Digestive α-L-fucosidase activity in Rhodnius prolixus after blood feeding: effect of secretagogue and nutritional stimuli.

Introduction: Rhodnius prolixus (Hemiptera: Reduviidae) is an important vector of Trypanosoma cruzi, the causative agent of Chagas Disease. This insect is a model for the study of insect physiology, especially concerning the digestion of blood. Among the enzymes produced in the midgut of R. prolixus after blood feeding there is a α-L-fucosidase activity. There are very few studies on α-L-fucosidase of insects, and the role of R. prolixus α-L-fucosidase is still not clear. In this work, we tested if the mechanism for production of this enzyme is similar to the observed for proteases, a secretatogue mechanism that respond to the protein contents of the meal. Methods: We tested if specific proteins or sugars elicit this response, which may help to understand the nature of the physiological substrate for this enzyme. Results: In general, our results showed that the Anterior Midgut was the only midgut fraction that responds to the blood meal in terms of α-L-fucosidase production. Besides that, this response was not triggered by midgut distension or by ingestion of the blood cell fraction. Conversely, the enzyme was produced after feeding with the plasma fraction. However, the production of α-L-fucosidase was also triggered by different biochemical stimuli, as protein or fucoidan ingestion. Discussion: This suggested that the production of the enzyme in the anterior midgut was a general physiological response under control of different convergent signals. Besides that, the comparison between different treatments for artificial blood feeding showed that heparinated blood was the choice with minor side effects for the study of the midgut α-L-fucosidase, when compared to defibrinated or citrated blood.

Structure and expression of Rhodnius prolixus GH18 chitinases and chitinase-like proteins: Characterization of the physiological role of RpCht7, a gene from subgroup VIII, in vector fitness and reproduction.

Chitinases are enzymes responsible for the hydrolysis of glycosidic linkages within chitin chains. In insects, chitinases are typically members of the multigenic glycoside hydrolase family 18 (GH18). They participate in the relocation of chitin during development and molt, and in digestion in detritivores and predatory insects, and they control the peritrophic membrane thickness. Chitin metabolism is a promising target for developing vector control strategies, and knowledge of the roles of chitinases may reveal new targets and illuminate unique aspects of their physiology and interaction with microorganisms. Rhodnius prolixus is an important vector of Chagas disease, which is caused by the parasite Trypanosoma cruzi. In this study, we performed annotation and structural characterization of nine chitinase and chitinase-like protein genes in the R. prolixus genome. The roles of their corresponding transcripts were studied in more depth; their physiological roles were studied through RNAi silencing. Phylogenetic analysis of coding sequences showed that these genes belong to different subfamilies of GH18 chitinases already described in other insects. The expression patterns of these genes in different tissues and developmental stages were initially characterized using RT-PCR. RNAi screening showed silencing of the gene family members with very different efficiencies. Based on the knockdown results and the general lack of information about subgroup VIII of GH18, the RpCht7 gene was chosen for phenotype analysis. RpCht7 knockdown doubled the mortality in starving fifth-instar nymphs compared to dsGFP-injected controls. However, it did not alter blood intake, diuresis, digestion, molting rate, molting defects, sexual ratio, percentage of hatching, or average hatching time. Nevertheless, female oviposition was reduced by 53% in RpCht7-silenced insects, and differences in oviposition occurred within 14-20 days after a saturating blood meal. These results suggest that RpCht7 may be involved in the reproductive physiology and vector fitness of R. prolixus.

Biochemical and Functional Characterization of Glycoside Hydrolase Family 16 Genes in Aedes aegypti Larvae: Identification of the Major Digestive ß-1,3-Glucanase.

Insect ß-1,3-glucanases belong to Glycoside Hydrolase Family 16 (GHF16) and are involved in digestion of detritus and plant hemicellulose. In this work, we investigated the role of GHF16 genes in Aedes aegypti larvae, due to their detritivore diet. Aedes aegypti genome has six genes belonging to GHF16 (Aae GH16.1 - Aae GH16.6), containing two to six exons. Sequence analysis suggests that five of these GHF16 sequences (Aae GH16.1, 2, 3, 5, and 6) contain the conserved catalytic residues of this family and correspond to glucanases. All genomes of Nematocera analyzed showed putative gene duplications corresponding to these sequences. Aae GH16.4 has no conserved catalytic residues and is probably a ß-1,3-glucan binding protein involved in the activation of innate immune responses. Additionally, Ae. aegypti larvae contain significant ß-1,3-glucanase activities in the head, gut and rest of body. These activities have optimum pH about 5-6 and molecular masses between 41 and 150 kDa. All GHF16 genes above showed different levels of expression in the larval head, gut or rest of the body. Knock-down of AeGH16.5 resulted in survival and pupation rates lower than controls (dsGFP and water treated). However, under stress conditions, severe mortalities were observed in AeGH16.1 and AeGH16.6 knocked-down larvae. Enzymatic assays of ß-1,3-glucanase in AeGH16.5 silenced larvae exhibited lower activity in the gut and no change in the rest of the body. Chromatographic activity profiles from gut samples after GH16.5 silencing showed suppression of enzymatic activity, suggesting that this gene codes for the digestive larval ß-1,3-glucanase of Ae. aegypti. This gene and enzyme are attractive targets for new control strategies, based on the impairment of normal gut physiology.

Caracterização funcional de quitinases de Rhodnius prolixus

Quitinases são enzimas responsáveis pela hidrólise de ligações glicosídicas no interior de cadeias de quitina. Em insetos, elas estão relacionadas ao remanejamento da quitina durante o desenvolvimento e ecdise, na digestão de insetos detritívoros e predadores, e no controle da espessura da membrana peritrófica intestinal. As quitinases de insetos descritas até o momento pertencem à família 18 das glicosídeo hidrolases. O Rhodnius prolixus é um dos principais vetores da doença de Chagas, causada pelo parasita Trypanosoma cruzi. No genoma de R. prolixus são encontrados 9 genes de quitinases, mas o papel dos transcritos correspondentes ainda não foi estudado em detalhe. O objetivo deste trabalho é o estudo detalhado da expressão dos transcritos de quitinase de R. prolixus e análise de seu papel fisiológico através do silenciamento por RNAi. O conhecimento do papel de quitinases neste inseto pode revelar novos alvos para controle do vetor, assim como revelar novos aspectos de sua fisiologia e da interação entre o inseto e microrganismos Através de análises filogenéticas das sequências codificantes de quitinases, foi constatado que esses genes pertencem à diferentes grupos da família 18 das glicosídeo hidrolases, já descritos em outros insetos. Para estudo funcional verificou-se o efeito do silenciamento da expressão desses genes utilizando RNAi. Foi realizada uma curva de concentração injetando quantidades crescentes de dsRNA nos insetos (1\03BCg, 3\03BCg, 6\03BCg e 2 x 3\03BCg) a fim de definir qual a menor concentração que permitiria obter uma maior porcentagem de silenciamento para os diferentes genes GH18. Com base nos resultados obtidos o gene RpCht7 foi escolhido para análise dos fenótipos, pois apresentou uma alta taxa de silenciamento. O silenciamento do gene RpCht7 dobrou a mortalidade de insetos em jejum quando comparado ao controle injetado com dsGFP, mas não alterou a ingestão sanguínea, diurese, digestão, porcentagem de muda, defeitos na muda, razão sexual, tempo médio de eclosão e porcentagem de eclosão dos ovos postos. Entretanto, foi observada uma alteração na oviposição, uma vez que as fêmeas silenciadas para esse gene colocam 34% ovos a menos que os controles. As diferenças na postura de ovos ocorrem de 14 a 20 dias após a alimentação sanguínea. Dessa forma, fica inferido que o gene RpCht7 tem um papel importante na reprodução e pode ser um alvo promissor pra manipulação da aptidão vetorial de R. prolixus