BackgroundAedes aegypti mosquitoes are the main vectors of dengue viruses to humans. Understanding their biology and interactions with the pathogen are prerequisites for development of dengue transmission control strategies. Mosquito salivary glands are organs involved directly in pathogen transmission to vertebrate hosts. Information on the spatial distribution of gene expression in these organs is expected to assist in the development of novel disease control strategies, including those that entail the release of transgenic mosquitoes with impaired vector competence.ResultsWe report here the hybridization in situ patterns of 30 transcripts expressed in the salivary glands of adult Ae. aegypti females. Distinct spatial accumulation patterns were identified. The products of twelve genes are localized exclusively in the proximal-lateral lobes. Among these, three accumulate preferentially in the most anterior portion of the proximal-lateral lobe. This pattern revealed a salivary gland cell type previously undescribed in Ae. aegypti, which was validated by transmission electron microscopy. Five distinct gene products accumulate in the distal-lateral lobes and another five localize in the medial lobe. Seven transcripts are found in the distal-lateral and medial lobes. The transcriptional product of one gene accumulates in proximal- and distal-lateral lobes. Seven genes analyzed by quantitative PCR are expressed constitutively. The most abundant salivary gland transcripts are those localized within the proximal-lateral lobes, while previous work has shown that the distal-lateral lobes are the most active in protein synthesis. This incongruity suggests a role for translational regulation in mosquito saliva production.ConclusionsTransgenic mosquitoes with reduced vector competence have been proposed as tools for the control of dengue virus transmission. Expression of anti-dengue effector molecules in the distal-lateral lobes of Ae. aegypti salivary glands has been shown to reduce prevalence and mean intensities of viral infection. We anticipate greater efficiency of viral suppression if effector genes are expressed in all lobes of the salivary glands. Based on our data, a minimum of two promoters is necessary to drive the expression of one or more anti-dengue genes in all cells of the female salivary glands.

Oenocytes are ectodermic cells that participate in a number of critical physiological roles such as detoxification and lipid storage and metabolism in insects. In light of the lack of information on oenocytes from Aedes aegypti and the potential role of these cells in the biology of this major yellow fever and dengue vector, we developed a protocol to purify and maintain Ae. aegypti pupa oenocytes in primary culture. Ae. aegypti oenocytes were cultured as clustered and as isolated ovoid cells with a smooth surface. Our results demonstrate that these cells remain viable in cell culture for at least two months. We also investigated their morphology in vivo and in vitro using light, confocal, scanning and transmission electron microscopes. This work is the first successful attempt in isolating and maintaining Ae. aegypti oenocytes in culture, and a significant step towards understanding the role of this cell type in this important disease vector. The purification and the development of primary cultures of insect oenocytes will allow future studies of their metabolism in producing and secreting compounds.

Leafcutter ants farm a fungal cultivar (Leucoagaricus gongylophorus) that converts inedible vegetation into food that sustains colonies with millions of workers. Like fruits of crops domesticated by humans, L. gongylophorus has evolved specialized nutritional rewards—tiny swollen hyphal cells called gongylidia that package metabolites eaten by ant farmers. Yet, little is known about how gongylidia form, and whether ants regulate this formation through plant-fragment provisioning. We used microscopy and in vitro manipulations to explain the cellular mechanisms governing gongylidium formation. First, L. gongylophorus is polykaryotic (up to 17 haploid nuclei/cell) and our results suggest intracellular nucleus distributions govern gongylidium morphology with their absence in expanding edges arresting apical growth and their presence mediating complex branching patterns. Second, nanoscale TEM imaging shows that the cultivar recycles its own cellular material (e.g. cytosol, mitochondria) through a process called ‘autophagy’ and stores the resulting metabolites in gongylidia. This autophagic pathway is further supported by gongylidium inhibition when isolated fungal cultures are grown on media with autophagy inhibitors (chloroquine, 3-methyladenine). We hypothesize that autophagic nutritional reward production is the ultimate cultivar service and reflects a higher-level organismality adaptation enabled by strict symmetric lifetime commitment between ant farmers and their fungal crop.