Knockdown of Sec16 causes early lethality and defective deposition of the protein Rp30 in the eggshell of the vector Rhodnius prolixus.

In nearly every species of insect, embryonic development takes place outside of the mother's body and is entirely dependent on the elements that the mother had previously stored within the eggs. It is well known that the follicle cells (FCs) synthesize the eggshell (chorion) components during the process of choriogenesis, the final step of oogenesis before fertilization. These cells have developed a specialization in the massive production of chorion proteins, which are essential for the protection and survival of the embryo. Here, we investigate the function of Sec16, a protein crucial for the endoplasmic reticulum (ER) to Golgi traffic, in the oocyte development in the insect Rhodnius prolixus. We discovered that Sec16 is strongly expressed in vitellogenic females' ovaries, particularly in the choriogenic oocyte and it is mainly associated with the FCs. Silencing of Sec16 by RNAi caused a sharp decline in oviposition rates, F1 viability, and longevity in adult females. In the FCs, genes involved in the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy were massively upregulated, whereas the mRNAs of Rp30 and Rp45-which code for the two major chorion proteins - were downregulated as a result of Sec16 silencing, indicating general proteostasis disturbance. As a result, the outer surface ultrastructure of Sec16-silenced chorions was altered, with decreased thickness, dityrosine crosslinking, sulfur signals, and lower amounts of the chorion protein Rp30. These findings collectively demonstrate the critical role Sec16 plays in the proper functioning of the FCs, which impacts the synthesis and deposition of particular components of the chorion as well as the overall reproduction of this vector.

Use of FGF21 analogs for the treatment of metabolic disorders: a systematic review and meta-analysis

ABSTRACT FGF21 is a hormone produced primarily by the liver with several metabolic functions, such as induction of heat production, control of glucose homeostasis, and regulation of blood lipid levels. Due to these actions, several laboratories have developed FGF21 analogs to treat patients with metabolic disorders such as obesity and diabetes. Here, we performed a systematic review and meta-analysis of randomized controlled trials that used FGF21 analogs and analyzed metabolic outcomes. Our search yielded 236 articles, and we included eight randomized clinical trials in the meta-analysis. The use of FGF21 analogs exhibited no effect on fasting blood glucose, glycated hemoglobin, HOMA index, blood free fatty acids or systolic blood pressure. However, the treatment significantly reduced fasting insulinemia, body weight and total cholesterolemia. None of the included studies were at high risk of bias. The quality of the evidence ranged from moderate to very low, especially due to imprecision and indirection issues. These results indicate that FGF21 analogs can potentially treat metabolic syndrome. However, more clinical trials are needed to increase the quality of evidence and confirm the effects seen thus far.

Use of FGF21 analogs for the treatment of metabolic disorders: a systematic review and meta-analysis.

FGF21 is a hormone produced primarily by the liver with several metabolic functions, such as induction of heat production, control of glucose homeostasis, and regulation of blood lipid levels. Due to these actions, several laboratories have developed FGF21 analogs to treat patients with metabolic disorders such as obesity and diabetes. Here, we performed a systematic review and meta-analysis of randomized controlled trials that used FGF21 analogs and analyzed metabolic outcomes. Our search yielded 236 articles, and we included eight randomized clinical trials in the meta-analysis. The use of FGF21 analogs exhibited no effect on fasting blood glucose, glycated hemoglobin, HOMA index, blood free fatty acids or systolic blood pressure. However, the treatment significantly reduced fasting insulinemia, body weight and total cholesterolemia. None of the included studies were at high risk of bias. The quality of the evidence ranged from moderate to very low, especially due to imprecision and indirection issues. These results indicate that FGF21 analogs can potentially treat metabolic syndrome. However, more clinical trials are needed to increase the quality of evidence and confirm the effects seen thus far.

Alterations in energy metabolism of Rhodnius prolixus induced by Trypanosoma rangeli infection.

Trypanosoma rangeli is a protozoan parasite that infects triatomines and mammals in the Americas, producing mixed infections with Trypanosoma cruzi, the etiological agent of Chagas disease. The former parasite is not pathogenic to humans, but has different levels of pathogenicity, as well as causing physiological and behavioral alterations, to its invertebrate hosts. In this study, we measured locomotory activity, and the glyceride accumulation profile in the hemolymph and fat body, as well as the expression of key genes related to triglyceride metabolism, of Rhodnius prolixus nymphs infected with T. rangeli. We found that the locomotory activity of the insects was correlated with the amount of triglycerides in the fat body. Infected nymphs had increased activity when starved, and also had an accumulation of glycerides in the fat body and hemolymph. These alterations were also associated with a higher expression of the diacylglycerol acyltransferase, lipophorin and lipophorin receptor genes in the fat body. We infer that T. rangeli is able to alter the energetic processes of its invertebrate host, in order to increase the availability of lipids to the parasite, which, in turn modifies the activity levels of the insect. These alterations are discussed with regard to their potential to increase the transmission rate of the parasite.

Gene identification and RNAi-silencing of p62/SQSTM1 in the vector Rhodnius prolixus reveals a high degree of sequence conservation but no apparent deficiency-related phenotypes in vitellogenic females.

Autophagy and the ubiquitin-proteasome system (UPS) are important cellular mechanisms that coordinate protein degradation essential for proteostasis. P62/SQSTM1 is a receptor cargo protein able to deliver ubiquitinated targets to the proteasome proteolytic complex and/or to the autophagosome. In the insect vector of Chagas disease, Rhodnius prolixus, previous works have shown that the knockdown of different autophagy-related genes (ATGs) and ubiquitin-conjugating enzymes resulted in abnormal oogenesis phenotypes and embryo lethality. Here, we investigate the role of the autophagy/UPS adaptor protein p62 during the oogenesis and reproduction of this vector. We found that R. prolixus presents one isoform of p62 encoded by a non-annotated gene. The predicted protein presents the domain architecture anticipated for p62: PB1 (N-term), ZZ-finger, and UBA (C-term) domains, and phylogenetic analysis showed that this pattern is highly conserved within insects. Using parental RNAi, we found that although p62 is expressed in the ovary, midgut, and fat body of adult females, systemic silencing of this gene did not result in any apparent phenotypes under in-house conditions. The insects' overall levels of blood meal digestion, lifespan, yolk protein production, oviposition, and embryo viability were not altered when compared to controls. Because it is known that autophagy and UPS can undergo compensatory mechanisms, we asked whether the silencing of p62 was triggering adaptative changes in the expression of genes of the autophagy, UPS, and the unfolded protein response (UPR) and found that only ATG1 was slightly up regulated in the ovaries of silenced females. In addition, experiments to further investigate the role of p62 in insects previously silenced for the E1-conjugating enzyme (a condition known to trigger the upregulation of p62), also did not result in any apparent phenotypes in vitellogenic females.

Knockdown of carnitine palmitoyltransferase I (CPT1) reduces fat body lipid mobilization and resistance to starvation in the insect vector Rhodnius prolixus.

The energy stored in fatty acids is essential for several critical activities of insects, such as embryogenesis, oviposition, and flight. Rhodnius prolixus is an obligatory hematophagous hemipteran and vector of Chagas disease, and it feeds infrequently on very large blood meals. As digestion slowly occurs, lipids are synthesized and accumulate in the fat body, mainly as triacylglycerol, in lipid droplets. Between feeding bouts, proper mobilization and oxidation of stored lipids are crucial for survival, and released fatty acids are oxidized by mitochondrial ß-oxidation. Carnitine palmitoyl transferase I (CPT1) is the enzyme that catalyzes the first reaction of the carnitine shuttle, where the activated fatty acid, acyl-CoA, is converted to acyl-carnitine to be transported into the mitochondria. Here, we investigated the role of CPT1 in lipid metabolism and in resistance to starvation in Rhodnius prolixus. The expression of the CPT1 gene (RhoprCpt1) was determined in the organs of adult females on the fourth day after a blood meal, and the flight muscle showed higher expression levels than the ovary, fat body, and anterior and posterior midgut. RhoprCpt1 expression in the fat body dramatically decreased after feeding, and started to increase again 10 days later, but no changes were observed in the flight muscle. ß-oxidation rates were determined in flight muscle and fat body homogenates with the use of 3H-palmitate, and in unfed females, they were higher in the flight muscle. In the fat body, lipid oxidation activity did not show any variation before or at different days after feeding, and was not affected by the presence of etomoxir or malonyl-CoA. We used RNAi and generated RhoprCPT1-deficient insects, which surprisingly did not show a decrease in measured 3H-palmitate oxidation rates. However, the RNAi-knockdown females presented increased amounts of triacylglycerol and larger lipid droplets in the fat body, but not in the flight muscle. When subjected to starvation, these insects had a shorter lifespan. These results indicated that the inhibition of RhoprCpt1 expression compromised lipid mobilization and affected resistance to starvation.

Silencing of the 20S proteasomal subunit-α6 triggers full oogenesis arrest and increased mRNA levels of the selective autophagy adaptor protein p62/SQSTM1 in the ovary of the vector Rhodnius prolixus.

The high reproductive rates of insects contribute significantly to their ability to act as vectors of a variety of vector-borne diseases. Therefore, it is strategically critical to find molecular targets with biotechnological potential through the functional study of genes essential for insect reproduction. The ubiquitin-proteasome system is a vital degradative pathway that contributes to the maintenance of regular eukaryotic cell proteostasis. This mechanism involves the action of enzymes to covalently link ubiquitin to proteins that are meant to be delivered to the 26S proteasome and broken down. The 26S proteasome is a large protease complex (including the 20S and 19S subcomplexes) that binds, deubiquitylates, unfolds, and degrades its substrates. Here, we used bioinformatics to identify the genes that encode the seven α and ß subunits of the 20S proteasome in the genome of R. prolixus and learned that those transcripts are accumulated into mature oocytes. To access proteasome function during oogenesis, we conducted RNAi functional tests employing one of the 20S proteasome subunits (Prosα6) as a tool to suppress 20S proteasomal activity. We found that Prosα6 silencing resulted in no changes in TAG buildup in the fat body and unaffected availability of yolk proteins in the hemolymph of vitellogenic females. Despite this, the silencing of Prosα6 culminated in the impairment of oocyte maturation at the early stages of oogenesis. Overall, we discovered that proteasome activity is especially important for the signals that initiate oogenesis in R. prolixus and discuss in what manner further investigations on the regulation of proteasome assembly and activity might contribute to the unraveling of oogenesis molecular mechanisms and oocyte maturation in this vector.

ATP synthase affects lipid metabolism in the kissing bug Rhodnius prolixus beyond its role in energy metabolism.

ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and ß family, including the α and ß subunits of ATP synthase (RpATPSynα and RpATPSynß), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynß knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynß increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.

Diet supplementation with egg yolk powder fattens the beetle Tribolium castaneum.

Insects have become essential models in studying human metabolic diseases, mainly due to their low maintenance cost and available tools. Both mutations and modified diets induce metabolic states similar to human obesity and diabetes. Here, we explore the effect of a high-calorie, high-fat diet on the metabolism of the beetle Tribolium castaneum. Supplementation of the wheat flour diet with powdered egg yolk for 3 weeks increased the total triacylglycerol and accelerated larval development. In addition, this diet increased the triacylglycerol levels of adult beetles. However, this egg yolk supplementation did not alter the larvae's total glucose levels or lipogenic capacity and ATP citrate lyase activity. The diet also did not change the expression profile of several lipid and carbohydrate metabolism genes and insulin-like peptides. Thus, we conclude that the diet supplemented with egg yolk induces increased fat without causing diabetes phenotypes, as seen in other hypercaloric diets in insects.

The Fate of Dietary Cholesterol in the Kissing Bug Rhodnius prolixus.

Insects are unable to synthesize cholesterol and depend on the presence of sterols in the diet for cell membrane composition and hormone production. Thus, cholesterol absorption, transport, and metabolism are potential targets for vector and pest control strategies. Here, we investigate the dietary cholesterol absorption and tissue distribution in the kissing bug Rhodnius prolixus using radiolabeled cholesterol. Both the anterior and posterior midguts absorbed cholesterol from the ingested blood, although the anterior midgut absorbed more. We also observed esterified cholesterol labeling in the epithelium, indicating that midgut cells can metabolize and store cholesterol. Only a small amount of labeled cholesterol was found in the hemolymph, where it was mainly in the free form and associated with lipophorin (Lp). The fat body transiently accumulated cholesterol, showing a labeled cholesterol peak on the fifth day after the blood meal. The ovaries also incorporated cholesterol, but cumulatively. The insects did not absorb almost half of the ingested labeled cholesterol, and radioactivity was present in the feces. After injection of 3H-cholesterol-labeled Lp into females, a half-life of 5.5 ± 0.7 h in the hemolymph was determined. Both the fat body and ovaries incorporated Lp-associated cholesterol, which was inhibited at low temperature, indicating the participation of active cholesterol transport. These results help describe an unexplored part of R. prolixus lipid metabolism.

dHNF4 regulates lipid homeostasis and oogenesis in Drosophila melanogaster.

The fly genome contains a single ortholog of the evolutionarily conserved transcription factor hepatocyte nuclear factor 4 (HNF4), a broadly and constitutively expressed member of the nuclear receptor superfamily. Like its mammalian orthologs, Drosophila HNF4 (dHNF4) acts as a critical regulator of fatty acid and glucose homeostasis. Because of its role in energy storage and catabolism, the insect fat body controls non-autonomous organs including the ovaries, where lipid metabolism is essential for oogenesis. The present paper used dHNF4 overexpression (OE) in the fat bodies and ovaries to investigate its potential roles in lipid homeostasis and oogenesis. When the developing fat body overexpressed dHNF4, animals exhibited reduced size and failed to pupariate, but no changes in body composition were observed. Conditional OE of dHNF4 in the adult fat body produced a reduction in triacylglycerol content and reduced oogenesis. Ovary-specific dHNF4 OE increased oogenesis and egg-laying, but reduced the number of adult offspring. The phenotypic effects on oogenesis that arise upon dHNF4 OE in the fat body or ovary may be due to its function in controlling lipid utilization.

Blood meal drives de novo lipogenesis in the fat body of Rhodnius prolixus.

In insects, lipids are stored in the fat body mainly as triacylglycerol. Lipids can be directly provided by digestion and incorporated from the hemolymph, or synthesized de novo from other substrates such as carbohydrates and amino acids. The first step in de novo lipid synthesis is catalyzed by acetyl-CoA carboxylase (ACC), which carboxylates acetyl-CoA to form malonyl-CoA. Rhodnius prolixus is a hematophagous insect vector of Chagas disease and feeds exclusively on large and infrequent blood meals. Adult females slowly digest the blood and concomitantly accumulate lipids in the fat body. In this study, we investigated the regulation of R. prolixus ACC (RhoprACC) expression and de novo lipogenesis activity in adult females at different nutritional and metabolic conditions. A phylogenetic analysis showed that insects, similar to other arthropods and unlike vertebrate animals, have only one ACC gene. In females on the fourth day after a blood meal, RhoprACC transcript levels were similar in the anterior and posterior midgut, fat body and ovary and higher in the flight muscles. In the fat body, gene expression was higher in fasted females and decreased after a blood meal. In the posterior midgut it increased after feeding, and no variation was observed in the flight muscle. RhoprACC protein content analysis of the fat body revealed a profile similar to the gene expression, with higher protein contents before feeding and in the first two days after a blood meal. Radiolabeled acetate was used to follow de novo lipid synthesis in the fat body and it was incorporated mainly into triacylglycerol, diacylglycerol and phospholipids. This lipogenic activity was inhibited by soraphen A, an ACC inhibitor, and it varied according to the insect metabolic status. De novo lipogenesis was very low in starved females and increased during the initial days after a blood meal. The flight muscles had a very low capacity to synthesize lipids when compared to the fat body. Radiolabeled leucine was also used as a substrate for de novo lipogenesis and the same lipid classes were formed. In conclusion, our results indicate that the blood meal induces the utilization of diet-derived amino acids by de novo lipogenesis in the fat body, and that the control of this activity does not occur at the RhoprACC gene or protein expression level.

Expression of acyl-CoA-binding protein 5 from Rhodnius prolixus and its inhibition by RNA interference.

The acyl-CoA-binding proteins (ACBP) act by regulating the availability of acyl-CoA in the cytoplasm and must have essential functions in lipid metabolism. The genome of the kissing-bug Rhodnius prolixus encodes five proteins of this family, but little is known about them. In this study we investigated the expression and function of RpACBP-5. Feeding induced RpACBP-5 gene expression in the posterior midgut, and an increase of about four times was observed two days after the blood meal. However, the amount of protein, which was only detected in this organ, did not change during digestion. The RpACBP-5 gene was also highly expressed in pre-vitellogenic and vitellogenic oocytes. Recombinant RpACBP-5 was shown to bind to acyl-CoA of different lengths, and it exhibited nanomolar affinity to lauroyl-CoA in an isothermal titration assay, indicating that RpACBP-5 is a functional ACBP. RpACBP-5 knockdown by RNA interference did not affect digestion, egg laying and hatching, survival, or accumulation of triacylglycerol in the fat body and oocytes. Similarly, double knockdown of RpACBP-1 and RpACBP-5 did not alter egg laying and hatching, survival, accumulation of triacylglycerol in the fat body and oocytes, or the neutral lipid composition of the posterior midgut or hemolymph. These results show that RpACBP-5 is a functional ACBP but indicate that the lack of a detectable phenotype in the knockdown insects may be a consequence of functional overlap of the proteins of the ACBP family found in the insect.

Pharmacotherapy of Obesity: Limits and Perspectives.

Obesity is a severe worldwide epidemic. Obesity comorbidities, such as type 2 diabetes mellitus, hypertension, and atherosclerosis, are costly for patients and governments. The treatment of obesity involves several facets, including lifestyle changes, bariatric surgery, and pharmacotherapy. As changes in lifestyle require considerable patient commitment that is sometimes unachievable, and surgery is expensive and invasive, pharmacotherapy is the primary option for most patients. This review describes the pharmacotherapy currently available in the USA, Europe, and Brazil, focusing on its limitations. We then analyze the results from clinical trials of new drug candidates. Most drugs cause weight loss of < 4 kg compared with controls, and severe adverse effects have caused a number of drugs to be withdrawn from the market in several countries. Drugs under development have not shown more significant weight loss or reduced adverse effects. We conclude that a significant portion of obese patients have few treatment options because of the adverse effects and minimal weight loss associated with current pharmacotherapy. However, drugs currently under development appear unable to change this scenario in the near future. Thus, it is essential that new compounds are developed and new molecular targets studied so obesity can be efficiently treated in all patients in the future.

Lipid metabolism in insect disease vectors.

More than a third of the world population is at constant risk of contracting some insect-transmitted disease, such as Dengue fever, Zika virus disease, malaria, Chagas' disease, African trypanosomiasis, and others. Independent of the life cycle of the pathogen causing the disease, the insect vector hematophagous habit is a common and crucial trait for the transmission of all these diseases. This lifestyle is unique, as hematophagous insects feed on blood, a diet that is rich in protein but relatively poor in lipids and carbohydrates, in huge amounts and low frequency. Another unique feature of these insects is that blood meal triggers essential metabolic processes, as molting and oogenesis and, in this way, regulates the expression of various genes that are involved in these events. In this paper, we review current knowledge of the physiology and biochemistry of lipid metabolism in insect disease vectors, comparing with classical models whenever possible. We address lipid digestion and absorption, hemolymphatic transport, and lipid storage by the fat body and ovary. In this context, both de novo fatty acid and triacylglycerol synthesis are discussed, including the related fatty acid activation process and the intracellular lipid binding proteins. As lipids are stored in order to be mobilized later on, e.g. for flight activity or survivorship, lipolysis and ß-oxidation are also considered. All these events need to be finely regulated, and the role of hormones in this control is summarized. Finally, we also review information about infection, when vector insect physiology is affected, and there is a crosstalk between its immune system and lipid metabolism. There is not abundant information about lipid metabolism in vector insects, and significant current gaps in the field are indicated, as well as questions to be answered in the future.

Insulin specifically regulates expression of liver and muscle phosphofructokinase isoforms.

Phosphofructokinase (PFK) is a key regulatory enzyme of glycolysis, being considered the pacemaker of this pathway. In mammals, this enzyme exists as three different isoforms, PFKM, PFKL and PFKP, presenting different regulatory and catalytic properties. The expression of these isoforms is tissue-specific and vary according to the cell differentiation and signalization. Although it is known that the expression of the different PFK isoforms directly affects cell function, the information regarding the regulation of PFK isoforms expression is scarce. In the present work, we evaluate the role of insulin signalization on the expression of three PFK isoforms on skeletal muscle, liver, and epididymal white adipose tissue (eWAT) of mice. For this, Swiss mice were treated with streptozotocin (STZ) to disrupt pancreatic ß-cells and, thus, insulin production. Control group were treated with citrate buffer (STZ vehicle). These groups were then treated with insulin or saline twice a day for ten consecutive days when animals were euthanized and tissues used for the evaluation of PFK isoforms expression by quantitative PCR (qPCR). Our results revealed that the lack of insulin significantly impacted the expression of PFKL, presenting mild effects on PFKM and no effects on PFKP. The decrease of PFKL and PFKM mRNA levels observed on the group treated with STZ was reversed by the treatment with insulin. In conclusion, insulin, the most known regulator of glucose consumption, specifically regulates the expression of PFKL and PFKM, which impact the regulation of glycolysis in the cell.

Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols.

Lipid metabolism in Rhodnius prolixus: Lessons from the genome.

The kissing bug Rhodnius prolixus is both an important vector of Chagas' disease and an interesting model for investigation into the field of physiology, including lipid metabolism. The publication of this insect genome will bring a huge amount of new molecular biology data to be used in future experiments. Although this work represents a promising scenario, a preliminary analysis of the sequence data is necessary to identify and annotate the genes involved in lipid metabolism. Here, we used bioinformatics tools and gene expression analysis to explore genes from different genes families and pathways, including genes for fat breakdown, as lipases and phospholipases, and enzymes from ß-oxidation, fatty acid metabolism, and acyl-CoA and glycerolipid synthesis. The R. prolixus genome encodes 31 putative lipase genes, including 21 neutral lipases and 5 acid lipases. The expression profiles of some of these genes were analyzed. We were able to identify nine phospholipase A2 genes. A variety of gene families that participate in fatty acid synthesis and modification were studied, including fatty acid synthase, elongase, desaturase and reductase. Concerning the synthesis of glycerolipids, we found a second isoform of glycerol-3-phosphate acyltransferase that was ubiquitously expressed throughout the organs. Finally, all genes involved in fatty acid ß-oxidation were identified, but not a long-chain acyl-CoA dehydrogenase. These results provide fundamental data to be used in future research on insect lipid metabolism and its possible relevance to Chagas' disease transmission.

Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan.

BACKGROUND: Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. RESULTS: Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. CONCLUSION: The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses.

The ACBP gene family in Rhodnius prolixus: Expression, characterization and function of RpACBP-1.

The acyl-CoA-binding proteins (ACBP) constitute a family of conserved proteins that bind acyl-CoA with high affinity and protect it from hydrolysis. Thus, ACBPs may have essential roles in basal cellular lipid metabolism. The genome of the insect Rhodnius prolixus encodes five ACBP genes similar to those described for other insect species. The qPCR analysis revealed that these genes have characteristic expression profiles in insect organs, suggesting that they have specific roles in insect physiology. Recombinant RpACBP-1 was able to bind acyl-CoA in an in vitro gel-shift assay. Moreover, heterologous RpACBP-1 expression in acb1Δ mutant yeast rescued the multi-lobed vacuole phenotype, indicating that RpACBP-1 acts as a bona fide acyl-CoA-binding protein. RpACBP-1 knockdown using RNAi caused triacylglycerol accumulation in the insect posterior midgut and a reduction in the number of deposited eggs. The amount of stored triacylglycerol was reduced in flight muscle, and the incorporation of fatty acids in cholesteryl esters was increased in the fat body. These results showed that RpACBP-1 participates in several lipid metabolism steps in R. prolixus.