Chitin is a component of the Rhodnius prolixus midgut.

Chitin is an essential component of the peritrophic matrix (PM), which is a structure that lines the insect's gut and protects against mechanical damage and pathogens. Rhodnius prolixus (Hemiptera: Reduviidae) does not have a PM, but it has an analogous structure, the perimicrovillar membrane (PMM); chitin has not been described in this structure. Here, we show that chitin is present in the R. prolixus midgut using several techniques. The FTIR spectrum of the KOH-resistant putative chitin-material extracted from the midgut bolus showed peaks characteristic of the chitin molecule at 3500, 1675 and 1085 cm(1). Both the midgut bolus material and the standard chitin NMR spectra showed a peak at 1.88 ppm, which is certainly due to methyl protons in the acetamide a group. The percentages of radioactive N-acetylglucosamine (CPM) incorporated were 2 and 4% for the entire intestine and bolus, respectively. The KOH-resistant putative chitin-material was also extracted and purified from the N-acetylglucosamine radioactive bolus, and the radioactivity was estimated through liquid scintillation. The intestinal CHS cDNA translated sequence was the same as previously described for the R. prolixus cuticle and ovaries. Phenotypic alterations were observed in the midgut of females with a silenced CHS gene after a blood meal, such as retarded blood meal digestion; the presence of fresh blood that remained red nine days after the blood meal; and reduced trachea and hemozoin content compared with the control. Wheat germ agglutinin (a specific probe that detects chitin) labeling proximal to the intestine (crop and midgut) was much lower in females with a silenced CHS gene, especially in the midgut region, where almost no fluorescence signal was detected compared with the control groups. Midguts from females with a CHS gene silenced by dsRNA-CHS and control midguts pre-treated with chitinase showed that the chitin-derived fluorescence signal decreased in the region around the epithelium, the region facing the midgut and projections towards the intestinal lumen when evaluated microscopically. The relative reduction in CHS transcripts by approximately 80% using an RNAi assay supports the phenotypical alterations in the midgut observed using fluorescence microscopy assays. These data show that chitin is present in the R. prolixus midgut epithelium and in its surface projections facing the lumen. The CHS gene expression and the presence of chitin in the R. prolixus midgut may suggest a target for controlling Chagas disease vectors and addressing this public health problem.

Effects of chitin synthase double-stranded RNA on molting and oogenesis in the Chagas disease vector Rhodnius prolixus.

In this study, we provided the demonstration of the presence of a single CHS gene in the Rhodnius prolixus (a blood-sucking insect) genome that is expressed in adults (integument and ovary) and in the integument of nymphs during development. This CHS gene appears to be essential for epidermal integrity and egg formation in R. prolixus. Because injection of CHS dsRNA was effective in reducing CHS transcript levels, phenotypic alterations in the normal course of ecdysis occurred. In addition, two phenotypes with severe cuticle deformations were observed, which were associated with loss of mobility and lifetime. The CHS dsRNA treatment in adult females affected oogenesis, reducing the size of the ovary and presenting a greater number of atresic oocytes and a smaller number of chorionated oocytes compared with the control. The overall effect was reduced oviposition. The injection of CHS dsRNA modified the natural course of egg development, producing deformed eggs that were dark in color and unable to hatch, distinct from the viable eggs laid by control females. The ovaries, which were examined under fluorescence microscopy using a probe for chitin detection, showed a reduced deposition on pre-vitellogenic and vitellogenic oocytes compared with control. Taken together, these data suggest that the CHS gene is fundamentally important for ecdysis, oogenesis and egg hatching in R. prolixus and also demonstrated that the CHS gene is a good target for controlling Chagas disease vectors.

Chitin deposition on the embryonic cuticle of Rhodnius prolixus: the reduction of CHS transcripts by CHS-dsRNA injection in females affects chitin deposition and eclosion of the first instar nymph.

In a previous study, we found that the embryonic cuticle of Rhodnius prolixus is a chitin-based structure that helps the first instar nymph to hatch from the chorion. Here, we investigated how the reduction of transcripts induced by CHS dsRNA injection affects R. prolixus embryogenesis and eclosion. Deposition of chitin in the embryonic cuticle begins later at embryogenesis, around day 8, and ends approximately at day 15, when the insects are ready for eclosion. In R. prolixus, chitin deposition follows pari passu with the synthesis of the chitin synthase mRNA, indicating a regulation at the transcriptional level. The reduction of the chitin synthase gene transcripts by the injection of CHS dRNA prevented chitin deposition during embryonic cuticle formation, being lethal to hatching nymphs, which end up dying while stuck in the chorionic border trying to leave the chorion. The successful eclosion rates were reduced by 60% in animals treated with CHS dsRNA when compared to animals injected with a control (dsRNA no related gene or water). We found that the harmful effects on oviposition and eclosion are possibly due to changes in the structure of the embryonic cuticle, as observed by directly comparing the morphology of control and chitin-deficient embryonic cuticles under the transmission electron microscope. The lack of chitin and changes in its morphological characteristics appears to alter the embryonic cuticle physiology and functionality. Additionally, we observed that the effects of CHS dRNA treatment on R. prolixus females lasted up to 3 egg-laying cycles (∼100 days), pointing to R. prolixus as a useful model for developmental studies.

A chitin-like component in Aedes aegypti eggshells, eggs and ovaries.

An insoluble white substance was prepared from extracts of eggshells of Aedes aegypti, the yellow fever mosquito and dengue vector. Its infrared and proton NMR spectra were similar to that of standard commercial chitin. This putative chitin-like material, also obtained from ovaries, newly laid and dark eggs, was hydrolyzed in acid and a major product was identified by HPLC to be glucosamine. The eggshell acid hydrolysate was also analyzed by ESI-MS and an ion identical to a glucosamine monoprotonated species was detected. The presence of chitin was also analyzed during different developmental stages of the ovary using a fluorescent microscopy technique and probes specific for chitin. The results showed that a chitin-like material accumulates in oocytes during oogenesis. Streptomyces griseus chitinase pre-treatment of oocytes greatly reduced the chitin-derived fluorescence. Chitinase activity was detected in newborn larvae and eggs prior to hatching. Feeding experiments indicated that the chitin synthesis inhibitor lufenuron inhibited chitin synthesis, either when mosquitoes were allowed to feed directly on lufenuron-treated chickens or when an artificial feeding system was used. Lufenuron inhibited egg hatch, larval development and reduced mosquito viability. These data demonstrate for the first time that (1) a chitin-like material is present in A. aegypti eggs, ovaries and eggshells; (2) a chitin synthesis inhibitor can be used to inhibit mosquito oogenesis; and (3) chitin synthesis inhibitors have potential for controlling mosquito populations.