Immo A. Hansen

GATA factor translation is the final downstream step in the amino acid/target-of-rapamycin-mediated vitellogenin gene expression in the anautogenous mosquito Aedes aegypti.

Ingestion of blood is required for vector mosquitoes to initiate reproductive cycles determining their role as vectors of devastating human diseases. Nutritional signaling plays a pivotal role in regulating mosquito reproduction. Transcription of yolk protein precursor genes is repressed until mosquitoes take blood. Previously, we have shown that to signal the presence of blood in the gut, mosquitoes utilize the target-of-rapamycin (TOR) pathway. The TOR signaling pathway transduces the amino acid signal activating the major yolk protein precursor gene, vitellogenin (Vg). Here we report the identification of a GATA factor (AaGATAa) that is synthesized after a blood meal and acts as a transcriptional activator of Vg. We showed that AaGATAa bound specifically to GATA-binding sites present in the proximal promoter region of the Vg gene and positively regulated Vg expression in transfection assays. RNA interference-mediated knock down of AaGATAa transcript resulted in a significant inhibition of Vg expression in both fat-body tissue culture and blood-fed mosquitoes. AaGATAa mRNA accumulated in the fat body prior to blood feeding. However, translation of GATA was activated by blood feeding because the GATA protein increased dramatically in the fat body of blood-fed mosquitoes. This increase was also reproduced in the fat-body culture stimulated with amino acids. GATA translation was inhibited by rapamycin and cycloheximide as well as by RNA interference-mediated knock down of S6 kinase. These experiments have revealed that the TOR signaling pathway induced by nutritional signaling regulates the translation of a GATA factor, which is the specific transcriptional activator of the Vg gene.

Identification of two cationic amino acid transporters required for nutritional signaling during mosquito reproduction.

SUMMARY The defining characteristic of anautogenous mosquitoes is their requirement for a blood meal to initiate reproduction. The need for blood drives the association of vector and host, and is the primary reason why anautogenous mosquitoes are effective disease vectors. During mosquito vitellogenesis, a key process in reproduction, yolk protein precursor (YPP) gene expression is activated specifically in the fat body, the insect analogue of the vertebrate liver. We have demonstrated that blood meal derived amino acids (AAs) activate YPP genes via the target of rapamycin (TOR)-signal transduction pathway. Here we show, by stimulating fat bodies with balanced AA solutions lacking individual AAs, that specific cationic and branched AAs are essential for activation of the vitellogenin (vg) gene, the major YPP gene. Treatment of fat bodies with AA uptake inhibitors results in a strong inhibition of AA-induced vg gene expression proving that an active transport mechanism is necessary to transduce the AA signal. We identified two cationic AA transporters (CATs) in the fat body of Aedes aegypti females - Aa slimfast and iCAT2. RNAi knockdown of slimfast and iCAT2 results in a strong decrease in the response to AAs by the vg gene similar to that seen due to TOR inhibition. These data demonstrate that active uptake of specific AAs plays a key role in nutritional signaling during the onset of vitellogenic gene expression in mosquitoes and it is mediated by two cationic AA transporters.

The Aquaporin Gene Family of the Yellow Fever Mosquito, Aedes aegypti

Background The mosquito, Aedes aegypti, is the principal vector of the Dengue and yellow fever viruses. During feeding, an adult female can take up more than its own body weight in vertebrate blood. After a blood meal females excrete large amounts of urine through their excretion system, the Malpighian tubules (MT). Diuresis starts within seconds after the mosquito starts feeding. Aquaporins (AQPs) are a family of membrane transporters that regulate the flow of water, glycerol and other small molecules across cellular membranes in both prokaryotic and eukaryotic cells. Our aim was to identify aquaporins that function as water channels, mediating transcellular water transport in MTs of adult female Ae. aegypti. Methodology/Principal Findings Using a bioinformatics approach we screened genome databases and identified six putative AQPs in the genome of Ae. aegypti. Phylogenetic analysis showed that five of the six Ae. aegypti AQPs have high similarity to classical water-transporting AQPs of vertebrates. Using microarray, reverse transcription and real time PCR analysis we found that all six AQPs are expressed in distinct patterns in mosquito tissues/body parts. AaAQP1, 4, and 5 are strongly expressed in the adult female MT. RNAi-mediated knockdown of the MT-expressed mosquito AQPs resulted in significantly reduced diuresis. Conclusions/Significance Our results support the notion that AQP1, 4, and 5 function as water transporters in the MTs of adult female Ae. aegypti mosquitoes. Our results demonstrate the importance of these AQPs for mosquito diuresis after blood ingestion and highlight their potential as targets for the development of novel vector control strategies.

The Fat Body Transcriptomes of the Yellow Fever Mosquito Aedes aegypti, Pre- and Post- Blood Meal

Background The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood feeding. Results We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in 204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the existing reference Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456 contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b, vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families. Conclusion The two fat body transcriptomes were considerably different from each other in terms of transcript expression in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body from a resting state to vitellogenic gene expression after feeding.

Four-way regulation of mosquito yolk protein precursor genes by juvenile hormone-, ecdysone-, nutrient-, and insulin-like peptide signaling pathways

Anautogenous mosquito females require a meal of vertebrate blood in order to initiate the production of yolk protein precursors by the fat body. Yolk protein precursor gene expression is tightly repressed in a state-of-arrest before blood meal-related signals activate it and expression levels rise rapidly. The best understood example of yolk protein precursor gene regulation is the vitellogenin-A gene (vg) of the yellow fever mosquito Aedes aegypti. Vg-A is regulated by (1) juvenile hormone signaling, (2) the ecdysone-signaling cascade, (3) the nutrient sensitive target-of-rapamycin signaling pathway, and (4) the insulin-like peptide (ILP) signaling pathway. A plethora of new studies have refined our understanding of the regulation of yolk protein precursor genes since the last review on this topic in 2005 (Attardo et al., 2005). This review summarizes the role of these four signaling pathways in the regulation of vg-A and focuses upon new findings regarding the interplay between them on an organismal level.

Functional characterization of aquaporins and aquaglyceroporins of the yellow fever mosquito, Aedes aegypti

After taking vertebrate blood, female mosquitoes quickly shed excess water and ions while retaining and concentrating the mostly proteinaceous nutrients. Aquaporins (AQPs) are an evolutionary conserved family of membrane transporter proteins that regulate the flow of water and in some cases glycerol and other small molecules across cellular membranes. In a previous study, we found six putative AQP genes in the genome of the yellow fever mosquito, Ae. aegypti, and demonstrated the involvement of three of them in the blood meal-induced diuresis. Here we characterized AQP expression in different tissues before and after a blood meal, explored the substrate specificity of AQPs expressed in the Malpighian tubules and performed RNAi-mediated knockdown and tested for changes in mosquito desiccation resistance. We found that AQPs are generally down-regulated 24 hrs after a blood meal. Ae. aegypti AQP 1 strictly transports water, AQP 2 and 5 demonstrate limited solute transport, but primarily function as water transporters. AQP 4 is an aquaglyceroporin with multiple substrates. Knockdown of AQPs expressed in the MTs increased survival of Ae. aegypti under dry conditions. We conclude that Malpighian tubules of adult female yellow fever mosquitoes utilize three distinct AQPs and one aquaglyceroporin in their osmoregulatory functions.

Aquaporins are critical for provision of water during lactation and intrauterine progeny hydration to maintain tsetse fly reproductive success.

Tsetse flies undergo drastic fluctuations in their water content throughout their adult life history due to events such as blood feeding, dehydration and lactation, an essential feature of the viviparous reproductive biology of tsetse. Aquaporins (AQPs) are transmembrane proteins that allow water and other solutes to permeate through cellular membranes. Here we identify tsetse aquaporin (AQP) genes, examine their expression patterns under different physiological conditions (blood feeding, lactation and stress response) and perform functional analysis of three specific genes utilizing RNA interference (RNAi) gene silencing. Ten putative aquaporins were identified in the Glossina morsitans morsitans (Gmm) genome, two more than has been previously documented in any other insect. All organs, tissues, and body parts examined had distinct AQP expression patterns. Two AQP genes, gmmdripa and gmmdripb ( = gmmaqp1a and gmmaqp1b) are highly expressed in the milk gland/fat body tissues. The whole-body transcript levels of these two genes vary over the course of pregnancy. A set of three AQPs (gmmaqp5, gmmaqp2a, and gmmaqp4b) are expressed highly in the Malpighian tubules. Knockdown of gmmdripa and gmmdripb reduced the efficiency of water loss following a blood meal, increased dehydration tolerance and reduced heat tolerance of adult females. Knockdown of gmmdripa extended pregnancy length, and gmmdripb knockdown resulted in extended pregnancy duration and reduced progeny production. We found that knockdown of AQPs increased tsetse milk osmolality and reduced the water content in developing larva. Combined knockdown of gmmdripa, gmmdripb and gmmaqp5 extended pregnancy by 4–6 d, reduced pupal production by nearly 50%, increased milk osmolality by 20–25% and led to dehydration of feeding larvae. Based on these results, we conclude that gmmDripA and gmmDripB are critical for diuresis, stress tolerance and intrauterine lactation through the regulation of water and/or other uncharged solutes.

AaCAT1 of the Yellow Fever Mosquito, Aedes aegypti: A NOVEL HISTIDINE-SPECIFIC AMINO ACID TRANSPORTER FROM THE SLC7 FAMILY*

Insect yolk protein precursor gene expression is regulated by nutritional and endocrine signals. A surge of amino acids in the hemolymph of blood-fed female mosquitoes activates a nutrient signaling system in the fat bodies, which subsequently derepresses yolk protein precursor genes and makes them responsive to activation by steroid hormones. Orphan transporters of the SLC7 family were identified as essential upstream components of the nutrient signaling system in the fat body of fruit flies and the yellow fever mosquito, Aedes aegypti. However, the transport function of these proteins was unknown. We report expression and functional characterization of AaCAT1, cloned from the fat body of A. aegypti. Expression of AaCAT1 transcript and protein undergoes dynamic changes during postembryonic development of the mosquito. Transcript expression was especially high in the third and fourth larval stages; however, the AaCAT1 protein was detected only in pupa and adult stages. Functional expression and analysis of AaCAT1 in Xenopus oocytes revealed that it acts as a sodium-independent cationic amino acid transporter, with unique selectivity to l-histidine at neutral pH (K0.5l-His = 0.34 ± 0.07 mm, pH 7.2). Acidification to pH 6.2 dramatically increases AaCAT1-specific His+-induced current. RNAi-mediated silencing of AaCAT1 reduces egg yield of subsequent ovipositions. Our data show that AaCAT1 has notable differences in its transport mechanism when compared with related mammalian cationic amino acid transporters. It may execute histidine-specific transport and signaling in mosquito tissues.

The Efficacy of Some Commercially Available Insect Repellents for Aedes aegypti (Diptera: Culicidae) and Aedes albopictus (Diptera: Culicidae)

Reducing the number of host-vector interactions is an effective way to reduce the spread of vector-borne diseases. Repellents are widely used to protect humans from a variety of protozoans, viruses, and nematodes. DEET (N,N-Diethyl-meta-toluamide), a safe and effective repellent, was developed during World War II. Fear of possible side effects of DEET has created a large market for “natural” DEET-free repellents with a variety of active ingredients. We present a comparative study on the efficacy of eight commercially available products, two fragrances, and a vitamin B patch. The products were tested using a human hand as attractant in a Y-tube olfactometer setup with Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), both major human disease vectors. We found that Ae. albopictus were generally less attracted to the test subject’s hand compared with Ae, aegypti. Repellents with DEET as active ingredient had a prominent repellency effect over longer times and on both species. Repellents containing p-menthane-3,8-diol produced comparable results but for shorter time periods. Some of the DEET-free products containing citronella or geraniol did not have any significant repellency effect. Interestingly, the perfume we tested had a modest repellency effect early after application, and the vitamin B patch had no effect on either species. This study shows that the different active ingredients in commercially available mosquito repellent products are not equivalent in terms of duration and strength of repellency. Our results suggest that products containing DEET or p-menthane-3,8-diol have long-lasting repellent effects and therefore provide good protection from mosquito-borne diseases.

Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti

Aedes aegypti is the major vector of dengue, yellow fever and chikungunya viruses that put millions of people in endemic countries at risk. Mass rearing of this mosquito is crucial for strategies that use modified insects to reduce vector populations and transmission of pathogens, such as sterile insect technique or population replacement. A major problem for vector mosquito mass rearing is the requirement of vertebrate blood for egg production since it poses significant costs as well as potential health hazards. Also, regulations for human and animal use as blood source can pose a significant obstacle. A completely artificial diet that supports egg production in vector mosquitoes can solve this problem. In this study, we compared different blood fractions, serum and red blood cells, as dietary protein sources for mosquito egg production. We also tested artificial diets made from commercially available blood proteins (bovine serum albumin (BSA) and hemoglobin). We found that Ae. aegypti performed vitellogenesis and produced eggs when given whole bovine blood, serum, or an artificial diet containing BSA. Conversely, egg production was impaired after feeding of the red blood cell fraction or an artificial diet containing only hemoglobin. We also found that egg viability of serum-fed mosquitoes were comparable to that of whole blood and an iron supplemented BSA meal produced more viable eggs than a meal containing BSA alone. Our results indicate that serum proteins, not hemoglobin, may replace vertebrate blood in artificial diets for mass mosquito rearing.