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Tag: Mark Brown

Reciprocal interactions between neuropeptide F and RYamide regulate host attraction in the mosquito Aedes aegypti

Donna Huber on July 1, 2024

NPF accumulates in EECs of the posterior midgut after adult emergence but rapidly depletes after blood feeding.
NPF accumulates in EECs of the posterior midgut after adult emergence but rapidly depletes after blood feeding.

Female mosquitoes produce eggs in gonadotrophic cycles that are divided between a previtellogenic and vitellogenic phase. Previtellogenic females consume water and sugar sources like nectar while also being attracted to hosts for blood feeding. Consumption of a blood meal activates the vitellogenic phase, which produces mature eggs and suppresses host attraction. In this study, we tested the hypothesis that neuropeptide Y-like hormones differentially modulate host attraction behavior in the mosquito Aedes aegypti. A series of experiments collectively indicated that enteroendocrine cells (EECs) in the posterior midgut produce and release neuropeptide F (NPF) into the hemolymph during the previtellogenic phase which stimulates attraction to humans and biting behavior. Consumption of a blood meal, which primarily consists of protein by dry weight, down-regulated NPF in EECs until mature eggs developed, which was associated with a decline in hemolymph titer. NPF depletion depended on protein digestion but was not associated with EEC loss. Other experiments showed that neurons in the terminal ganglion extend axons to the posterior midgut and produce RYamide, which showed evidence of increased secretion into circulation after a blood meal. Injection of RYamide-1 and -2 into previtellogenic females suppressed host attraction, while coinjection of RYamides with or without short NPF-2 also inhibited the host attraction activity of NPF. Overall, our results identify NPF and RYamide as gut-associated hormones in A. aegypti that link host attraction behavior to shifts in diet during sequential gonadotrophic cycles.

Xiaoyi Dou, Kangkang Chen, Mark R Brown, Michael R Strand. Proc Natl Acad Sci U S A. 2024 Jul 9;121(28):e2408072121. doi: 10.1073/pnas.2408072121.

The mosquito Aedes aegypti requires a gut microbiota for normal fecundity, longevity and vector competence

Donna Huber on November 13, 2023

Mosquitoes shift from detritus-feeding larvae to blood-feeding adults that can vector pathogens to humans and other vertebrates. The sugar and blood meals adults consume are rich in carbohydrates and protein but are deficient in other nutrients including B vitamins. Facultatively hematophagous insects like mosquitoes have been hypothesized to avoid B vitamin deficiencies by carryover of resources from the larval stage. However, prior experimental studies have also used adults with a gut microbiota that could provision B vitamins. Here, we used Aedes aegypti, which is the primary vector of dengue virus (DENV), to ask if carryover effects enable normal function in adults with no microbiota. We show that adults with no gut microbiota produce fewer eggs, live longer with lower metabolic rates, and exhibit reduced DENV vector competence but are rescued by provisioning B vitamins or recolonizing the gut with B vitamin autotrophs. We conclude carryover effects do not enable normal function.

Ruby E Harrison, Xiushuai Yang, Jai Hoon Eum, Vincent G Martinson, Xiaoyi Dou, Luca Valzania, Yin Wang, Bret M Boyd, Mark R Brown, Michael R Strand. Commun Biol. 2023 Nov 13;6(1):1154. doi: 10.1038/s42003-023-05545-z.

Insulin-like peptides and ovary ecdysteroidogenic hormone differentially stimulate physiological processes regulating egg formation in the mosquito Aedes aegypti

Donna Huber on October 30, 2023

graphical abstract

Mosquitoes including Aedes aegypti are human disease vectors because females must blood feed to produce and lay eggs. Blood feeding triggers insulin-insulin growth factor signaling (IIS) which regulates several physiological processes required for egg development. A. aegypti encodes 8 insulin-like peptides (ILPs) and one insulin-like receptor (IR) plus ovary ecdysteroidogenic hormone (OEH) that also activates IIS through the OEH receptor (OEHR). In this study, we assessed the expression of A. aegypti ILPs and OEH during a gonadotropic cycle and produced each that were functionally characterized to further understand their roles in regulating egg formation. All A. aegypti ILPs and OEH were expressed during a gonadotropic cycle. Five ILPs (1, 3, 4, 7, 8) and OEH were specifically expressed in the head, while antibodies to ILP3 and OEH indicated each was released after blood feeding from ventricular axons that terminate on the anterior midgut. A subset of ILP family members and OEH stimulated nutrient storage in previtellogenic females before blood feeding, whereas most IIS-dependent processes after blood feeding were activated by one or more of the brain-specific ILPs and/or OEH. ILPs and OEH with different biological activities also exhibited differences in IIS as measured by phosphorylation of the IR, phosphoinositide 3-kinase/Akt kinase (AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK). Altogether, our results provide the first results that compare the functional activities of all ILP family members and OEH produced by an insect.

Kangkang Chen, Xiaoyi Dou, Jai Hoon Eum, Ruby A Harrison, Mark R Brown, Michael R Strand. Insect Biochem Mol Biol. 2023 Oct 30:104028. doi: 10.1016/j.ibmb.2023.104028.

Multiple endocrine factors regulate nutrient mobilization and storage in Aedes aegypti during a gonadotrophic cycle

Donna Huber on September 2, 2022

Anautogenous mosquitoes must blood feed on a vertebrate host to produce eggs. Each gonadotrophic cycle is subdivided into a sugar-feeding previtellogenic phase that produces primary follicles and a blood meal-activated vitellogenic phase in which large numbers of eggs synchronously mature and are laid. Multiple endocrine factors including juvenile hormone (JH), insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) coordinate each gonadotrophic cycle. Egg formation also requires nutrients from feeding that are stored in the fat body. Regulation of egg formation is best understood in Aedes aegypti but the role different endocrine factors play in regulating nutrient mobilization and storage remains unclear. In this study, we report that adult female Ae. aegypti maintained triacylglycerol (TAG) stores during the previtellogenic phase of the first gonadotrophic cycle while glycogen stores declined. In contrast, TAG and glycogen stores were rapidly mobilized during the vitellogenic phase and then replenishment. Several genes encoding enzymes with functions in TAG and glycogen metabolism were differentially expressed in the fat body, which suggested regulation was mediated in part at the transcriptional level. Gain of function assays indicated that stored nutrients were primarily mobilized by adipokinetic hormone (AKH) while juvenoids and OEH regulated replenishment. ILP3 further showed evidence of negatively regulating certain lipolytic enzymes. Loss of function assays further indicated AKH depends on the AKH receptor (AKHR) for function. Altogether, our results indicate that the opposing activities of different hormones regulate nutrient stores during a gonadotrophic cycle in Ae. aegypti. This article is protected by copyright. All rights reserved.

Xiaoyi Dou, Kangkang Chen, Mark R Brown, Michael R Strand. Insect Sci. 2022 Sep 2. doi: 10.1111/1744-7917.13110.

Ad libitum consumption of protein- or peptide-sucrose solutions stimulates egg formation by prolonging the vitellogenic phase of oogenesis in anautogenous mosquitoes

Donna Huber on April 12, 2022

Background: Anautogenous mosquitoes commonly consume nectars and other solutions containing sugar but are thought to only produce eggs in discrete gonadotrophic cycles after blood-feeding on a vertebrate host. However, some anautogenous species are known to produce eggs if amino acids in the form of protein are added to a sugar solution. Unclear is how different sources of amino acids in sugar solutions affect the processes that regulate egg formation and whether responses vary among species. In this study, we addressed these questions by focusing on Aedes aegypti and conducting some comparative assays with Aedes albopictus, Anopheles gambiae, Anopheles stephensi and Culex quinquefasciatus.

Methods: Adult female mosquitoes were fed sugar solutions containing amino acids, peptides or protein. Markers for activation of a gonadotrophic cycle including yolk deposition into oocytes, oviposition, ovary ecdysteroidogenesis, expression of juvenile hormone and 20-hydroxyecdysone-responsive genes, and adult blood-feeding behavior were then measured.

Results: The five anautogenous species we studied produced eggs when fed two proteins (bovine serum albumin, hemoglobin) or a mixture of peptides (tryptone) in 10% sucrose but deposited only small amounts of yolk into oocytes when fed amino acids in 10% sucrose. Focusing on Ae. aegypti, cultures were maintained for multiple generations by feeding adult females protein- or tryptone-sugar meals. Ad libitum access to protein- or tryptone-sugar solutions protracted production of ecdysteroids by the ovaries, vitellogenin by the fat body and protease activity by the midgut albeit at levels that were lower than in blood-fed females. Females also exhibited semi-continual oogenesis and repressed host-seeking behavior.

Conclusions: Several anautogenous mosquitoes produce eggs when provided ad libitum access to protein- or peptide-sugar meals, but several aspects of oogenesis also differ from females that blood-feed.

Ruby E Harrison, Kangkang Chen, Lilith South, Ange Lorenzi, Mark R Brown, Michael R Strand. Parasit Vectors. 2022 Apr 12;15(1):127. doi: 10.1186/s13071-022-05252-4.

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes

Donna Huber on March 8, 2022

Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti, where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle, but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes, but few genes – including almost no transcripts for immune factors – were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny.

Ellen O Martinson, Kangkang Chen, Luca Valzania, Mark R Brown, Michael R Strand. J Exp Biol. 2022 Mar 1;225(5):jeb243460. doi: 10.1242/jeb.243460.

The Peptide Hormone CNMa Influences Egg Production in the Mosquito Aedes aegypti

Donna Huber on February 25, 2022

Mosquito reproduction is regulated by a suite of hormones, many acting through membrane-bound receptor proteins. The Aedes aegypti G protein-coupled receptors AAEL024199 (AeCNMaR-1a) and AAEL018316 (AeCNMaR-1b) were identified as orthologs of the Drosophila melanogaster CNMa receptor (DmCNMaR). The receptor was duplicated early in the evolution of insects, and subsequently in Culicidae, into what we refer to as CNMaR-1a and CNMaR-1b. AeCNMaR-1a is only detected in male mosquito antennae while AeCNMaR-1b is expressed at high levels in mosquito ovaries. Using a heterologous cell assay, we determined that AeCNMa activates AeCNMaR-1a with a ~10-fold lower concentration than it does AeCNMaR-1b, though both receptors displayed half maximal effective concentrations of AeCNMa in the low nanomolar range. Finally, we show that injections of AeCNMa into blood-fed mated female Ae. aegypti resulted in fewer eggs laid.

Nia I Keyes-Scott, Aryan Lajevardi, Kyle R Swade, Mark R Brown, Jean-Paul Paluzzi, Kevin J Vogel. Insects. 2022 Feb 25;13(3):230. doi: 10.3390/insects13030230.

Insulin-like peptide 3 stimulates hemocytes to proliferate in anautogenous and facultatively autogenous mosquitoes

Donna Huber on February 7, 2022

Most mosquito species are anautogenous, which means they must blood feed on a vertebrate host to produce eggs, while a few are autogenous and can produce eggs without blood feeding. Egg formation is best understood in the anautogenous mosquito Aedes aegypti where insulin-like peptides (ILPs), ovary ecdysteroidogenic hormone (OEH) and 20-hydroxyecdysone (20E) interact to regulate gonadotrophic cycles. Circulating hemocytes also approximately double in abundance in conjunction with a gonadotrophic cycle but the factors responsible for stimulating this increase remain unclear. Focusing on Ae. aegypti, we determined that hemocyte abundance similarly increased in intact blood-fed females and decapitated blood-fed females that were injected with ILP3, whereas OEH, 20E, or heat-killed bacteria had no stimulatory activity. ILP3 upregulated insulin-insulin growth factor signaling in hemocytes but few genes, including almost no transcripts for immune factors, were differentially expressed. ILP3 also stimulated circulating hemocytes to increase in two other anautogenous (Anopheles gambiae and Culex quinquefasciatus) and two facultatively autogenous mosquitoes (Aedes atropalpus and Culex pipiens molestus), but had no stimulatory activity in the obligately autogenous mosquito Toxorhynchites amboinensis. Altogether, our results identify ILPs as the primary regulators of hemocyte proliferation in association with egg formation, but also suggest this response has been lost in the evolution of obligate autogeny.

Ellen O Martinson, Kangkang Chen, Luca Valzania, Mark R Brown, Michael R Strand. J Exp Biol. 2022 Feb 7;jeb.243460. doi: 10.1242/jeb.243460.

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development

Donna Huber on April 9, 2021

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Yin Wang, Jai Hoon Eum, Ruby E. Harrison, Luca Valzania, Xiushuai Yang, Jena A. Johnson, Derek T. Huck, Mark R. Brown, Michael R. Strand Proceedings of the National Academy of Sciences Apr 2021, 118 (15) e2101080118; DOI: 10.1073/pnas.2101080118

Whole blood and blood components from vertebrates differentially affect egg formation in three species of anautogenous mosquitoes

Donna Huber on February 24, 2021

Background: Most female mosquitoes are anautogenous and must blood feed on a vertebrate host to produce eggs. Prior studies show that the number of eggs females lay per clutch correlates with the volume of blood ingested and that protein is the most important macronutrient for egg formation. In contrast, how whole blood, blood fractions and specific blood proteins from different vertebrates affect egg formation is less clear. Since egg formation is best understood in Aedes aegypti, we examined how blood and blood components from different vertebrates affect this species and two others: the malaria vector Anopheles gambiae and arbovirus vector Culex quinquefasciatus.

Methods: Adult female mosquitoes were fed blood, blood fractions and purified major blood proteins from different vertebrate hosts. Markers of reproductive response including ovary ecdysteroidogenesis, yolk deposition into oocytes and number of mature eggs produced were measured.

Results: Ae. aegypti, An. gambiae and C. quinquefasciatus responded differently to meals of whole blood, plasma or blood cells from human, rat, chicken and turkey hosts. We observed more similarities between the anthropophiles Ae. aegypti and An. gambiae than the ornithophile C. quinquefasciatus. Focusing on Ae. aegypti, the major plasma-derived proteins (serum albumin, fibrinogen and globulins) differentially stimulated egg formation as a function of vertebrate host source. The major blood cell protein, hemoglobin, stimulated yolk deposition when from pigs but not humans, cows or sheep. Serum albumins from different vertebrates also variably affected egg formation. Bovine serum albumin (BSA) stimulated ovary ecdysteroidogenesis, but more weakly induced digestive enzyme activities than whole blood. In contrast, BSA-derived peptides and free amino acids had no stimulatory effects on ecdysteroidogenesis or yolk deposition into oocytes.

Conclusions: Whole blood, blood fractions and specific blood proteins supported egg formation in three species of anautogenous mosquitoes but specific responses varied with the vertebrate source of the blood components tested.

Harrison, R.E., Brown, M.R. & Strand, M.R. Whole blood and blood components from vertebrates differentially affect egg formation in three species of anautogenous mosquitoes. Parasites Vectors 14, 119 (2021). https://doi.org/10.1186/s13071-021-04594-9