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Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The adult females of Aedes aegypti mosquitoes are facultative hematophagous insects but they are unable to feed on blood right after pupae emergence. The maturation process that takes place during the first post-emergence days, hereafter named hematophagic and gonotrophic capacitation, comprises a set of molecular and physiological changes that prepare the females for the first gonotrophic cycle. Notwithstanding, the molecular bases underlying mosquito hematophagic and gonotrophic capacitation remain obscure. Here, we investigated the molecular and biochemical changes in adult Ae. aegypti along the first four days post-emergence, prior to a blood meal. We performed a RNA-Seq analysis of the head and body, comparing male and female gene expression time courses. A total of 811 and 203 genes were differentially expressed, respectively in the body and head, and both body parts showed early, mid, and late female-specific expression profiles. Female-specific up-regulation of genes involved in muscle development and the oxidative phosphorylation pathway were remarkable features observed in the head. Functional assessment of mitochondrial oxygen consumption in heads showed a gradual increase in respiratory capacity and ATP-linked respiration as a consequence of induced mitochondrial biogenesis and content over time. This pattern strongly suggests that boosting oxidative phosphorylation in heads is a required step towards blood sucking habit. Several salivary gland genes, proteases, and genes involved in DNA replication and repair, ribosome biogenesis, and juvenile hormone signaling were up-regulated specifically in the female body, which may reflect the gonotrophic capacitation. This comprehensive description of molecular and biochemical mechanisms of the hematophagic and gonotrophic capacitation in mosquitoes unravels potentially new targets for vector control., Competing Interests: The authors have declared that no competing interests exist. Author Mario Alberto C da Silva-Neto, was unable to confirm their authorship contributions. On their behalf, the corresponding author has reported their contributions to the best of their knowledge.

In the midgut of the mosquito Aedes aegypti, a vector of dengue and yellow fever, an intense release of heme and iron takes place during the digestion of a blood meal. Here, we demonstrated via chromatography, light absorption and mass spectrometry that xanthurenic acid (XA), a product of the oxidative metabolism of tryptophan, is produced in the digestive apparatus after the ingestion of a blood meal and reaches milimolar levels after 24 h, the period of maximal digestive activity. XA formation does not occur in the White Eye (WE) strain, which lacks kynurenine hydroxylase and accumulates kynurenic acid. The formation of XA can be diminished by feeding the insect with 3,4-dimethoxy-N-[4-(3-nitrophenyl)thiazol-2-yl] benzenesulfonamide (Ro-61-8048), an inhibitor of XA biosynthesis. Moreover, XA inhibits the phospholipid oxidation induced by heme or iron. A major fraction of this antioxidant activity is due to the capacity of XA to bind both heme and iron, which occurs at a slightly alkaline pH (7.5-8.0), a condition found in the insect midgut. The midgut epithelial cells of the WE mosquito has a marked increase in occurrence of cell death, which is reversed to levels similar to the wild type mosquitoes by feeding the insects with blood supplemented with XA, confirming the protective role of this molecule. Collectively, these results suggest a new role for XA as a heme and iron chelator that provides protection as an antioxidant and may help these animals adapt to a blood feeding habit.

Once mosquito midgut barrier was crossed malaria parasite faces a extensive metabolic developmental program in order to ensure its transmission. In the hemolymph of the mosquito the dynamics of lipid metabolism is conducted by a major lipoprotein, lipophorin (Lp). It was recently shown that Lp is engaged in the mosquito immune response to parasite infection. However, it is not clear if Lp is uptaken by the parasite. Here, we show that oocysts are able to uptake mosquito Lp. The uptake of FITC-labeled Lp was demonstrated in midgut-associated oocysts. Alternatively, to confirm Lp incorporation by oocysts we have conducted another set of experiments with iodinated Lp ((125)I-Lp). Oocysts were able to incorporate (125)I-Lp and the process is both time and temperature dependent. This set of results indicated that no matter oocysts are attached to mosquito midgut wall they bear a lipid sequestering machinery from its surroundings. Phospholipid transfer to sporozoites was also demonstrated. In conclusion, these results demonstrate for the first time that malaria parasite undergoes lipid uptake while in the invertebrate host.

Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes.