The influence of larval density on triacylglycerol content in Aedes aegypti (Linnaeus) (Diptera: Culicidae).

Given the importance of Aedes aegypti as an arbovirus vector, understanding the impact of population density during larval development is fundamental to improve control methods. The aims of this study were to analyze the effects of larval density on the triacylglycerol (TAG) reserves, fecundity, and the size of mosquitos. To evaluate the influence of density on the amount of TAG in the different stages of development of A. aegypti, the larvae were reared in densities of 0.04, 0.32, and 0.8 larvae/ml, represented by D1, D2, and D3, respectively. Larval rearing density affected the amount of TAG in the different developmental stages of this insect. The group reared with the highest population density (D3) presented greater storage of TAG, except when analyzing the amount of TAG µg/protein in the fat body of blood-fed females. In addition, it was possible to observe higher resistance to fasting and a greater number of eggs laid. Insects reared in D2 and D3 were larger than those in the D1 group. This study provides interesting data for future investigations on how the density of larval rearing affects the lipid metabolism for synthesis and mobilization of TAG stored in A. aegypti and how it influences size and reproduction.

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.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas´ disease vector Rhodnius prolixus

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as “alecrim-pimenta”. Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas’ disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Lethal and sublethal effects of essential oil of Lippia sidoides (Verbenaceae) and monoterpenes on Chagas' disease vector Rhodnius prolixus.

The aim of this study was to identify the composition of the essential oil from leaves of Lippia sidoides (EOLS), a typical shrub commonly found in the dry northeast of Brazil, popularly known as "alecrim-pimenta". Additionally, we investigated the nymphicidal, ovicidal, phagoinhibitory and excretion effects of EOLS, its major constituent thymol and its isomer carvacrol, on fourth instar nymphs and eggs of Rhodnius prolixus, the Chagas' disease vector. The nymphicidal and ovicidal activity of thymol, carvacrol, and EOLS was assessed by tests using impregnated Petri dishes. The lethal concentration values (LC50) for EOLS, carvacrol, and thymol were 54.48, 32.98, and 9.38 mg/cm2, respectively. The ovicidal test showed that both carvacrol and thymol (50 mg/cm2) inhibited hatching (50% and 23.3%, respectively), while treatments with 10 mg/cm2 or 50 mg/cm2 EOLS did not affect the hatching rate at all (80% and 90%, respectively). We observed an anti-feeding effect in insects fed with blood containing natural products at the higher concentrations (100 µg/mL). Finally, excretion rate was affected by EOLS and carvacrol, but not by thymol. These findings offer novel insights into basic physiological processes that make the tested natural compounds interesting candidates for new types of insecticides.

Neutral lipid composition changes in the fat bodies of engorged females Rhipicephalus microplus ticks in response to fungal infections.

The tick's fat body plays an essential role in energy storage and utilization. This study aimed to analyze the fat body neutral lipid composition in Rhipicephalus microplus engorged females. In the first study (physiological profile of untreated ticks), the lipid analysis took place over the course of 4 days; the engorged females were incubated at optimal conditions and their fat bodies were dissected daily. Fat body lipid analysis after fungal infection with Metarhizium anisopliae sensu lato (s.l.) or Beauveria bassiana s.l. was performed with four groups: one without any treatment, one that was inoculated with a solution of 0.1 % Tween 80 in water, and two groups that were inoculated with M. anisopliae or B. bassiana conidial suspensions. The fat bodies were dissected 24 and 48 h after infection. Lipid analysis was conducted by thin-layer chromatography on a silica plate. The results of the physiological profile showed that the amounts of triacylglycerol (TAG) and free cholesterol (CHO) decreased with time, whereas cholesterol ester (CHOE) increased on the second and fourth days. Following M. anisopliae or B. bassiana infection, there was an increase in the amount of CHO after 24 h, whereas the other lipid classes were not altered. M. anisopliae caused an increase in CHOE and TAG and a reduction in CHO at 48 h after infection; however, B. bassiana infection did not cause significant alterations in the concentrations of these lipids. M. anisopliae and B. bassiana infection changed the fat body metabolism of engorged female R. microplus ticks. This study provides the first report of changes in the neutral lipid composition of the R. microplus fat body.

Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading cysteine endopeptidase.

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites.

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.

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.