Unsaturated glycerophospholipids mediate heme crystallization: biological implications for hemozoin formation in the kissing bug Rhodnius prolixus

Hemozoin (Hz) is a heme crystal produced by some blood-feeding organisms, as an efficient way to detoxify heme derived from hemoglobin digestion. In the triatomine insect Rhodnius prolixus , Hz is essentially produced by midgut extracellular phospholipid membranes known as perimicrovillar membranes (PMVM). Here, we investigated the role of commercial glycerophospholipids containing serine, choline and ethanolamine as headgroups and R. prolixus midgut lipids (RML) in heme crystallization. All commercial unsaturated forms of phospholipids, as well as RML, mediated fast and efficient β-hematin formation by means of two kinetically distinct mechanisms: an early and fast component, followed by a late and slow one. The fastest reactions observed were induced by unsaturated forms of phosphatidylethanolamine (uPE) and phosphatidylcholine (uPC), with half-lives of 0.04 and 0.7 minutes, respectively. β-hematin crystal morphologies were strikingly distinct among groups, with uPE producing homogeneous regular brick-shaped crystals. Interestingly, uPC-mediated reactions resulted in two morphologically distinct crystal populations: one less representative group of regular crystals, resembling those induced by uPE, and the other largely represented by crystals with numerous sharp edges and tapered ends. Heme crystallization reactions induced by RML were efficient, with a heme to β-hematin conversion rate higher than 70%, but clearly slower (t1/2 of 9.9-17.7 minutes) than those induced by uPC and uPE. Interestingly, crystals produced by RML were homogeneous in shape and quite similar to those mediated by uPE. Thus, β-hematin formation can be rapidly and efficiently induced by unsaturated glycerophospholipids, particularly uPE and uPC, and may play a role on biological heme crystallization in R. prolixus midgut

Fat body fructose-2,6-bisphosphate content and phosphorylase activity correlate with changes in hemolymph glucose concentration during fasting and re-feeding in larval Manduca sexta

Abstract Fasting of second-day fifth instar larval Manduca sexta leads to a rapid decrease in hemolymph glucose concentration from 3.39±0.29 to 0.33±0.06 mM in 1 h, along with a decrease in the fructose-2,6-bisphosphate content in the fat body (from 5.92±0.31 to 2.80±0.47 nmol fructose-2,6-bisphosphate/g fat body in 3 h) and activation of fat body glycogen phosphorylase (from 16% to 55-65% phosphorylase a). During re-feeding an increase in the glucose level in the hemolymph was observed (from 0.36±0.05 to 3.91±0.36 mM in 3 h), along with an increase in the fructose-2,6-bisphosphate level in the fat body (from 2.88±0.47 to 6.66±0.42 nmol fructose-2,6-bisphosphate/g fat body in 3 h) and inactivation of fat body glycogen phosphorylase (from 56% to 16% phosphorylase a). These data are consistent with the hypothesis that a decrease in hemolymph glucose both activates fat body glycogen phosphorylase and causes a decrease in fat body fructose-2,6-bisphosphate content. Both of these changes would favor conversion of stored glucose to trehalose in the fat body. When second-day larvae were decapitated, the changes in hemolymph glucose and fat body fructose-2,6-bisphosphate were very similar to those observed in fasting whole insects. These data are consistent with a direct role for glucose in controlling carbohydrate metabolism in Manduca sexta

Oogenesis and egg development in triatomines: a biochemical approach

In triatomines, as well as in other insects, accumulation of yolk is a process in which an extra-ovarian tissue, the fat body, produces yolk proteins that are packed in the egg. The main protein, synthesized by the fat body, which is accumulated inside the oocyte, is vitellogenin. This process is also known as vitellogenesis. There are growing evidences in triatomines that besides fat body the ovary also produces yolk proteins. The way these yolk proteins enter the oocyte will be discussed. Yolk is a complex material composed of proteins, lipids, carbohydrates and other minor components which are packed inside the oocyte in an organized manner. Fertilization triggers embryogenesis, a process where an embryo will develop. During embryogenesis the yolk will be used for the construction of a new individual, the first instar nymph. The challenge for the next decade is to understand how and where these egg proteins are used up together with their non-protein components, in pace with the genetic program of the embryo, which enables cell differentiation (early phase of embryogenesis) and embryo differentiation (late phase) inside the egg.<br>Em triatomíneos, assim como em outros insetos, o acúmulo de vitelo é um processo no qual um tecido extraovariano, o corpo gorduroso, produz proteínas que são empacotadas no interior de um ovo. A principal proteína, sintetizada pelo corpo gorduroso, que é acumulada no interior de um ovócito, é a vitelogenina. Este processo é também conhecido por vitelogênese. Existem crescentes evidências em triatomíneos, que além do corpo gorduroso, o ovário também produz proteínas de vitelo. A forma como estas proteínas de vitelo entram nos ovócitos será aqui comentada. O vitelo é um material complexo composto por proteínas, lipídeos, carboidratos e outros compostos minoritários que são empacotados de uma maneira organizada no interior dos ovócitos. A fertilização dispara a embriogênese, um processo que culmina com o desenvolvimento do embrião. Durante a embriogênese o vitelo será utilizado para a construção de um novo indivíduo, a ninfa de primeiro estádio. O desafio para a próxima década é entender onde e como estas proteínas de vitelo são utilizadas junto com os seus componentes não protéicos, em compasso com o programa genético do embrião, que comanda a diferenciação celular (fase inicial da embriogênese) e diferenciação do embrião (fase final da embriogênese) no interior do ovo

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 β 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

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

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 β-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 β-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.Fil: Majerowicz, David. Universidade Federal do Rio de Janeiro; BrasilFil: Calderón Fernández, Gustavo Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Alves Bezerra, Michelle. Universidade Federal do Rio de Janeiro; BrasilFil: De Paula, Iron F.. Universidade Federal do Rio de Janeiro; BrasilFil: Cardoso, Livia S.. Universidade Federal do Rio de Janeiro; BrasilFil: Juarez, Marta Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Atella, Georgia C.. Universidade Federal do Rio de Janeiro; Brasil. Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular; BrasilFil: Gondim, Katia C.. Universidade Federal do Rio de Janeiro; Brasi

Image1_In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.pdf

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%–50% higher autophagic activation in the first 2 weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.</p

Table1_In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.DOCX

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%–50% higher autophagic activation in the first 2 weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.</p

Interaction between Trypanosoma rangeli and the Rhodnius prolixus salivary gland depends on the phosphotyrosine ecto-phosphatase activity of the parasite

Trypanosoma rangeli is the trypanosomatid that colonizes the salivary gland of its insect vector, with a profound impact on the feeding capacity of the insect. In this study we investigated the role of the phosphotyrosine (P-Tyr) ecto-phosphatase activity of T. rangeli in its interaction with Rhodnius prolixus salivary glands. Long but not short epimastigotes adhered to the gland cells and the strength of interaction correlated with the enzyme activity levels in different strains. Differential interference contrast microscopy demonstrated that clusters of parasites are formed in most cases, suggesting cooperative interaction in the adhesion process. The tightness of the correlation was evidenced by modulating the P-Tyr ecto-phosphatase activity with various concentrations of inhibitors. Sodium orthovanadate, ammonium molybdate and zinc chloride decreased the interaction between T. rangeli and R. prolixus salivary glands in parallel. Levamisole, an inhibitor of alkaline phosphatases, affected neither process. EDTA strongly inhibited adhesion and P-Tyr ecto-phosphatase activity to the same extent, an effect that was no longer seen if the parasites were pre-incubated with the chelator and then washed. When the P-Tyr ecto-phosphatase of living T. ranged epimastigotes was irreversibly inactivated with sodium orthovanadate and the parasite cells were then injected into the insect thorax, colonization of the salivary glands was greatly depressed for several days after blood feeding. Addition of P-Tyr ecto-phosphatase substrates such as p-nitrophenyl phosphate (pNPP) and P-Tyr inhibited the adhesion of T. rangeli to salivary glands, but P-Ser, P-Thr and beta-glycerophosphate were completely ineffective. Immunoassays using anti-P-Tyr-residues revealed a large number of P-Tyr-proteins in extracts of R. prolixus salivary glands, which could be potentially targeted by T. rangeli during adhesion. These results indicate that dephosphorylation of structural P-Tyr residues on the gland cell surfaces, mediated by a P-Tyr ecto-phosphatase of the parasite, is a key event in the interaction between T. rangeli and R. prolixus salivary glands. (C) 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.Brazilian National Research Council (CNPq)Brazilian National Research Council (CNPq)Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ)Carlos Chagas Filho Rio de Janeiro State Research Foundation (FAPERJ)Sao Paulo State Research Foundation (FAPESP)Sao Paulo State Research Foundation (FAPESP)Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES)Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES)National Institutes of Science and Technology, BrazilNational Institutes of Science and Technology, Brazi

Structural and physico-chemical features of the glycerophospholipids investigated in the present work.

a<p>T_m values were obtained from the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088976#pone.0088976-Silvius1" target="_blank">[67]</a>.</p