Role of Methoprene-tolerant in the regulation of oogenesis in Dipetalogaster maxima.

Juvenile hormone (JH) signalling, via its receptor Methoprene-tolerant (Met), controls metamorphosis and reproduction in insects. Met belongs to a superfamily of transcription factors containing the basic Helix Loop Helix (bHLH) and Per Arnt Sim (PAS) domains. Since its discovery in 1986, Met has been characterized in several insect species. However, in spite of the importance as vectors of Chagas disease, our knowledge on the role of Met in JH signalling in Triatominae is limited. In this study, we cloned and sequenced the Dipetalogaster maxima Met transcript (DmaxMet). Molecular modelling was used to build the structure of Met and identify the JH binding site. To further understand the role of the JH receptor during oogenesis, transcript levels were evaluated in two main target organs of JH, fat body and ovary. Functional studies using Met RNAi revealed significant decreases of transcripts for vitellogenin (Vg) and lipophorin (Lp), as well as their receptors. Lp and Vg protein amounts in fat body, as well as Vg in hemolymph were also decreased, and ovarian development was impaired. Overall, these studies provide additional molecular insights on the roles of JH signalling in oogenesis in Triatominae; and therefore are relevant for the epidemiology of Chagas´ disease.

Morphological and Ultrastructural Characterization of Hemocytes in an Insect Model, the Hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae).

Hemocytes, the cells present in the hemolymph of insects and other invertebrates, perform several physiological functions, including innate immunity. The current classification of hemocyte types is based mostly on morphological features; however, divergences have emerged among specialists in triatomines, the insect vectors of Chagas' disease (Hemiptera: Reduviidae). Here, we have combined technical approaches in order to characterize the hemocytes from fifth instar nymphs of the triatomine Dipetalogaster maxima. Moreover, in this work we describe, for the first time, the ultrastructural features of D. maxima hemocytes. Using phase contrast microscopy of fresh preparations, five hemocyte populations were identified and further characterized by immunofluorescence, flow cytometry and transmission electron microscopy. The plasmatocytes and the granulocytes were the most abundant cell types, although prohemocytes, adipohemocytes and oenocytes were also found. This work sheds light on a controversial aspect of triatomine cell biology and physiology setting the basis for future in-depth studies directed to address hemocyte classification using non-microscopy-based markers.

Juvenile hormone mediates lipid storage in the oocytes of Dipetalogaster maxima.

Triatomines are vectors of Chagas disease and important model organisms in insect physiology. "Kissing bugs" are obligatory hematophagous insects. A blood meal is required to successfully complete oogenesis, a process primarily controlled by juvenile hormone (JH). We used Dipetalogaster maxima as an experimental model to further understand the roles of JH in the regulation of vitellogenesis and oogenesis. A particular focus was set on the role of JH controlling lipid and protein recruitment by the oocytes. The hemolymph titer of JH III skipped bisepoxide increased after a blood meal. Following a blood meal there were increased levels of mRNAs in the fat body for the yolk protein precursors, vitellogenin (Vg) and lipophorin (Lp), as well as of their protein products in the hemolymph; mRNAs of the Vg and Lp receptors (VgR and LpR) were concomitantly up-regulated in the ovaries. Topical administration of JH induced the expression of Lp/LpR and Vg/VgR genes, and prompted the uptake of Lp and Vg in pre-vitellogenic females. Knockdown of the expression of LpR by RNA interference in fed females did not impair the Lp-mediated lipid transfer to oocytes, suggesting that the bulk of lipid acquisition by oocytes occurred by other pathways rather than by the endocytic Lp/LpR pathway. In conclusion, our results strongly suggest that JH signaling is critical for lipid storage in oocytes, by regulating Vg and Lp gene expression in the fat body as well as by modulating the expression of LpR and VgR genes in ovaries.

The entomotoxin Jack Bean Urease changes cathepsin D activity in nymphs of the hematophagous insect Dipetalogaster maxima (Hemiptera: Reduviidae).

In insects, cathepsin D is a lysosomal aspartic endopeptidase involved in several functions such as digestion, defense and reproduction. Jack Bean Urease (JBU) is the most abundant urease isoform obtained from the seeds of the plant Canavalia ensiformis. JBU is a multifunctional protein with entomotoxic effects unrelated to its catalytic activity, by mechanisms not yet fully understood. In this work, we employed nymphs of the hematophagous insect Dipetalogaster maxima as an experimental model in order to study the effects of JBU on D. maxima CatD (DmCatD). In insects without treatment, immunofluorescence assays revealed a conspicuous distribution pattern of DmCatD in the anterior and posterior midgut as well as in the fat body and hemocytes. Western blot assays showed that the active form of DmCatD was present in the fat body, the anterior and posterior midgut; whereas the proenzyme was visualized in hemocytes and hemolymph. The transcript of DmCatD and its enzymatic activity was detected in the anterior and posterior midgut as well as in fat body and hemocytes. JBU injections induced a significant increase of DmCatD activity in the posterior midgut (at 3 h post-injection) whereas in the hemolymph, such an effect was observed after 18 h. These changes were not correlated with modifications in DmCatD mRNA and protein levels or changes in the immunofluorescence pattern. In vitro experiments might suggest a direct effect of the toxin in DmCatD activity. Our findings indicated that the tissue-specific increment of cathepsin D activity is a novel effect of JBU in insects.

The Fat Body of the Hematophagous Insect, Panstrongylus megistus (Hemiptera: Reduviidae): Histological Features and Participation of the ß-Chain of ATP Synthase in the Lipophorin-Mediated Lipid Transfer.

In insects, lipid transfer to the tissues is mediated by lipophorin, the major circulating lipoprotein, mainly through a nonendocytic pathway involving docking receptors. Currently, the role of such receptors in lipid metabolism remains poorly understood. In this work, we performed a histological characterization of the fat body of the Chagas' disease vector, Panstrongylus megistus (Burmeister), subjected to different nutritional conditions. In addition, we addressed the role of the ß-chain of ATP synthase (ß-ATPase) in the process of lipid transfer from lipophorin to the fat body. Fifth-instar nymphs in either fasting or fed condition were employed in the assays. Histological examination revealed that the fat body was composed by diverse trophocyte phenotypes. In the fasting condition, the cells were smaller and presented a homogeneous cytoplasmic content. The fat body of fed insects increased in size mainly due to the enlargement of lipid stores. In this condition, trophocytes contained abundant lipid droplets, and the rough endoplasmic reticulum was highly developed and mitochondria appeared elongated. Immunofluorescence assays showed that the ß-ATPase, a putative lipophorin receptor, was located on the surface of fat body cells colocalizing partially with lipophorin, which suggests their interaction. No changes in ß-ATPase expression were found in fasting and fed insects. Blocking the lipophorin-ß-ATPase interaction impaired the lipophorin-mediated lipid transfer to the fat body. The results showed that the nutritional status of the insect influenced the morphohistological features of the tissue. Besides, these findings suggest that ß-ATPase functions as a lipophorin docking receptor in the fat body.

The process of lipid storage in insect oocytes: The involvement of ß-chain of ATP synthase in lipophorin-mediated lipid transfer in the chagas' disease vector Panstrongylus megistus (Hemiptera: Reduviidae).

Lipophorin is the main lipoprotein in the hemolymph of insects. During vitellogenesis, lipophorin delivers its hydrophobic cargo to developing oocytes by its binding to non-endocytic receptors at the plasma membrane of the cells. In some species however, lipophorin may also be internalized to some extent, thus maximizing the storage of lipid resources in growing oocytes. The ectopic ß chain of ATP synthase (ß-ATPase) was recently described as a putative non-endocytic lipophorin receptor in the anterior midgut of the hematophagous insect Panstrongylus megistus. In the present work, females of this species at the vitellogenic stage of the reproductive cycle were employed to investigate the role of ß-ATPase in the transfer of lipids to the ovarian tissue. Subcellular fractionation and western blot revealed the presence of ß-ATPase in the microsomal membranes of the ovarian tissue, suggesting its localization in the plasma membrane. Immunofluorescence assays showed partial co-localization of ß-ATPase and lipophorin in the membrane of oocytes as well as in the basal domain of the follicular epithelial cells. Ligand blotting and co-immunoprecipitation approaches confirmed the interaction between lipophorin and ß-ATPase. In vivo experiments with an anti-ß-ATPase antibody injected to block such an interaction demonstrated that the antibody significantly impaired the transfer of fatty acids from lipophorin to the oocyte. However, the endocytic pathway of lipophorin was not affected. On the other hand, partial inhibition of ATP synthase activity did not modify the transfer of lipids from lipophorin to oocytes. When the assays were performed at 4°C to diminish endocytosis, the results showed that the antibody interfered with lipophorin binding to the oocyte plasma membrane as well as with the transfer of fatty acids from the lipoprotein to the oocyte. The findings strongly support that ß-ATPase plays a role as a docking lipophorin receptor at the ovary of P. megistus, similarly to its function in the midgut of such a vector. In addition, the role of ß-ATPase as a docking receptor seems to be independent of the enzymatic ATP synthase activity.