Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Blood meal digestion and changes in lipid reserves are associated with the post-ecdysis development of the flight muscle and ovary in young adults of Rhodnius prolixus.

Rhodnius prolixus is a hemimetabolous, hematophagous insect, and both nymphs and adults feed exclusively on blood. The blood feeding triggers the molting process and, after five nymphal instar stages, the insect reaches the winged adult form. After the final ecdysis, the young adult still has a lot of blood in the midgut and, thus, we have investigated the changes in protein and lipid contents that are observed in the insect organs as digestion continues after molting. Total midgut protein content decreased during the days after the ecdysis, and digestion was finished fifteen days later. Simultaneously, proteins and triacylglycerols present in the fat body were mobilized, and their contents decreased, whereas they increased in both the ovary and the flight muscle. In order to evaluate the activity of de novo lipogenesis of each organ, the fat body, ovary and flight muscle were incubated in the presence of radiolabeled acetate, and the fat body showed the highest efficiency rate (around 47%) to convert the taken up acetate into lipids. The levels of de novo lipid synthesis in the flight muscle and ovary were very low. When 3H-palmitate was injected into the young females, its incorporation by the flight muscle was higher than by the ovary or the fat body. In the flight muscle, the 3H-palmitate was similarly distributed amongst triacylglycerols, phospholipids, diacylglycerols and free fatty acids, while in the ovary and fat body it was mostly found in triacylglycerols and phospholipids. The flight muscle was not fully developed after the molt, and at day two no lipid droplets were observed. At day five, very small lipid droplets were present, and they increased in size up to day fifteen. The diameter of the muscle fibers also increased from day two to fifteen, as well as the internuclear distance, indicating that muscle hypertrophy occurred along these days. The lipid droplets from the fat body showed a different pattern, and their diameter decreased after day two, but started to increase again at day ten. The data presented herein describes the development of the flight muscle after the final ecdysis, and modifications that occur regarding lipid stores. We show that, after molting, substrates that are present in the midgut and fat body are mobilized and directed to the ovary and flight muscle, for the adults of R. prolixus to be ready to feed and reproduce.

Deficiency of Acetyl-CoA Carboxylase Impairs Digestion, Lipid Synthesis, and Reproduction in the Kissing Bug Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, vector of Chagas disease. After feeding, as blood is slowly digested, amino acids are used as substrates to fuel lipid synthesis, and adult females accumulate lipids in the fat body and produce eggs. In order to evaluate the importance of de novo fatty acid synthesis for this insect metabolism, we generated acetyl-CoA carboxylase (ACC) deficient insects. The knockdown (AccKD) females had delayed blood digestion and a shorter lifespan. Their fat bodies showed reduced de novo lipogenesis activity, did not accumulate triacylglycerol during the days after blood meal, and had smaller lipid droplets. At 10 days after feeding, there was a general decrease in the amounts of neutral lipids and phospholipids in the fat body. In the hemolymph, no difference was observed in lipid composition at 5 days after blood meal, but at day ten, there was an increase in hydrocarbon content and a decrease in phospholipids. Total protein concentration and amino acid composition were not affected. The AccKD females laid 60% fewer eggs than the control ones, and only 7% hatched (89% for control), although their total protein and triacylglycerol contents were not different. Scanning electron microscopy of the egg surface showed that chorion (eggshell) from the eggs laid by the AccKD insects had an altered ultrastructural pattern when compared to control ones. These results show that ACC has a central role in R. prolixus nutrient homeostasis, and its appropriate activity is important to digestion, lipid synthesis and storage, and reproductive success.

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.

Inhibition of energy metabolism by 3-bromopyruvate in the hard tick Rhipicephalus microplus.

The cattle tick R. microplus is the biggest obstacle to livestock rearing in tropical countries. It is responsible for billions of dollars in losses every year, affecting meat and milk production, beef and dairy cattle, and the leather industry. The lack of knowledge and strategies to combat the tick only increases the losses, it leads to successive and uncontrolled applications of acaricides, favouring the selection of strains resistant to commercially available chemical treatments. In this paper, we tested 3­bromopyruvate (3­BrPA), an alkylating agent with a high affinity for cysteine residues, on the R. microplus metabolism. We found that 3-BrPA was able to induce cell death in an assay using BME26 strain cell cultures derived from embryos, it was also able to reduce cellular respiration in developing embryos. 3-BrPA is a nonspecific inhibitor, affecting enzymes of different metabolic pathways in R. microplus. In our experiments, we demonstrated that 3-BrPA was able to affect the glycolytic enzyme hexokinase, reducing its activity by approximately 50%; and it strongly inhibited triose phosphate isomerase, which is an enzyme involved in both glycolysis and gluconeogenesis. Also, the mitochondrial respiratory chain was affected, NADH cytochrome c reductase (complex I-III) and succinate cytochrome c reductase (complex II-III) were strongly inhibited by 3-BrPA. Glutamate dehydrogenase was also affected by 3-BrPA, showing a gradual inhibition of activity in all the 3-BrPA concentrations tested. Altogether, these results show that 3-BrPA is a harmful compound to the tick organism.