Research

Mosquitoes transmit more devastating human diseases than all other blood feeding arthropods combined 



Malaria causes 300 million clinical cases/year and over 0.7 million death/year, mostly children.

Dengue fever – 50-100 million cases/year, 0.5 million with severe illness2.5% mortalityOver 2.5 billion people are at risk.     

 

                                         Yellow fever - 200 000 cases/year and 30 000 death/year.

                                         West Nile Virus causes serious illness and numerous deaths in the United States; 2013 total cases 2,271, 100 death; California – 357 cases, 14 deaths.


Chikungunya fever is an emerging mosquito-borne viral disease in Asia, Africa and Southern Europe. Co-infection with Dengue fever is particularly severe

Lymphatic Filariasis - 120 millions are infected with over 40 millions seriously incapacitated, 1.4 billion people at risk.


          


Molecular basis of mosquito reproduction and immunity  

Female mosquitoes require vertebrate blood for egg development. As a consequence, mosquitoes are vectors of numerous disease pathogens of human and domestic animals. Obligatory blood feeding is an evolutionary adaptation of mosquitoes for rapid and massive egg production. Disease pathogens co-evolved exploiting mosquito egg maturation cycles to their advantage; they invade a mosquito organism in a process of the initial blood feeding, multiply and are transmitted to a vertebrate host during subsequent blood feedings. Due to this intimate relationship between mosquito gonadotrophic cycles and transmission of disease pathogens, it is of outmost significance to understand the molecular basis of egg development in these insects. Elucidating mechanisms governing mosquito reproduction is of critical importance for devising novel control approaches.

My laboratory explores the molecular basis of mosquito reproduction and immunity. We utilize a variety of molecular genetics, transgenics, genomics and bioinformatics tools. We are deciphering the role of small non-coding RNAs (microRNAs) in mosquito blood feeding and reproduction. Two large projects deal with understanding of molecular mechanisms controlling activating and repressing actions of hormones (see below for short summaries of lab projects).


The role of microRNAs in regulating blood digestion and egg development (Click here for publications) 

MicroRNAs (miRNAs) are short non-coding RNAs 21 to 22 nucleotides in length that control developmental timing, stem cell maintenance and other developmental and physiological processes in plants and animals. miRNAs have been implicated in numerous human diseases including cancers. miRNAs are negative regulators that function as specificity determinants that promote degradation of mRNA targets. In my laboratory, we work to functionally characterize miRNAs in mosquitoes by their specific depletion using specific antagomirs and transgenic techniques, bioinformatics target predictions, cell transfection verification and phenotype rescue experiments. Our research has identified several miRNAs that play significant roles in regulating vital functions in mosquitoes such as blood digestion and egg maturation. Mosquito-specific miRNAs are of particular interest as they could provide a foundation for developing novel mosquito control approaches utilizing these small molecules.

      

Female mosquitoes are unable to digest blood after miR-275 depletion. Shown are isolated digestive systems of female mosquitoes post blood meal from antagomir depletion of miR-275 (miR-275-ant) and wild type untreated female mosquitoes 24 h postblood meal. Cr – crop, Mg – midgut or stomach.  Egg development is prevented in these miR-275 deprived mosquitoes (Bryant and Raikhel, 2010, PNAS)


                 

Egg development: genes, proteins and receptors (Click here for publications) 

Vitellogenesis is a process central to egg maturation. It involves massive production of yolk protein precursors (YPP) by the fat body, the insect metabolic tissue analogous to vertebrate liver and adipose tissue. We have characterized regulation of genes encoding YPPs and biosynthesis of these proteins. YPPs, including vitellogenin, vitellogenic carboxypeptidase, vitellogenic cathepsin-like protein and lipophorin, are secreted by the fat body and internalized by developing oocytes.

The major YPPs, vitellogenin (Vg) and lipophorin (Lp), are lipoproteins that are internalized by their specific receptors in the ovary in the process of receptor-mediated endocytosis.  My laboratory was first to characterize the vitellogenin receptor in an insect ovary (Sappinton et al. 1998, PNAS). We have identified the molecular structure of the Vg receptor and its intracellular route of delivering its ligand load and recycling. Mosquito vitellogenin (AaVgR) and lipophorin (AaLpR) receptors belong to the family of low-density lipoprotein receptors. In contrast to vertebrate (GgVgR) or the nematode C. elegans (CeVgR) VgRs, AaVgR contains two ligand binding modules.

Receptor-mediated endocytosis in the mosquito oocyte


The role of the insect-specific juvenile hormone (JH) in regulation of gene expression (Click here for publications) 

In the yellow fever mosquito Aedes aegypti, the first cycle of egg development is divided into two periods: previtellogenic and vitellogenic. The first, previtellogenic period is controlled by the insect-specific hormone, juvenile hormone III (JH III), while the second, vitellogenic period by a steroid hormone 20-hydroxyecdysone (20E). My laboratory is investigating how these hormones control differential gene expression during mosquito reproduction. Because of the clear separation of reproductive periods controlled by these two different hormones, mosquitoes represent an excellent system for studying molecular basis of hormone action.  Insect-specific sesquiterpenoid, juvenile hormones (JH), control numerous essential physiological functions in immature and adult insects, including development, reproduction, pheromone production, and cast differentiation in social insects. Methoprene-tolerant (Met), a member of the family of basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) transcription factors, has been recognized as the JH receptor. A single Met gene exists in Aedes aegypti genome and JH is a sole hormone regulating mosquito previtellogenic period of egg development, making this insect an excellent model for studying JH action. For its transcriptional activity, Met requires the bHLH-PAS domain-containing steroid receptor coactivator SRC (also known as FISC). Met also dimerizes with the bHLH-PAS circadian clock protein Cycle and binds to specific binding sites in promoters of genes regulated in a JH- and circadian rhythm-dependent manner (Shin et al. 2012. PNAS). We have only a scant understanding of the JH regulatory hierarchy governing differential gene expression. Our recent work has indicated certain genes are activated via direct binding of Met to their promoters (Zou et al. 2013. PNAS). However, how  JH and Met repress gene  expression remains to be determined. Bioinformatics analysis of 5’ regulatory regions of JH/Met-regulated genes. Met binding sites have been found in Met-activated genes but not in Met-repressed ones.


Molecular basis of the insect steroid hormone 20-hydroxyecdysone action in regulation of differential gene expression (Click here for publications) 

Insect steroid hormone 20-hydroxyecdysone (20E) plays a major role in regulating gene expression during the vitellogenic period of female mosquito reproduction. We are investigating how the 20E gene regulatory pathway controls differential gene expression in female mosquitoes.  

In the presence of 20E, the nuclear receptor βFTZ-F1 (Zhu et al. 2003. PNAS) facilitates the formation of the complex of ecdysone receptor (EcR), its heterodimer partner USP (an orthologue of vertebrate RXR) and the steroid receptor coactivator SRC/FISC.

βFTZ-F1 aids hormone-dependent loading of transcription-activating complex onto target promoters, where histone acetylation facilitates target gene transcription (Zhu et al. 2006. Mol. Cell. Biol).

Although the 20E pathway is conserved, specific isoforms of the 20E hierarchy factors (early genes) play unique roles in regulating different biological processes. How is the 20E genetic hierarchy adapted for the specific requirements of the blood-meal triggered cyclic egg development  in mosquitoes? Our research has demonstrated that a unique set of 20E-activated transcription factor isoforms is reponsible for activating vitellogenic genes, while another set is involved in a timely downregulation of their expression.

Microarray gene expression profiling in the female mosquito fat body has revealed that about 8,000 genes are differentially expressed during vitellogenic period Further bioinformatics analysis has permitted us to distinguish regulatory motifs common for co-regulated gene clusters. We are elucidating molecular basis of activation and repression of genes by liganded EcR.We are conductiing RNAi/RNA-seq analysis for specific factors of the 20E gene hierarchy to decifer how this hormone orchestrates a complex developmental program during mosquito reproduction.   


Nutritional control of egg development (Click here for publications)


There has been a long standing controversy concerning the nature of blood-meal activation of vitellogenic events in mosquitoes. Hansen et al. (2004, PNAS) have elucidated the molecular mechanism underlying this activation showing that it is regulated by several signaling amino acids from ingested blood. Mosquitoes, utilize an evolutionally conserved nutritional signaling - the TOR (Target of Rapamycin) pathway. This pathway that is ubiquitously expressed in eukaryotes (from yeast to humans) mediates nutritional signals, playing a key role in cell growth, proliferation and cancer. However, mosquitoes utilize amino acid-based TOR signaling in a unique way, adapting it for their lifestyle as blood-feeding insects and pathogen vectors. Using various techniques including targeted RNA interference gene knockdowns, we have characterized this pathway in Aedes aegypti.


                                                             

                                                                Genomics (Click here for publications) 


My laboratory has been involved in studies associated with analysis of mosquito genomes (Nene et al. 2007. Science; Arensburger et al. 2010. Science). In particular, we have been interested in evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes (Waterhouse et al. 2007. Science; Bartholomay et al. 2010. Science). Transcriptome analysis of Aedes aegypti transgenic mosquitoes with altered immunity has lead to identification of repertories of genes controlled by the Toll and IMD pathways (Zou et al. 2011. Plos Pathogen). RNAi screens for transcription factors coupled with either microarray or RNAseq analyses are being used to decipher regulatory cascades controlling hormone-mediated gene expression (Zou et al. 2013. PNAS).  



Mosquito transgenesis (Click here for publications)



My laboratory was among the first to establish mosquito genetic transformation. We have pioneered generating transgenic mosquitoes with altered immunity. Recently, we established the first mosquito binary Gal4-UAS system that enables researchers to study cell/tissue- stage- and sex-specific expression of mosquito genes. It has opened the door for the refinement of tools for investigating mosquito-vector interactions.

Transgenic Vg-Gal4>UAS-EGFP mosquitoes with sex-, tissue (fat body) and stage-specific gene expression.

We are currently utilizing the Gal4-UAS system for creating mosquitoes with loss-of-function microRNA phenotypes. 

                               


Mosquito immunity and interaction with pathogens (Click here for publications) 

Exploration of the mosquito immune system in my laboratory was initiated with creating transgenic mosquitoes in which elements of innate immunity were altered by their specific over-expression (Kokoza et al. 2000. PNAS). 

We tested a hypothesis that transgenic activation of endogenous immune genes at the time of pathogen entry to a mosquito organism during blood feeding could interfere with pathogen dissemination and block its transmission to a vertebrate host.

To engineer such mosquitoes, we utilized the fat body-, female-specific Vg gene promoter that we characterized as being activated by blood feeding. We selected this fat body-specific gene because the fat body is a central immune organ in insects, including mosquitoes. Using this system, we characterized the effect of antimicrobial proteins, defensins and cecropins, on the malaria pathogen Plasmodium. We have shown that simultaneous transgenic activation of cecropins and defensins blocks Plasmodium development and completely prevents its transmission (Kokoza et al. 2011. PNAS). Thus, this study provided the proof-of-principal for creating transgenic mosquitoes with altered immunity capable to resist pathogen infection.

In insects, including mosquitoes, the Toll and IMD are major pathways of innate immunity. We characterized mosquito orthologues of vertebrate NF-kappaB factors, Rel1 and Rel2, which are downstream gene activators of the Toll and IMD pathways, respectively (Shin et al. 2002 PNAS; Shin et al. 2003 PNAS). Using the Vg promoter driver for activating Rel1 and Rel2 genes, we have created transgenic mosquitoes with the stage-specific activation of either Toll or IMD pathways (Bian et al. 2005 PNAS). Transcriptome analysis has revealed repertoires of genes activated by these pathways. Utilizing transgenic lines co-expressing Rel1 and Rel2, we are investigating the cross talk between these two pathways.

Melanization is the Arthropod-specific immune response that involves formation of melanin capsules either at a wound site or around invading pathogens. The role of melanization in resistance to Plasmodium is a matter of debate. We have shown that the melanization pathway is more complex and specific that it has previously been anticipated (Zou et al. 2010 Immunity).  In this study, we have demonstrated that there are at least two independent melanization pathways, one responsible for a wound repair and another for pathogen melanization.      

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