Reverse transcription-PCR detection of LaCrosse virus in mosquitoes and comparison with enzyme immunoassay and virus isolation.

A reverse transcription-PCR (RT-PCR) assay was developed and compared with enzyme immunoassay (EIA) and virus isolation for detecting LaCrosse virus (LAC) in mosquito pools. All three techniques were able to detect a single LAC-infected mosquito in a pool of 99 negative mosquitoes. Virus isolation was the most sensitive of the three techniques; it was possible to isolate virus immediately following intrathoracic inoculation of mosquitoes. RT-PCR was second in sensitivity; LAC RNA was detected 1 day postinfection. EIA detected LAC antigen 2 days postinfection. Additionally, RT-PCR and EIA were able to detect LAC RNA and protein, respectively, from mosquito samples which were subjected to seven freeze-thaw cycles, and RT-PCR was able to detect LAC RNA from mosquito samples which remained at room temperature for up to 7 days

Inhibition of luciferase expression in transgenic Aedes aegypti mosquitoes by Sindbis virus expression of antisense luciferase RNA

A rapid and reproducible method of inhibiting the expression of specific genes in mosquitoes should further our understanding of gene function and may lead to the identification of mosquito genes that determine vector competence or are involved in pathogen transmission. We hypothesized that the virus expression system based on the mosquito-borne Alphavirus, Sindbis (Togaviridae), may efficiently transcribe effector RNAs that inhibit expression of a targeted mosquito gene. To test this hypothesis, germ-line-transformed Aedes aegypti that express luciferase (LUC) from the mosquito Apyrase promoter were intrathoracically inoculated with a double subgenomic Sindbis (dsSIN) virus TE/3′2J/anti-luc (Anti-luc) that transcribes RNA complementary to the 5′ end of the LUC mRNA. LUC activity was monitored in mosquitoes infected with either Anti-luc or control dsSIN viruses expressing unrelated antisense RNAs. Mosquitoes infected with Anti-luc virus exhibited 90% reduction in LUC compared with uninfected and control dsSIN-infected mosquitoes at 5 and 9 days postinoculation. We demonstrate that a gene expressed from the mosquito genome can be inhibited by using an antisense strategy. The dsSIN antisense RNA expression system is an important tool for studying gene function in vivo

Characterization of Phlebotomus papatasi peritrophins, and the role of PpPer1 in Leishmania major survival in its natural vector

Citation: Coutinho-Abreu IV, Sharma NK, Robles-Murguia M, Ramalho-Ortigao M (2013) Characterization of Phlebotomus papatasi Peritrophins, and the Role of PpPer1 in Leishmania major Survival in its Natural Vector. PLoS Negl Trop Dis 7(3): e2132. doi:10.1371/journal.pntd.0002132The peritrophic matrix (PM) plays a key role in compartmentalization of the blood meal and as barrier to pathogens in many disease vectors. To establish an infection in sand flies, Leishmania must escape from the endoperitrophic space to prevent excretion with remnants of the blood meal digestion. In spite of the role played regarding Leishmania survival, little is known about sand fly PM molecular components and structural organization. We characterized three peritrophins (PpPer1, PpPer2, and PpPer3) from Phlebotomus papatasi. PpPer1 and PpPer2 display, respectively, four and one chitin-binding domains (CBDs). PpPer3 on the other hand has two CBDs, one mucin-like domain, and a putative domain with hallmarks of a CBD, but with changes in key amino acids. Temporal and spatial expression analyses show that PpPer1 is expressed specifically in the female midgut after blood feeding. PpPer2 and PpPer3 mRNAs were constitutively expressed in midgut and hindgut, with PpPer3 also being expressed in Malpighian tubules. PpPer2 was the only gene expressed in developmental stages. Interestingly, PpPer1 and PpPer3 expression are regulated by Le. major infection. Recombinant PpPer1, PpPer2 and PpPer3 were obtained and shown to display similar biochemical profiles as the native; we also show that PpPer1 and PpPer2 are able to bind chitin. Knockdown of PpPer1 led to a 44% reduction in protein, which in spite of producing an effect on the percentage of infected sand flies, resulted in a 39% increase of parasite load at 48 h. Our data suggest that PpPer1 is a component for the P. papatasi PM and likely involved in the PM role as barrier against Le. major infection

Evaluation of toxic effects with transition metal ions, EDTA, SBTI and acrylic polymers on Aedes aegypti (L., 1762) (Culicidae) and Artemia salina (Artemidae)

This work aimed to evaluate the toxicity of some insecticides compounds on Aedes aegypti and Artemia salina larvae. Bioassays were carried out to evaluate the toxic effect after of 24 and 72 h using the compounds or associations. The LC10, LC50 and LC90 values were obtained and utilized for toxicity comparations. For Ae. aegypti, LC50 were 32.65 mg L-1 in 24 h for Na2[EDTA-Cu(II)] and total mortality in 72 h for SAP-Na2[EDTA-Cu(II)]