Induction of a peptide with activity against a broad spectrum of pathogens in the Aedes aegypti salivary gland, following Infection with Dengue Virus.

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquito's salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vector's innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.

Natural transovarial dengue virus infection rate in both sexes of dark and pale forms of Aedes aegypti from an urban area of Bangkok, Thailand.

Transovarial dengue virus infection status of two forms of adult Aedes aegypti (dark or Ae. aegypti type form and pale or form queenslandensis), reared from field-collected larval and pupal stages, was determined by one-step RT-PCR and dengue viral serotype by nested-PCR. Natural transovarial transmission (TOT) of dengue virus was detected in the two Ae. aegypti forms, and in both adult males and females. Male Ae. aegypti had a higher rate of TOT dengue virus infection than female. The overall minimum infection rate among the male and female populations was 19.5 and 12.3 per 1,000 mosquitoes, respectively. All four dengue serotypes were detected in mosquito samples, with DEN-4 being the predominant serotype. Thus, both male and female Ae. aegypti have influences on the epidemiology of dengue virus transmission.

Protein expression in the salivary glands of dengue-infected Aedes aegypti mosquitoes and blood-feeding success.

Mosquito salivary glands (SG) play an essential role in food digestion and pathogen transmission. The function of the salivary components during infection is poorly understood. In this study, female Aedes aegypti mosquitoes were infected with dengue virus serotype 2 (DENV-2) via an artificial membrane feeding apparatus. The mosquito SGs were examined for DENV-2 infection for 14 days post-infection (dpi). The amount of dengue virus increased throughout the 14 dpi. Three different meals were provided for the Ae. aegypti mosquitoes. SG protein expression was compared among sugar-fed (SF), blood-fed (BF), and dengue-infected blood-fed (DF) mosquitoes using SDS-PAGE coupled with densitometric analysis. The SG of SF mosquitoes had fewer protein bands than those of BF and DF mosquitoes. The major SG proteins seen among BF and DF mosquitoes had molecular weights of 12-15, 25-30, 35-40, 45-50, 55-60 kDa and 61-67 kDa. We compared the SG protein band expression profiles in BF and DF mosquitoes. Two bands (35-40 and 61-67 kDa) were expressed more by DF mosquitoes and 3 different bands (25-30, 45-50, and 55-60 kDa) were expressed more by BF mosquitoes. These SG proteins may have some role in facilitating blood-feeding and dengue infection. We speculate these specific SG proteins in dengue-infected mosquitoes may increase the chance of blood-feeding and virus transmission by infected mosquitoes. These results may be useful for designing additional tools to investigate the interaction between Ae. aegypti SG and the dengue virus.

Blood-feeding and immunogenic Aedes aegypti saliva proteins.

Mosquito-transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar-fed and blood-fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar- or blood-based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar-fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up-regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2-DE profiles of immunogenic Ae. aegypti SG BF-related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.

Isolating and identifying fungi to determine whether their biological properties have the potential to control the population density of mosquitoes.

Mosquitoes transmit diseases such as dengue, chikungunya, Zika, and yellow fever to humans. Biological control methods are required for these insects because they can be environmentally friendlier, safer, and more cost-effective than chemical or physical methods currently available. The aim of this research is to identify fungi found in mosquito breeding containers that have the potential to control the population density of mosquitoes. For the identification, water samples were taken from mosquito breeding containers situated in seven districts of Bangkok to obtain pure cultures. Deoxyribonucleic acid (DNA) was extracted from the cultures then sent for sequencing and analyzing. The results show that fourteen strains of fungi were isolated. The most common strain found was Aspergillus spp., which was present in 31 of the 78 fungi samples. The strains Metarhizium anisopliae and Penicilium citrinum were found to be interesting because they may have the potential to act as entomopathogenic fungi. The biological properties of these strains should be further investigated because they could help in the fight against mosquito-borne diseases.

Susceptibility and transovarial transmission of dengue virus in Aedes aegypti: a preliminary study of morphological variations.

Two types of morphological variants, the dark form and the pale form of Aedes aegypti were selected from wild-caught mosquitos. Ascertaining any differences between the two forms for susceptibility to dengue type 2 virus was performed by oral feeding. Transovarial transmission was further determined from the progenies of infected mosquitos by tracing them to the third generation. Significant differences in oral infection were not observed between these two forms of mosquitos. Transovarial transmission was found in the progenies of infected females of both forms, and the filial infection rates (FIRs) were also similar. However, there was a trend of declining FIR in the later generation. In order to achieve an accurate result, more tests are currently underway to obtain a larger number of progeny. Although the FIR was low in the present study under laboratory conditions, higher rates might occur under field conditions, which could have a significant impact on the maintenance of dengue viruses in nature.

Prospective field study of transovarial dengue-virus transmission by two different forms of Aedes aegypti in an urban area of Bangkok, Thailand.

A prospective field study was conducted to determine transovarial dengue-virus transmission in two forms of Aedes aegypti mosquitoes in an urban district of Bangkok, Thailand. Immature Aedes mosquitoes were collected monthly for one year and reared continuously until adulthood in the laboratory. Mosquitoes assayed for dengue virus were processed in pools and their dengue virus infection status was determined by one-step RT-PCR and nested-PCR methods. Of a total 15,457 newly emerged adult Ae. aegypti, 98.2% were dark and 1.8% of the pale form. The results showed that the minimum infection rate (MIR) by transovarial transmission (TOT) of dengue virus during the one-year study ranged between 0 to 24.4/1,000 mosquitoes. Dengue virus TOT increased gradually during the hot summer months, reaching a peak in April-June, while dengue cases peaked in September, a rainy month near the end of the rainy season. Therefore, mosquito infections due to TOT were prevalent four months before a high incidence of human infections. TOT dengue virus infections occurred in both forms of Ae. aegypti. All four dengue serotypes were detected, with DEN-4 predominant, followed by DEN-3, DEN-1, and DEN-2, respectively.