Salvador Hernández-Martínez

The dengue virus is an arbovirus of public health importance transmitted by Aedes aegypti mosquitoes. The NS1 protein is a multifunctional glycoprotein, highly conserved among Orthoflaviviruses, that is secreted from infected cells, circulate in the sera of dengue patients at high concentrations and has been involved with pathogenesis by several mechanisms, including vascular leakage and immune evasion. However, the role of NS1 within the mosquito vector is not fully understood. In this study, we observed that feeding female Aedes aegypti mosquitoes with blood meals supplemented with recombinant NS1 resulted in increased midgut permeability, as evaluated by the diffusion of the non-toxic dye Brilliant Blue FCF into the hemocoel. Histological and transmission electron microscopy analysis revealed epithelial damage and disruption of the midgut cellular junctions. Immunofluorescence assays further demonstrated the delocalization of septate junction proteins essential for epithelial integrity. In addition, metalloproteinase expression in the midgut was also reduced. All these effects were abolished when NS1 was heat-inactivated indicating that they were NS1-dependent. Significantly, virus blood meals containing NS1 resulted in enhanced dissemination of DENV to secondary organs and earlier virus presence in the mosquito salivary glands, in comparison with meals treated with specific NS1 antibodies. Our findings reveal a novel function for NS1 in mosquitoes, and expand the understanding of NS1 functions beyond its human pathophysiological role. In addition, highlight NS1 as a strategic intervention point to reduce transmission efficiency in mosquitoes. Importance: Dengue is the most important virus disease transmitted by mosquitoes to humans. Thus, a deeper understanding of the virus-vector interaction is required for the development of control measures and the mosquito transmission capacity. In this work, we present evidence indicating that the NS1 protein plays a role in the establishment and dissemination of the dengue virus infection in Aedes aegypti mosquitoes. Ingested NS1 was found to disrupt the septate junctions and to inhibit metalloproteinase expression of the midgut, allowing the virus to escape the digestive tract. In addition, NS1 was found to promote virus dissemination into the hemocoel, carcass and salivary glands. These findings uncover new functions for NS1 in the mosquito and highpoint ways to interfere with vector competence.

Anophelinae mosquitoes are vectors of human malaria, a disease that infects hundreds of millions of people and causes almost 600,000 fatalities annually. Despite their medical importance, laboratory studies on key aspects of Anophelinae reproductive biology have been limited, and in particular, relatively little is known about the role of juvenile hormone (JH) in the control of female reproduction. The study presented here attempts to fill a gap of knowledge in our understanding of the JH control of ovarian development in female Anophelinae mosquitoes, using Anopheles albimanus as a model. Our studies revealed that JH controls the tempo of maturation of primary follicles in An. albimanus in a similar manner to that previously described in Aedes aegypti. At adult eclosion JH hemolymph titer was low, increased in 1-day old sugar-fed insects, and decreased in blood fed individuals. JH titers decreased if An. albimanus females were starved, and were reduced if insects emerged with low teneral reserves, precluding previtellogenic ovarian development. However, absolute hemolymph titers were lower than Ae. aegypti. Decapitation experiments suggested that if teneral reserves are sufficient, factors from the head activate JH synthesis by the corpora allata (CA) during the first 9–12 h after adult emergence. In conclusion, our studies support the hypothesis that JH controls previtellogenic ovarian development in female An. albimanus mosquitoes, in a similar manner that have been described in Culicinae.

Endoreplication is a cell cycle program in which cells replicate their genomes without undergoing mitosis and cytokinesis. For the normal development of many organisms (from fungi to humans) and the formation of their organs, endoreplication is indispensable. The aim of the present study was to explore whether endoreplication and DNA synthesis are relevant processes during the induction of trained innate immunity in human monocytes and in the Anopheles albimanus mosquito cell line. During the induction of trained immunity in both models, endoreplication markers were overexpressed and we observed an increase in DNA synthesis with an augmented copy number of genes essential for trained immunity. Blocking DNA synthesis prevented trained immunity from being established. Overall, these findings suggest that DNA synthesis and endoreplication are important mechanisms involved in inducing innate immune memory. They have probably been conserved throughout evolution from invertebrates to humans.

Aedes aegypti is the main vector of Dengue Virus, carrying the virus during the whole mosquito life post-infection. Few mosquito fitness costs have been associated to the virus infection, thereby allowing for a swift dissemination. In order to diminish the mosquito population, public health agency use persistent chemicals with environmental impact for disease control. Most countries barely use biological controls, if at all. With the purpose of developing novel Dengue control strategies, a detailed understanding of the unexplored virus-vector interactions is urgently needed. Damage induced (through tissue injury or bacterial invasion) DNA duplication (endoreplication) has been described in insects during epithelial cells renewal. Here, we delved into the mosquito midgut tissue ability to synthesize DNA de novo; postulating that Dengue virus infection could trigger a protective endoreplication mechanism in some mosquito cells. We hypothesized that the Aedes aegypti orthologue of the Drosophila melanogaster hindsight gene (not previously annotated in Aedes aegypti transcriptome/genome) is part of the Delta-Notch pathway. The activation of this transcriptional cascade leads to genomic DNA endoreplication. The amplification of the genomic copies of specific genes ultimately limits the viral spreading during infection. Conversely, inhibiting DNA synthesis capacity, hence endoreplication, leads to a higher viral replication.

Allatotropins (AT) are neuropeptides with pleotropic functions on a variety of insect tissues. They affect processes such as juvenile hormone biosynthesis, cardiac rhythm, oviduct and hindgut contractions, nutrient absorption and circadian cycle. The present work provides experimental evidence that AT elicits immune responses in two important mosquito disease vectors, Anopheles albimanus and Aedes aegypti. Hemocytes and an immune-competent mosquito cell line responded to AT by showing strong morphological changes and increasing bacterial phagocytic activity. Phenoloxidase activity in hemolymph was also increased in Ae. aegypti mosquitoes treated with AT but not in An. albimanus, suggesting differences in the AT-dependent immune activation in the two species. In addition, two important insect immune markers, nitric oxide levels and expression of antimicrobial peptide genes, were increased in An. albimanus guts after AT treatment. AT conjugated to quantum dot nanocrystals (QDots) specifically labeled hemocytes in vivo in both mosquito species, implying molecular interactions between AT and hemocytes. The results of our studies suggest a new role for AT in the modulation of the immune response in mosquitoes.