The mosquito genome: perspectives and possibilities

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| Research Focus

The mosquito genome: perspectives and possibilities Kirkwood M. Land Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, CA 94143, USA

Anopheles gambiae is the mosquito vector responsible for transmitting Plasmodium falciparum, a malaria parasite of humans. With the emergence of genome projects for a variety of prokaryotic and eukaryotic microorganisms, there has been a long-standing interest in sequencing the genomes of the malaria parasite and its insect vector. This tour de force effort has now been completed and reported. The alignment of putative orthologs in An. gambiae with those of Drosophila melanogaster highlights several similarities and differences. These findings could have implications in: (1) identifying new targets for insecticide development; (2) strengthening our understanding of the developmental biology of mosquitoes; and (3) possibly controlling pathogen transmission. A brief overview of these interesting findings and the implications for further studies will be discussed here. Mosquitoes have long-plagued humankind, especially those individuals living in tropical regions of the world. Such mosquito-borne human illnesses include malaria, dengue and yellow fever. Anopheles gambiae is the principal mosquito vector of malaria, a disease that afflicts . 500 million people and causes .2 – 3 million deaths each year. The genomes of An. gambiae and of Plasmodium falciparum, a malaria parasite of humans, have been sequenced and reported [1,2]. With the genome of An. gambiae in hand, scientists are now armed with a panoply of molecular information to understand the biology of this arthropod vector better, and to possibly combat malaria transmission and other mosquito-borne human illnesses. Overview of the mosquito genome project To ensure that a complete coverage of the mosquito genome would be accomplished, a tenfold shotgun sequence coverage was achieved using genomic DNA from both males and females of the PEST strain of An. gambiae. These DNA sequences were assembled into a final map that encompasses .278 million base pairs (bp) [2]. A total of 91% of the genome was organized into 303 scaffolds, with the largest scaffold containing , 23.1 million bp. The resulting genome sequence suggests the presence of , 14 000 protein-encoding genes [2]. However, the assembly of the Y-chromosome has lagged behind the other chromosomes due to the large number of transposable elements present in this sex chromosome [2]. Mosquito strain chosen for analysis The PEST strain of An. gambiae, which was derived from a cross between a laboratory strain and a field-collected Corresponding author: Kirkwood M. Land ([email protected]). http://trepar.trends.com

isolate, was chosen for this sequencing project for several reasons. (1) The PEST strain was technically more feasible for sequencing because investigators had previously constructed two different bacterial artificial chromosome (BAC) libraries with DNA from this mosquito strain [2]. (2) This strain has a homogeneous chromosome arrangement, a feature that is not consistently observed in wildtype mosquito populations. (3) The original PEST strain possesses an X-linked, pink-eye mutation that is used to screen mosquito populations for evidence of cross-colony contamination [3]. (4) From a biological standpoint, the PEST strain was ideal for sequencing based on its prior use in studies of human reservoirs of malaria (see Ref. [2]). (5) This strain is naturally susceptible to infection with P. falciparum [4], a crucial prerequisite for studying malaria transmission. For more information on the PEST strain, see: http://www.malaria.mr4.org Comparison of An. gambiae with Drosophila melanogaster Complete sequencing of the An. gambiae genome now begs the question: how similar or dissimilar is the An. gambiae genome sequence to that of its well-studied cousin Drosophila melanogaster? Such an analysis was undertaken to address this question [5]. Despite evolutionarily diverging . 250 million years ago, the two insects reveal striking similarities in their DNA sequences. However, there are also highlighted differences in their genomes. Almost half of the genes in both insect genomes are presently identified as orthologs (genes in different species that have evolved from a common ancestral gene by speciation) and show an average sequence identity of , 56%, and these orthologous genes retained only half of their intron– exon structure [5]. This could be attributed to successive adaptation of these insects to different life strategies and ecological niches. These observations might also stimulate further discussion on the evolution of higher organisms and, in particular, the evolutionary adaptation of pathogens to insect vectors or vice versa. The discovery of new genes Are there genes that regulate mosquito development and reproduction? A bloodmeal is necessary for egg production in An. gambiae, and multiple blood-feedings assist in the transmission of malaria parasites to their vertebrate hosts. To this end, a series of regulatory peptides encoded by .35 genes from the An. gambiae genome was identified, [6]. These include genes encoding neuropeptides, hormones and myoregulatory peptides (see Table 1 of Ref. [6]) that could regulate mosquito developmental and other physiological processes. The presence of Plasmodium in the

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mosquito might also play a role in the expression and activity of several newly identified insulin-like peptides, which in turn might coordinate growth and reproduction of An. gambiae [6]; the underlying mechanism for this is unclear. However, the identification of these genes should expand our understanding of hematophagy and pathogen transmission in this mosquito. Another set of interesting genes present in An. gambiae is the G-protein coupled receptors (GPCRs) genes. GPCRs are proteins that probably function in second messengermediated pathways (such as those involving Ca2þ and cAMP) which might, consequently, affect many aspects of the mosquito life cycle. In the search of these genes, 276 GPCRs were identified from the An. gambiae genome [7]. Seventy-nine candidate odorant receptors were further characterized for tissue expression, along with 76 putative gustatory receptors for their molecular evolutionary relationship relative to D. melanogaster [7]. Examples of both lineage-specific gene expansions and unusually high sequence conservation were also observed. There is current interest in understanding the basic roles of phototransduction and olfaction in insects, and whether these pathways are distinct or interdependent [8]. Another area of long-standing interest in insect biology is immunity. Molecular features of the insect immune response to bacteria have strongly suggested that the insect immune system might represent an evolutionary precursor to the innate immune system in vertebrates [9,10]. The mechanisms involved in the insect immune response to eukaryotic pathogens have only been examined in detail recently. Nevertheless, mosquitoes infected with malaria parasites have served as an excellent model system for these investigations. To date, a variety of methods have been useful in identifying genes involved in insect immunity; these include degenerate PCR [11,12], differential display analysis [13] and, more recently, complementary DNA (cDNA) microarrays [14,15]. Orthologous genes involved in mosquito immunity can be identified by sequence homology with other insect genes: for instance, 242 An. gambiae genes from 18 gene families implicated in innate immunity have been identified, which are distinct from the D. melanogaster genes [14]. A search for immune-related gene families involved in recognition, signal modulation and effector systems also revealed an absence of obvious orthologs and the presence of excessive expansions of genes [14], which could reflect different selective pressures from the different pathogens encountered by these insects. By contrast, components of the multifunctional Toll signal-transduction pathway are present and substantially conserved [14]. Expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges [14]. The identification of new genes involved in insect immunity should stimulate further debate on the evolution of the vertebrate immune system, as well as the potential to identify new insecticide targets against mosquitoes. On this note, the emergence of insecticide resistance in the mosquito poses a serious threat to the efficacy of many malaria control programs. To address this issue, the An. gambiae genome was searched for members of the three major enzyme families implicated in insecticide http://trepar.trends.com

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resistance: (1) carboxylesterases; (2) glutathione transferases; and (3) cytochrome P450s [14]. A comparative genomic analysis with D. melanogaster reveals a considerable expansion of these supergene families in the mosquito [14]. The biological role of such gene expansions is not clear. However, they might reflect the diversification of these insects, analogous to the major differences in their life history and ecology. These data could also provide insight into identifying resistance-associated enzymes in other insects; perhaps enabling the resistance status of mosquitoes, flies and other insects to be monitored. Future directions and perspectives The availability of a genome sequence for An. gambiae has created a plethora of opportunities for mosquito and malaria research. Genomic expression profiling should now be possible using standard whole-genome DNA microarrays. Available tools for reverse genetic analysis will also be important in verifying the functions of these newly identified genes. Hemocyte-like cell lines [16] coupled with in vitro transient transfections, and stable, transposonmediated transfections are already available [17]. Germline transformation has also been accomplished for An. gambiae [18] and, more recently, RNA interference methods have been described [19]. Genes identified from the An. gambiae genome project will assist in parasite refractoriness studies [20,21], an area possibly linked to control of parasite transmission. Using a combination of molecular genetic and expression profiling approaches, phenotypic analyses can now be accomplished in An. gambiae. This combination of genetics with insect physiology should provide a powerful means to understand the biology of mosquitoes and the diseases they transmit. Acknowledgements I apologize to all of my colleagues whose work was not cited here as a result of space constraints. K.M.L. is supported by a fellowship from the Giannini Family Foundation for Medical Research.

References 1 Gardner, M.J. et al. (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498 – 511 2 Holt, R.A. et al. (2002) The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129 – 149 3 Mason, G.F. (1967) Genetic studies on mutations in species A and B of the Anopheles gambiae complex. Genet. Res. 10, 205 – 217 4 Githeko, A.K. et al. (1992) The reservoir of Plasmodium falciparum malaria in a holoendemic area of western Kenya. Trans. R. Soc. Trop. Med. Hyg. 86, 355 – 358 5 Zdobnov, E.M. et al. (2002) Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298, 149 – 159 6 Riehle, M.A. et al. (2002) Neuropeptides and peptide hormones in Anopheles gambiae. Science 298, 172 – 175 7 Hill, C.A. et al. (2002) G protein-coupled receptors in Anopheles gambiae. Science 298, 176 – 178 8 Merrill, C.E. et al. (2002) Visual arrestins in olfactory pathways of Drosophila and the malaria vector mosquito Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 99, 1633 – 1638 9 Gillespie, J.P. et al. (1997) Biological mediators of insect immunity. Annu. Rev. Entomol. 42, 611 – 643 10 Hoffmann, J.A. et al. (1999) Phylogenetic perspectives in innate immunity. Science 284, 1313 – 1318 11 Luckhart, S. et al. (1998) The mosquito Anopheles stephensi limits

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malaria parasite development with inducible synthesis of nitric oxide. Proc. Natl. Acad. Sci. U. S. A. 95, 5700 – 5705 Gorman, M.J. et al. (2000) Molecular characterization of five serine protease genes cloned from Anopheles gambiae hemolymph. Insect Biochem. Mol. Biol. 30, 35 – 46 Dimopoulos, G. et al. (1996) Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 93, 13066 – 13071 Christophides, G.K. et al. (2002) Immunity-related genes and gene families in Anopheles gambiae. Science 298, 59 – 65 Dimopoulos, G. et al. (2002) Genome expression analysis of Anopheles gambiae: responses to injury, bacterial challenge, and malaria infection. Proc. Natl. Acad. Sci. U. S. A. 99, 8814 – 8819 Muller, H.M. et al. (1999) A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes. J. Biol. Chem. 274, 11727 – 11735

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17 Catteruccia, F. et al. (2000) Stable germline transformation of the malaria mosquito Anopheles stephensi. Nature 405, 959– 962 18 Grossman, G.L. et al. (2001) Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element. Insect Mol. Biol. 10, 597 – 604 19 Blandin, S. et al. (2002) Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene. EMBO Rep. 3, 852– 856 20 Ito, J. et al. (2002) Transgenic anopheline mosquitoes impaired in transmission of a malaria parasite. Nature 417, 452 – 455 21 Thomasova, D. et al. (2002) Comparative genomic analysis in the region of a major Plasmodium-refractoriness locus of Anopheles gambiae. Proc. Natl. Acad. Sci. U. S. A. 99, 8179– 8184 1471-4922/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1471-4922(03)00021-7

Aping Jane Goodall: insights into human lymphatic filariasis Balachandran Ravindran Division of Immunology, Regional Medical Research Centre, Indian Council of Medical Research, Nandankanan Road, Bhubaneswar-751023, India

The relationship between infection and disease has been a subject of intense debate in human filariasis. Most patients with chronic disease such as lymphedema or elephantiasis are free of demonstrable current infection. Based on cross-sectional data and a few assumptions and presumptions, two models of natural history of filariasis have been in vogue during the past two decades. The only (but arduous) way to understand the sequence of events is to follow up subjects with and without patent infections over a period of several years. This article offers critical comments and highlights the insights acquired from such studies. Lymphatic filariasis, a chronic disease characterized by clinical manifestations such as lymphedema, elephantiasis and/or hydrocele, is caused by the vector-borne nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori. About 90% of the 120 million globally prevalent cases are caused by W. bancrofti. Human infections are initiated by the bite of infected mosquitoes, releasing infective larvae that home to the lymphatic system and develop into adult male and female worms (measuring , 2 – 5 cm in length). After mating, the female worms release several million microfilariae (Mf), which appear in the blood circulation, and these Mf are passed on to mosquitoes during bloodfeeding. Typically, in filariasis-endemic areas, there are three groups of subjects: (1) those who are often free of overt chronic disease manifestations, but with an active patent infection [adult worms that are present in the lymphatic system, which is indicated by the presence of circulating filarial antigens (CFA)]; (2) patients who display one or more of the disease manifestations such as hydrocele and/or lymphedema, with or often without demonstrable filarial Corresponding author: Balachandran Ravindran ([email protected]). http://trepar.trends.com

infections; and (3) subjects who are free of patent infection and disease [1]. The relationship between infection and disease (Box 1) has been a contentious issue that several investigators have attempted to address in human filariasis. In the absence of comparable clinical features in experimental animal models, the progression of infection leading to development of chronic disease manifestations has been deduced by analysis of cross-sectional data on subjects living in endemic areas. Sequencing the events that take place over a period of several decades by using such ‘snapshots’ of cross-sectional data has been the mainstay for development of models [1– 6]. Broadly, two models (both speculative) have been put forward. (1) A static immunological viewpoint proposes that the clinical outcome of infection could take one of two routes (either patent infection without overt pathology, or a form of inflammatory pathology without microfilaraemia) and that differing immune responses could predispose individuals towards either harboring the infection or developing the disease [2]. (2) The ‘dynamic model’ proposes that there is a sequential progression from infection, microfilaraemia and amicrofilaraemia to obstructive disease in all individuals who experience microfilaraemia [4] and/or that the lymphatic-dwelling adult worms mediate pathology and disease [5,6]. Thus, the dynamic model (unlike the immunological perspective) presumes a phase of patent infection as a pre-requisite for development of chronic filariasis. Box 2 shows a simplified representation of the two models and their limitations. An alternative model that assumes a role for adult worm, as well as inflammatory host response-mediated mechanisms of disease development, has been proposed very recently [1]. The other engaging issue in human filariasis has been the association observed between patent filarial infections and immunological hyporesponsiveness or tolerance,