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1.  Artificial Diets for Mosquitoes 
Mosquito-borne diseases are responsible for more than a million human deaths every year. Modern mosquito control strategies such as sterile insect technique (SIT), release of insects carrying a dominant lethal (RIDL), population replacement strategies (PR), and Wolbachia-based strategies require the rearing of large numbers of mosquitoes in culture for continuous release over an extended period of time. Anautogenous mosquitoes require essential nutrients for egg production, which they obtain through the acquisition and digestion of a protein-rich blood meal. Therefore, mosquito mass production in laboratories and other facilities relies on vertebrate blood from live animal hosts. However, vertebrate blood is expensive to acquire and hard to store for longer times especially under field conditions. This review discusses older and recent studies that were aimed at the development of artificial diets for mosquitoes in order to replace vertebrate blood.
PMCID: PMC5201408  PMID: 28009851
artificial blood meal; Aedes aegypti; mosquito; diet
2.  Substrate specificity and transport mechanism of amino acid transceptor Slimfast from Aedes aegypti 
Nature communications  2015;6:8546.
Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino acid transporter Slimfast (Slif) from the yellow fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signaling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+-dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction.
PMCID: PMC4608377  PMID: 26449545
3.  “Highly evolvable malaria vectors: the genomes of 16 Anopheles mosquitoes” 
Neafsey, Daniel E. | Waterhouse, Robert M. | Abai, Mohammad R. | Aganezov, Sergey S. | Alekseyev, Max A. | Allen, James E. | Amon, James | Arcà, Bruno | Arensburger, Peter | Artemov, Gleb | Assour, Lauren A. | Basseri, Hamidreza | Berlin, Aaron | Birren, Bruce W. | Blandin, Stephanie A. | Brockman, Andrew I. | Burkot, Thomas R. | Burt, Austin | Chan, Clara S. | Chauve, Cedric | Chiu, Joanna C. | Christensen, Mikkel | Costantini, Carlo | Davidson, Victoria L.M. | Deligianni, Elena | Dottorini, Tania | Dritsou, Vicky | Gabriel, Stacey B. | Guelbeogo, Wamdaogo M. | Hall, Andrew B. | Han, Mira V. | Hlaing, Thaung | Hughes, Daniel S.T. | Jenkins, Adam M. | Jiang, Xiaofang | Jungreis, Irwin | Kakani, Evdoxia G. | Kamali, Maryam | Kemppainen, Petri | Kennedy, Ryan C. | Kirmitzoglou, Ioannis K. | Koekemoer, Lizette L. | Laban, Njoroge | Langridge, Nicholas | Lawniczak, Mara K.N. | Lirakis, Manolis | Lobo, Neil F. | Lowy, Ernesto | MacCallum, Robert M. | Mao, Chunhong | Maslen, Gareth | Mbogo, Charles | McCarthy, Jenny | Michel, Kristin | Mitchell, Sara N. | Moore, Wendy | Murphy, Katherine A. | Naumenko, Anastasia N. | Nolan, Tony | Novoa, Eva M. | O'Loughlin, Samantha | Oringanje, Chioma | Oshaghi, Mohammad A. | Pakpour, Nazzy | Papathanos, Philippos A. | Peery, Ashley N. | Povelones, Michael | Prakash, Anil | Price, David P. | Rajaraman, Ashok | Reimer, Lisa J. | Rinker, David C. | Rokas, Antonis | Russell, Tanya L. | Sagnon, N'Fale | Sharakhova, Maria V. | Shea, Terrance | Simão, Felipe A. | Simard, Frederic | Slotman, Michel A. | Somboon, Pradya | Stegniy, Vladimir | Struchiner, Claudio J. | Thomas, Gregg W.C. | Tojo, Marta | Topalis, Pantelis | Tubio, José M.C. | Unger, Maria F. | Vontas, John | Walton, Catherine | Wilding, Craig S. | Willis, Judith H. | Wu, Yi-Chieh | Yan, Guiyun | Zdobnov, Evgeny M. | Zhou, Xiaofan | Catteruccia, Flaminia | Christophides, George K. | Collins, Frank H. | Cornman, Robert S. | Crisanti, Andrea | Donnelly, Martin J. | Emrich, Scott J. | Fontaine, Michael C. | Gelbart, William | Hahn, Matthew W. | Hansen, Immo A. | Howell, Paul I. | Kafatos, Fotis C. | Kellis, Manolis | Lawson, Daniel | Louis, Christos | Luckhart, Shirley | Muskavitch, Marc A.T. | Ribeiro, José M. | Riehle, Michael A. | Sharakhov, Igor V. | Tu, Zhijian | Zwiebel, Laurence J. | Besansky, Nora J.
Science (New York, N.Y.)  2014;347(6217):1258522.
Variation in vectorial capacity for human malaria among Anopheles mosquito species is determined by many factors, including behavior, immunity, and life history. To investigate the genomic basis of vectorial capacity and explore new avenues for vector control, we sequenced the genomes of 16 anopheline mosquito species from diverse locations spanning ~100 million years of evolution. Comparative analyses show faster rates of gene gain and loss, elevated gene shuffling on the X chromosome, and more intron losses, relative to Drosophila. Some determinants of vectorial capacity, such as chemosensory genes, do not show elevated turnover, but instead diversify through protein-sequence changes. This dynamism of anopheline genes and genomes may contribute to their flexible capacity to take advantage of new ecological niches, including adapting to humans as primary hosts.
PMCID: PMC4380271  PMID: 25554792
4.  Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti 
Nature Communications  2015;6:8546.
Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+ dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction.
Anautogenous mosquitoes need to obtain essential amino acids from a blood meal for reproduction. Here, the authors examine the amino acid transporter Slimfast from the yellow-fever mosquito and describe both its specificity and mechanism of action.
PMCID: PMC4608377  PMID: 26449545
5.  Blood serum and BSA, but neither red blood cells nor hemoglobin can support vitellogenesis and egg production in the dengue vector Aedes aegypti 
PeerJ  2015;3:e938.
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.
PMCID: PMC4435475  PMID: 26020000
Artificial blood meal; Aedes aegypti; Vitellogenesis; Egg development; Mosquito culture; Mass rearing
6.  Small mosquitoes, large implications: crowding and starvation affects gene expression and nutrient accumulation in Aedes aegypti 
Parasites & Vectors  2015;8:252.
Environmental factors such as temperature, nutrient availability, and larval density determine the outcome of postembryonic development in mosquitoes. Suboptimal temperatures, crowding, and starvation during the larval phase reduce adult mosquito size, nutrient stores and affect vectorial capacity.
In this study we compared adult female Aedes aegypti, Rockefeller strain, raised under standard laboratory conditions (Large) with those raised under crowded and nutritionally deprived conditions (Small). To compare the gene expression and nutritional state of the major energy storage and metabolic organ, the fat body, we performed transcriptomics using Illumina based RNA-seq and metabolomics using GC/MS on females before and 24 hours following blood feeding.
Analysis of fat body gene expression between the experimental groups revealed a large number of significantly differentially expressed genes. Transcripts related to immunity, reproduction, autophagy, several metabolic pathways; including amino acid degradation and metabolism; and membrane transport were differentially expressed. Metabolite profiling identified 60 metabolites within the fat body to be significantly affected between small and large mosquitoes, with the majority of detected free amino acids at a higher level in small mosquitoes compared to large.
Gene expression and metabolites in the adult fat body reflect the individual post-embryonic developmental history of a mosquito larva. These changes affect nutritional storage and utilization, immunity, and reproduction. Therefore, it is apparent that changes in larval environment due to weather conditions, nutrition availability, vector control efforts, and other factors can affect adult vectorial capacity in the field.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0863-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4415286  PMID: 25924822
Aedes aegypti; fat body; RNAseq; Nutrition; Transcriptome; Metabolome; Immunity; Starvation
7.  The Efficacy of Some Commercially Available Insect Repellents for Aedes aegypti (Diptera: Culicidae) and Aedes albopictus (Diptera: Culicidae) 
Journal of Insect Science  2015;15(1):140.
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.
PMCID: PMC4667684  PMID: 26443777
repellents; Aedes; yellow fever mosquito; Asian tiger mosquito; DEET
8.  The Odorant Receptor Co-Receptor from the Bed Bug, Cimex lectularius L 
PLoS ONE  2014;9(11):e113692.
Recently, the bed bug, Cimex lectularius L. has re-emerged as a serious and growing problem in many parts of the world. Presence of resistant bed bugs and the difficulty to eliminate them has renewed interest in alternative control tactics. Similar to other haematophagous arthropods, bed bugs rely on their olfactory system to detect semiochemicals in the environment. Previous studies have morphologically characterized olfactory organs of bed bugs’ antenna and have physiologically evaluated the responses of olfactory receptor neurons (ORNs) to host-derived chemicals. To date, odorant binding proteins (OBPs) and odorant receptors (ORs) associated with these olfaction processes have not been studied in bed bugs. Chemoreception in insects requires formation of heteromeric complexes of ORs and a universal OR coreceptor (Orco). Orco is the constant chain of every odorant receptor in insects and is critical for insect olfaction but does not directly bind to odorants. Orco agonists and antagonists have been suggested as high-value targets for the development of novel insect repellents. In this study, we have performed RNAseq of bed bug sensory organs and identified several odorant receptors as well as Orco. We characterized Orco expression and investigated the effect of chemicals targeting Orco on bed bug behavior and reproduction. We have identified partial cDNAs of six C. lectularius OBPs and 16 ORs. Full length bed bug Orco was cloned and sequenced. Orco is widely expressed in different parts of the bed bug including OR neurons and spermatozoa. Treatment of bed bugs with the agonist VUAA1 changed bed bug pheromone-induced aggregation behavior and inactivated spermatozoa. We have described and characterized for the first time OBPs, ORs and Orco in bed bugs. Given the importance of these molecules in chemoreception of this insect they are interesting targets for the development of novel insect behavior modifiers.
PMCID: PMC4239089  PMID: 25411789
9.  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.
Author Summary
Glossina sp. are responsible for transmission of African trypanosomes, the causative agents of sleeping sickness in humans and Nagana in cattle. Blood feeding and nutrient provisioning through lactation during intrauterine progeny development are periods when considerable water movement occurs within tsetse flies. With the completion of the tsetse fly genome, we sought to characterize the role of aquaporins in relation water homeostasis during blood feeding, stress tolerance and the lactation cycle. We provide evidence that specific AQPs are 1. critical during diuresis following a bloodmeal, 2. important in the regulation of dehydration resistance and heat tolerance and 3. crucial in the allocation of water within tsetse milk that is necessary for progeny hydration. Specifically, we discovered a novel tsetse AQP that is imperative to lactation and may represent a potential target for population control of this disease vector.
PMCID: PMC3998938  PMID: 24762803
10.  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.
PMCID: PMC3960487  PMID: 24688471
mosquito; vitellogenesis; insulin; juvenile hormone; ecdysone; target of rapamycin; yolk proteins
11.  The effect of the radio-protective agents ethanol, trimethylglycine, and beer on survival of X-ray-sterilized male Aedes aegypti 
Parasites & Vectors  2013;6:211.
Sterile Insect Technique (SIT) has been successfully implemented to control, and in some cases, eradicate, dipteran insect populations. SIT has great potential as a mosquito control method. Different sterilization methods have been used on mosquitoes ranging from chemosterilization to genetically modified sterile male mosquito strains; however, sterilization with ionizing radiation is the method of choice for effective sterilization of male insects for most species. The lack of gentle radiation methods has resulted in significant complications when SIT has been applied to mosquitoes. Several studies report that irradiating mosquitoes resulted in a decrease in longevity and mating success compared to unirradiated males.
The present study explored new protocols for mosquito sterilization with ionizing radiation that minimized detrimental effects on the longevity of irradiated males.
We tested three compounds that have been shown to act as radioprotectors in the mouse model system - ethanol, trimethylglycine, and beer. Male Aedes aegypti were treated with one of three chosen potential radioprotectors and were subsequently irradiated with identical doses of long-wavelength X-rays. We evaluated the effect of these radioprotectors on the longevity of male mosquito after irradiation.
We found that X-ray irradiation with an absorbed dose of 1.17 gy confers complete sterility. Irradiation with this dose significantly shortened the lifespan of male mosquitoes and all three radioprotectors tested significantly enhanced the lifespan of irradiated mosquito males.
Our results suggest that treatment with ethanol, beer, or trimethylglycine before irradiation can be used to enhance longevity in mosquitoes.
PMCID: PMC3723957  PMID: 23866939
Sterile insect technique; Mosquitoes; Aedes aegypti; X-rays; Radioprotectors; Trimethylglycine; Ethanol; Beer; Survival; Longevity; Sterility; Fecundity
12.  SLC7 amino acid transporters of the yellow fever mosquito Aedes aegypti and their role in fat body TOR signaling and reproduction 
Journal of Insect Physiology  2012;58(4):513-522.
An important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.
Methodology/Principal Findings
We identified eleven putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All eleven Aedes aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these ‘knockdowns’ reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.
Our analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.
PMCID: PMC3322257  PMID: 22266018
SLC7 transporter; Mosquito; Target of rapamycin; Amino acid; Aedes aegypti; RNA interference
13.  The Fat Body Transcriptomes of the Yellow Fever Mosquito Aedes aegypti, Pre- and Post- Blood Meal 
PLoS ONE  2011;6(7):e22573.
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.
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.
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.
PMCID: PMC3144915  PMID: 21818341
14.  The Aquaporin Gene Family of the Yellow Fever Mosquito, Aedes aegypti 
PLoS ONE  2010;5(12):e15578.
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.
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.
PMCID: PMC3014591  PMID: 21249121
Journal of insect physiology  2007;54(1):231-239.
Anautogenous mosquitoes require blood meals to promote egg development. If adequate nutrients are not obtained during larval development, the resulting “small” sized adult mosquitoes require multiple blood meals for egg development; markedly increasing host-vector contacts and the likelihood of disease transmission. Nutrient-sensitive target-of-rapamycin (TOR) signaling is a key signaling pathway that links elevated hemolymph amino acid levels derived from the blood meal to the expression of yolk protein precursors in the fat body. Here we report that the blood-meal-induced activation of the TOR-signaling pathway and subsequent egg maturation depends on the accumulation of adequate nutritional reserves during larval development. We have established well-nourished, “standard” mosquitoes and mal-nourished, “small” mosquitoes as models to address this nutrient sensitive pathway. This regulatory mechanism involves juvenile hormone (JH), which acts as a mediator of fat body competence, permitting the response to amino acids derived from the blood meal. We demonstrate that treatment with JH results in recovery of the TOR molecular machinery, AaiCAT2, TOR, and S6K, in fat bodies of small mosquitoes, enabling them to complete their first gonotrophic cycle after a single blood meal. These findings establish a direct link between nutrient reserves and the establishment of TOR signaling in mosquitoes.
PMCID: PMC2242809  PMID: 17981294
amino acid signaling; fat body; vitellogenesis; competence; S6 Kinase; iCAT2
16.  Forkhead transcription factors regulate mosquito reproduction 
Forkhead box (Fox) genes encode a family of transcription factors defined by a ‘winged helix’ DNA-binding domain. In this study we aimed to identify Fox factors that are expressed within the fat body of the yellow fever mosquito Aedes aegypti, and determine whether any of these are involved in the regulation of mosquito yolk protein gene expression. The Ae. aegypti genome contains eighteen loci that encode putative Fox factors. Our stringent cladistic analysis has profound implications for the use of Fox genes as phylogenetic markers. Twelve Ae. aegypti Fox genes are expressed within various tissues of adult females, six of which are expressed within the fat body. All six Fox genes expressed in the fat body displayed dynamic expression profiles following a blood meal. We knocked down the ’fat body Foxes’ through RNAi to determine whether these “knockdowns” hindered amino acid-induced vitellogenin gene expression. We also determined the effect of these knockdowns on the number of eggs deposited following a blood meal. Knockdown of FoxN1, FoxN2, FoxL, and FoxO, had a negative effect on amino acid- induced vitellogenin gene expression and resulted in significantly fewer eggs laid. Our analysis stresses the importance of Fox transcription factors in regulating mosquito reproduction.
PMCID: PMC2441594  PMID: 17681238
forkhead; Aedes aegypti; fat body; vitellogenin; yolk protein; RNAi
17.  Effect of insulin and 20-hydroxyecdysone in the fat body of the yellow fever mosquito, Aedes aegypti 
In mosquitoes, yolk protein precursor (YPP) gene expression is activated after a blood meal through the synergistic action of a steroid hormone and the amino acid/Target of Rapamycin (TOR) signaling pathway in the fat body. We investigated the role of insulin signaling in the regulation of YPP gene expression. The presence of mosquito insulin receptor (InR) and the Protein kinase B (PKB/Akt) in the adult fat body of female mosquitoes was confirmed by means of the RNA interference (RNAi). Fat bodies stimulated with insulin were able to promote the phosphorylation of ribosomal S6 Kinase, a key protein of the TOR signaling pathway. Importantly, insulin in combination with 20-hydroxyecdysone activated transcription of the YPP gene vitellogenin (Vg), and this process was sensitive to the Phosphoinositide-3 kinase (PI-3k) inhibitor LY294002 as well as the TOR inhibitor rapamycin. RNAi-mediated knockdown of the mosquito InR, Akt, and TOR inhibited insulin-induced Vg gene expression as well as S6 Kinase phosphorylation in in vitro fat body culture assays.
PMCID: PMC2104489  PMID: 17967350
insulin receptor; Akt; S6K; target of rapamycin; RNAi

Results 1-17 (17)