Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain.

The vector competence (VC) of Aedes aegypti (Linnaeus) (Diptera: Culicidae) varies geographically and is affected by both genetic and environmental factors. Understanding the molecular mechanisms that influence VC may help develop novel control strategies. The selection of susceptible and refractory strains is the first step in this process. We collected immature A. aegypti in the field and established strains that were susceptible and refractory to Dengue-2 virus by isofamily selection through several generations. Infection was detected by immunofluorescence of head or midgut tissues to determine infection barriers and the % of VC by tissue. We selected three strains: Susceptible (Cali-S) (96.4% susceptible at F 19 ), Refractory with a midgut escape barrier (Cali-MEB) (44.1% refractory at F 15 ), and Refractory with a midgut infection barrier (Cali-MIB) (40% refractory at F 16 ). The effects of the infection were measured using Kaplan-Meier survival rates over the first seven generations. All selected strains showed a similar decrease in survival and in the number of eggs laid/female through the seven generations, suggesting that changes were a result of the selection process rather than the virus infection. The results of this study suggest that VC is associated with multiple genes, which have additive effects on susceptibility.Résumé-La compétence vectorielle (CV) d'Aedes aegypti (Linnaeus) (Diptera: Culicidae) varie géographiquement et est influencée par des facteurs génétiques et environnementaux. Comprendre les mécanismes moléculaires qui affectent la CV pourrait aider à développer de nouvelles stratégies de contrôle des moustiques. La sélection de souches susceptibles et réfractaires est la première étape dans ce processus. Nous avons recueilli des A. aegypti immatures sur le terrain et des souches susceptibles et réfractaires au virus dengue-2 on été établies par la sélection d'isofamilles sur plusieurs générations. L'infection a été détectée par immunofluorescence sur des tissus de la tête ou de l'intestin moyen pour déterminer les barrières d'infection et le % de CV par tissu. Nous avons sélectionné trois souches: Susceptible (Cali-S) (96,4% sensibles à F 19 ), Réfractaire avec une barrière contre la dissémination dans l'intestin moyen (Cali-MEB) (44,1% réfractaire à F 15 ) et Réfractaires avec une barrière contre l'infection dans l'intestin moyen (Cali-MIB) (40% réfractaire à F 16 ). Les effets de l'infection ont été mesurés en utilisant les taux de survie de Kaplan-Meier au cours des sept premières générations. Toutes les souches sélectionnées ont montré une diminution similaire du taux de survie et du nombre d'oeufs pondus/ femelle à travers les sept générations, ce qui suggère que les changements sont le résultat du processus de sélection plutôt que l'infection par le virus. Les résultats de cette étude suggèrent que la CV est associée à de multiples gènes qui ont des effets additifs sur la susceptibilité.

Tropical ecosystems hold an extremely diverse array of endophytic fungi, but their potential use still remains to be explored. In this study, we isolated an endophytic fungus from the leaves of Otoba gracilipes, a medicinal tree from a tropical rainforest in Colombia. Following isolation and cultivation, we evaluated its extracellular crude extract for antioxidant activity. Using traditional and molecular methods (ITS1, NL1 regions), the endophyte was identified as Fusarium oxysporum. Fresh spores from the fungal isolate were inoculated in liquid media (potato dextrose broth [PDB] and potato dextrose–yeast extract broth [PDYB]) and centrifuged for recovering extracellular polysaccharides from the exhausted medium after 30 days of cultivation. Crude extracts were recovered, purified, lyophilized, and evaluated for their ability to inactivate the free radical 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH). The extracts obtained from PDB culture media had a 51.5% of scavenging effect on DPPH after 5 min of reaction compared with the extracts from PDBY (26.4%), which suggests a high antioxidant potential of these fungal extracts. Thus, our results suggest other fungi from tropical ecosystems should be explored as potential sources of novel enzymes and other metabolites with bioactivity.

Aedes (Stegomyia) aegypti, the principal global vector of dengue viruses, has differences in its susceptibility to dengue virus infection. We compared the global expression of genes in the midguts of Colombian Ae. aegypti dengue-susceptible (Cali-S) and dengue-refractory (Cali-MIB) field derived strains after ingesting either a sugarmeal, a bloodmeal, or a bloodmeal containing dengue virus serotype 2 (DENV-2). Microarray-based transcriptome analysis among treatments indicated a total of 4725 transcripts with differential expression between the two strains. Eleven genes were selected from different functional groups based on their significant up or down expression levels as well as reports in the literature suggesting they are associated with dengue virus elimination. We measured mRNA abundance of these 11 genes at 0, 8, 24, and 36 h postinfection using quantitative real time PCR (qPCR) to confirm the microarray results and assess any temporal patterns. Four genes were selected (Gram-negative binding protein-GNBP [AAEL009176], Niemann Pick Type-C2-NPC2 [AAEL015136], Keratinocyte lectin [AAEL009842], and Cathepsin-b [AAEL007585]) for knockdown experiments using RNA interference (RNAi) methodology to determine the phenotype (DENV-2 susceptible or refractory). Silencing GNBP, Cathepsin-b and Keratinocyte lectin reduced the percentage of mosquitoes with disseminated virus in the Cali-S strain to 8%, 20%, and 12% respectively compared with 96% in the controls. Silencing of NPC2 increased the percentage of mosquitos with disseminated virus infections in Cali-MIB to 66% compared with 35% in the controls. This study provides insight into genes that may contribute to the Cali-S susceptible and Cali-MIB refractory phenotypes in Ae. aegypti.

Control of dengue virus (DenV) transmission, primarily based on strategies to reduce populations of the principle vector Stegomya aegypti (= Aedes aegypti) (Diptera: Culicidae), is difficult to sustain over time. Other potential strategies aim to manipulate characteristics such as vector competence (VC), the innate capacity of the vector to transmit the virus. Previous studies have identified genetic factors, including differential expression of apoptosis-related genes, associated with the refractory and susceptible phenotypes in selected strains of S. aegypti from Cali, Colombia. The present study was designed to evaluate the variability of VC in selected strains against different DenV serotypes and to determine whether field-collected mosquitoes respond similarly to selected laboratory strains in terms of enhanced or reduced expression of apoptosis-related genes. Vector competence differed between strains, but did not differ in response to different DenV serotypes. Differences in VC were observed among mosquitoes collected from different localities in Cali. The overexpression of the pro-apoptosis genes, caspase 16 and Aedronc, was conserved in field-collected refractory mosquitoes and the selected laboratory refractory strain. The results suggest that the apoptosis response is conserved among all refractory mosquitoes to inhibit the development of all DenV serotypes.