervous system. Since the central nervous system-fat body-ovary axis is essential for successful reproduction, we studied the influence of gonadulin on vitellogenesis and oocyte growth. A reduction in the gonadulin transcript (via RNA interference) led to a significant reduction in vitellogenin mRNA levels in the fat body and a strong oocyte growth inhibition, thus suggesting an important role for gonadulin in reproduction in this species.