Characterization of cultivable intestinal microbiota in Rhynchophorus palmarum Linnaeus (Coleoptera: Curculionidae) and determination of its cellulolytic activity.

Rhynchophorus palmarum Linnaeus is an agricultural pest that affects various palm crops, including coconut (Cocos nucifera) plantations which are prominent in the economy of Northeastern Brazil. Characterization of the intestinal microbiota of R. palmarum, as well as elucidation of aspects related to the biochemistry and physiology of the insect's digestion, is essential for intervention in specific metabolic processes as a form of pest control. Thus, this study aimed to characterize the intestinal microbiota of R. palmarum and investigate its ability to degrade cellulosic substrates, to explore new biological control measures. Intestinal dissection of eight adult R. palmarum insects was performed in a laminar flow chamber, and the intestines were homogenized in sterile phosphate-buffered saline solution. Subsequently, serial dilution aliquots of these solutions were spread on nutritive agar plates for the isolation of bacteria and fungi. The microorganisms were identified by matrix-assisted laser desorption/ionization with a time-of-flight mass spectrometry and evaluated for their ability to degrade cellulose. Fourteen bacterial genera (Acinetobacter, Alcaligenes, Arthrobacter, Bacillus, Citrobacter, Enterococcus, Kerstersia, Lactococcus, Micrococcus, Proteus, Providencia, Pseudomonas, Serratia, and Staphylococcus) and two fungal genera (Candida and Saccharomyces)-assigned to the Firmicutes, Actinobacteria, Proteobacteria, and Ascomycota phyla-were identified. The cellulolytic activity was exhibited by six bacterial and one fungal species; of these, Bacillus cereus demonstrated the highest enzyme synthesis (enzymatic index = 4.6). This is the first study characterizing the R. palmarum intestinal microbiota, opening new perspectives for the development of strategies for the biological control of this insect.

Chemical composition and antimicrobial activity of cuticular and internal lipids of the insect Rhynchophorus palmarum.

Insect cuticle lipids are involved in various types of chemical communication between species, and reduce the penetration of insecticides, chemicals, and toxins, as well as provide protection against the attack of microorganisms, parasitic insects, and predators. Ecological studies related to the insect Rhynchophorus palmarum are well-known; however, very little is known about its resistance mechanisms, which includes its lipid composition and its importance, specifically the cuticle layer. This study aimed to characterize the cuticle and internal lipid compounds of the male and female R. palmarum adult insects and to evaluate the presence of antimicrobial activity. We performed by gas chromatography coupled to mass spectrometry (GC-MS) analyzes of lipid extracts fractions and we identified 10 methyl esters of fatty acids esters of C14 to C23, with variation between the sexes of C22:0, C21:0, present only in male cuticle, and C20:2 in female. The lipid content of this insect showed relevant amount of C16:1, C18:1, and C18:2. The antimicrobial activity of the cuticular and internal fractions obtained was tested, which resulted in minimum inhibitory concentrations between 12.5 and 20 µg/ml against Gram-positive bacteria (Staphylococcus epidermidis, Enterococcus faecalis), Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumonia), and fungal species (Candida albicans e Candida tropicalis). The antimicrobial effect of the R. palmarum cuticle open perspectives for a new source to bioinsecticidal strategies, in addition to elucidating a bioactive mixture against bacteria and fungi.

Biogenic synthesis of silver nanoparticles using Brazilian propolis.

Biological methods have been used to synthesize silver nanoparticles through materials such as bacteria, fungi, plants, and propolis due to their reducing properties, stabilizer role and environmentally friendly characteristic. Considering the antimicrobial activity of propolis as well as the broad-spectrum antibacterial effects of silver nanoparticles, this study aim to describe the use of Brazilian propolis to synthesize silver nanoparticles (AgNP-P) and investigate its antimicrobial activity. The synthesis was optimized by factorial design, choosing the best conditions for smaller size particles. AgNP-P demonstrated a maximum absorbance at 412 nm in ultraviolet-visible spectra, which indicated a spherical format and its formation. Dynamic light scattering demonstrated a hydrodynamic size of 109 nm and polydispersity index less than 0.3, showing a good size distribution and stability. After its purification via centrifugation, microscopy analysis corroborates the format and showed the presence of propolis around silver nanoparticle. X-ray diffraction peaks were attributed to the main planes of the metallic silver crystalline structure; meanwhile infrared spectroscopy demonstrated the main groups responsible for silver reduction, represented by ∼22% of AgNP-P indicates by thermal analysis. Our product revealed an important antimicrobial activity indicating a synergism between propolis and silver nanoparticles as expected and promising to be an effective antimicrobial product to be used in infections.

Lipase Activity in the Larval Midgut of Rhynchophorus palmarum: Biochemical Characterization and the Effects of Reducing Agents.

Lipases have key roles in insect lipid acquisition, storage, and mobilization and are also fundamental to many physiological processes in insects. Lipids are an important component of insect diets, where they are hydrolyzed in the midgut lumen, absorbed, and used for the synthesis of complex lipids. The South American palm weevil Rhynchophorus palmarum is one of the most important pests on commercial palm plantations. However, there are few studies about lipid digestion for this insect. In this work, we have described the biochemical characterization of the lipase activity in the posterior midgut of the R. palmarum palm weevil. Lipase activity was highest between the temperatures of 37 °C and 45 °C and at pH 6.5. Lipase activity was also sensitive to variations in salt and calcium concentrations. Lipases have been described structurally as enzymes with the Ser-His-Asp Catalytic Triad, containing an active serine. The serine protease inhibitor PMSF (phenylmethane sulfonyl fluoride) inhibited the lipases from R. palmarum, demonstrating the importance of a serine residue for this activity. The ability of the lipases to hydrolyze p-Nitrophenyl esters with different chain lengths has revealed the activities of a broad range of substrates. The lipase activities of R. palmarum increased in the presence of reduced glutathione (GSH) and dithiothreitol (DTT), while in the presence of oxidized glutathione (GSSG), activities were drastically reduced. To our knowledge, this study has provided the first information about lipase activity in the R. palmarum palm weevil.

The Influence of Fatty Acid Methyl Esters (FAMEs) in the Biochemistry and the Na(+)/K(+)-ATPase Activity of Culex quinquefasciatus Larvae.

Culex quinquefasciatus is the main vector of lymphatic filariasis and combating this insect is of great importance to public health. There are reports of insects that are resistant to the products currently used to control this vector, and therefore, the search for new products has increased. In the present study, we have evaluated the effects of fatty acid methyl esters (FAMEs) that showed larvicidal activity against C. quinquefasciatus, on glucose, total protein, and triacylglycerol contents and Na(+)/K(+)-ATPase activity in mosquito larvae. The exposure of the fourth instar larvae to the compounds caused a decrease in the total protein content and an increase in the activity of the Na(+)/K(+)-ATPase. Furthermore, the direct effect of FAMEs on cell membrane was assessed on purified pig kidney Na(+)/K(+)-ATPase membranes, erythrocyte ghost membranes, and larvae membrane preparation. No modifications on total phospholipids and cholesterol content were found after FAMEs 20 min treatment on larvae membrane preparation, but only 360 µg/mL FAME 2 was able to decrease total phospholipid of erythrocyte ghost membrane. Moreover, only 60 and 360 µg/mL FAME 3 caused an activation of purified Na(+)/K(+)-ATPase, that was an opposite effect of FAMEs treatment in larvae membrane preparation, and caused an inhibition of the pump activity. These data together suggest that maybe FAMEs can modulate the Na(+)/K(+)-ATPase on intact larvae for such mechanisms and not for a direct effect, one time that the direct effect of FAMEs in membrane preparation decreased the activity of Na(+)/K(+)-ATPase. The biochemical changes caused by the compounds were significant and may negatively influence the development and survival of C. quinquefasciatus larvae.

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.

Embryonic development of Rhynchophorus palmarum (Coleoptera: Curculionidae): dynamics of energy source utilization.

Energy homeostasis is an essential process during oogenesis, nutrients are required for suitable embryonic development, and recently, studies have investigated metabolic activity during this process. This work aims the investigation of dynamics of energy source utilization of Rhynchophorus palmarum during embryogenesis. For this, we first evaluated the mobilization kinetics of the lipids and glycogen. Thereafter, the synthesis of RNA, protein, and the involvement of enzyme of the glycolytic and pentose-phosphate pathways. Results showed that lipid content decreased in contrast with the lipase activity. The total glycogen amounts it was partly consumed and the glucose content increased, but then values remained stable until hatching. Total RNA content increased, and no significant changes in total protein content were observed. A study of the glycolytic pathway data showed activity of hexokinase and pyruvate kinase at the beginning of embryogenesis. Furthermore, glucose-6-phosphate formed is driven into the pentose-phosphate pathway viewed the high activity of glucose-6-phosphate dehydrogenase. Finally, these results showed that mobilization of different energy sources together with different enzymatic activities has an important role in embryonic development of R. palmarum.

Serotonin regulates an acyl-CoA-binding protein (ACBP) gene expression in the midgut of Rhodnius prolixus.

Acyl-CoA esters have many intracellular functions, acting as energy source, substrate for metabolic processes and taking part in cell signaling. The acyl-CoA-binding protein (ACBP), a highly conserved 10 kDa intracellular protein, binds long- and medium-chain acyl-CoA esters with very high affinity, directing them to specific metabolic routes and protecting them from hydrolysis. An ACBP gene sequence was identified in the genome of Rhodnius prolixus. This ACBP gene (RpACBP-1) was expressed in all analyzed tissues and quantitative PCR showed that expression was highest in posterior midgut. In this tissue, ACBP gene expression increased in the first day after blood meal ( approximately 10-fold) and then decreased to unfed levels in the seventh day after meal. Injection of serotonin (5-hydroxytryptamine; 5-HT), a neuroamine released in the hemolymph after the start of feeding, increased the expression of this gene in the midgut of unfed females, reaching levels similar to those observed in fed insects. This effect of injected 5-HT was inhibited by spiperone, an antagonist of 5-HT mammalian receptors, that was also able to block the physiological increase in RpACBP-1 expression observed after feeding. Injection of cholera toxin or dibutyryl-cAMP also resulted in the stimulation of this gene expression. These data reveal a transcriptional regulatory mechanism in R. prolixus, that is triggered by 5-HT. In this way, a novel role for 5-HT is proposed, as a regulator of ACBP gene expression and, consequently, taking part in the control of lipid metabolism.

Lipid metabolism in Rhodnius prolixus (Hemiptera: Reduviidae): role of a midgut triacylglycerol-lipase.

The utilization of dietary lipids was studied in adult females of Rhodnius prolixus with the use of radiolabeled triacylglycerol (TAG). It was shown that (3)H-triolein, when added to blood meal, was hydrolyzed to free fatty acids in the posterior midgut lumen. Subsequently, free fatty acids were absorbed by posterior midgut epithelium and used in the synthesis of phospholipids, diacylglycerol (DAG) and TAG. Phospholipids, DAG and free fatty acids were then found in hemolymph, from where they were rapidly cleared, and label was found in the fat body, mainly associated with TAG. Radioactive lipids, especially TAG and phospholipids, also accumulated in the ovaries. The TAG-lipase activities of posterior midgut luminal content and tissue were characterized by incubation of these samples with (3)H-triolein in the presence of the detergent Triton X-100 and determination of the amounts of released radioactive free fatty acids. Under the conditions employed here, the release of free fatty acids was proportional to the incubation time and to the amount of sample obtained from insect midgut (enzyme source) that was added. TAG-lipase activities were affected by pH and posterior midgut tissue showed optimum activity around pH 7.0-7.5, but the luminal content had the highest activities as pH decreased. Differences in activities were observed according to calcium concentration in the medium. TAG-lipase activities were also affected by the concentration of NaCl and were activated in the presence of increasing salt concentrations. These activities were inhibited by phenylmethylsulphonyl fluoride (PMSF). On the second day after blood meal, when digestion is very intense, TAG-lipase activities were maximal and then gradually decreased.

Characterization of lipophorin binding to the fat body of Rhodnius prolixus.

In insects, lipids are transported by a hemolymphatic lipoprotein, lipophorin. The binding of lipophorin to the fat body of the hematophagous insect Rhodnius prolixus was characterized in a fat body membrane preparation, obtained from adult females. For the binding assay, purified lipophorin was radiolabelled in the protein moiety (125I-HDLp), and it was shown that iodination did not affect the affinity of the membrane preparation for lipophorin. Under incubation conditions used, lipophorin binding to membranes achieved equilibrium after 40-60 min, but this time was longer when a low concentration of lipophorin was present in the medium. The capacity of the fat body membrane preparation to bind lipophorin was abolished when membranes were pre-treated with trypsin, and it was also affected by heat. When 125I-HDLp was incubated with increasing concentrations of membrane protein, corresponding increases in binding were observed. Lipophorin binding was sensitive to pH, and it was maximal between pH 6.0 and 7.0. The specific binding of lipophorin to the fat body membrane preparation was a saturable process, with a Kd of 2.1 +/- 0.4 x 10(-7)M and a maximal binding capacity of 289 +/- 88 ng lipophorin/microgram of membrane protein. Binding to the fat body membranes did not depend on calcium, but it was affected by ionic strength, being totally inhibited at high salt concentrations. Suramin also interfered with lipophorin binding and it was abolished in the presence of 2 mM suramin, but at concentrations of 0.05 and 0.1 mM it seemed to increase binding activity slightly. Fat body membrane preparation from Rhodnius prolixus was able to bind lipophorin from Manduca sexta larvae.