Trypsin-Like Serine Proteases in Lutzomyia longipalpis – Expression, Activity and Possible Modulation by Leishmania infantum chagasi

Background: Midgut enzymatic activity is one of the obstacles that Leishmania must surpass to succeed in establishing infection. Trypsins are abundant digestive enzymes in most insects. We have previously described two trypsin cDNAs of L. longipalpis: one (Lltryp1) with a bloodmeal induced transcription pattern, the other (Lltryp2) with a constitutive transcription pattern. We have now characterized the expression and activity of trypsin-like proteases of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil. Methodology and Principal Findings: In order to study trypsin expression profiles we produced antibodies against peptides specific for Lltryp1 and Lltryp2. The anti-Lltryp1-peptide antibody revealed a band of 28 kDa between 6 and 48 hours. The anti-Lltryp2 peptide antibody did not evidence any band. When proteinaceous substrates (gelatin, hemoglobin, casein or albumin) were co-polymerized in polyacrylamide gels, insect midguts obtained at 12 hours after feeding showed a unique proteolytic pattern for each substrate. All activity bands were strongly inhibited by TLCK, benzamidine and 4-amino-benzamidine, indicating that they are trypsin-like proteases. The trypsin-like activity was also measured in vitro at different time points after ingestion of blood or blood containing Leishmania infantum chagasi, using the chromogenic substrate BArNA. L. longipalpis females fed on blood infected with L. i. chagasi had lower levels of trypsin activity after 12 and 48 hours than non-infected insects, suggesting that the parasite may have a role in this modulation. Conclusions and Significance: Trypsins are important and abundant digestive enzymes in L. longipalpis. Protein production and enzymatic activity followed previously identified gene expression of a blood modulated trypsin gene. A decrease of enzymatic activity upon the parasite infection, previously detected mostly in Old World vectors, was detected for the first time in the natural vector-parasite pair L. longipalpis-L. i. chagasi

Prevenção domiciliar da dengue: avaliação preliminar de tela protetora para pratos de vasos de planta

Avaliou-se em laboratório a eficácia de um protótipo de capa de tela de poliéster (evidengue®) destinada a vedar o acesso de fêmeas do mosquito Aedes aegypti a pratos de vasos de planta. Dois pratos de vasos com água foram envolvidos individualmente com a capa e colocados com os seus respectivos vasos em duas gaiolas entomológicas, um em cada gaiola. Numa terceira gaiola foi colocado um conjunto idêntico de prato e vasos sem a capa. Cada gaiola recebeu 20 fêmeas copuladas do mosquito, alimentadas com sangue de camundongo. Os resultados mostram que a capa foi eficaz como barreira ao acesso de fêmeas. Novos testes são necessários para se avaliar a eficácia da capa como dispositivo de prevenção da ovipostura nos pratos

Lutzomyia umbratilis from an area south of the Negro River is refractory to in vitro interaction with Leishmania guyanensis

BACKGROUND Lutzomyia umbratilis, the vector for Leishmania guyanensis in northern South America, has been found naturally infected with L. guyanensis only in areas north of the Negro and Amazon rivers. While populations of this sand fly species are also found in areas south of these rivers, these populations have never been reported to be infected and/or transmitting L. guyanensis. However, no studies on the corresponding host-parasite interactions are available. OBJECTIVES This study evaluated the interaction between Lu. guyanensis promastigotes and field-collected Lu. umbratilis sand flies from Rio Preto da Eva and Manacapuru, which are located to the north and south, respectively, of the Negro River. METHODS Procyclic and metacyclic attachment was quantified using an in vitro system. FINDINGS Low attachment of parasites to the midguts of insects collected from Manacapuru was detected. Conversely, greater binding of metacyclic parasites was observed in the midguts of insects collected from Rio Preto da Eva, and this attachment was more pronounced than that observed for procyclics (p < 0.03). MAIN CONCLUSIONS The Lu. umbratilis population from an area south of the Negro River has lower in vitro interaction with L. guyanensis. The higher attachment of L. guyanensis to midguts of insects from Rio Preto da Eva may suggest better vector competence. These findings are in accordance with previously reported epidemiological information of American cutaneous leishmaniasis (ACL) transmission in the Amazon

Enzymatic assay performed in solution with trypsin synthetic substrate BAρNA.

<p>Horizontal axis indicates insect midgut samples obtained at different hours ABF. Vertical axis indicate enzyme units detected per midgut. <b>0</b>: sugar-fed females; <b>6</b> to <b>72</b>: females obtained at 6, 12, 24, 48 and 72 hours ABF. White bars indicate samples obtained from non infected insect midguts. Gray bars indicate samples obtained from infected insect midguts. Samples corresponding to half of one midgut were used in each assay. Statistically significant difference (<i>P</i><0.05) is marked with an asterisk.</p