Department of Entomology

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Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation
Miller, Jason R.; Koren, Sergey; Dilley, Kari A.; Puri, Vinita; Brown, David M.; Harkins, Derel M.; Thibaud-Nissen, Françoise; Rosen, Benjamin D.; Xiao-Guang, Chen; Tu, Zhijian Jake; Sharakhov, Igor V.; Sharakhova, Maria V.; Sebra, R.; Stockwell, T. B.; Bergman, N. H.; Sutton, G. G.; Phillippi, A. M.; Pieemarini, P. M.; Shabman, R. S. (2018-03)
The 50-year old Aedes albopictus C6/36 cell line is a resource for the detection, amplification, and analysis of mosquito-borne viruses including Zika, dengue, and chikungunya. The cell line is derived from an unknown number of larvae from an unspecified strain of Aedes albopictus mosquitoes. Toward improved utility of the cell line for research in virus transmission, we present an annotated assembly of the C6/36 genome.
The Beginning of the End: A Chromosomal Assembly of the New World Malaria Mosquito Ends with a Novel Telomere
Compton, Austin; Liang, Jiangtao; Chen, Chujia; Lukyanchikova, Varvara; Qi, Yumin; Potters, Mark B.; Settlage, Robert; Miller, Dustin; Deschamps, Stephane; Mao, Chunhong; Llaca, Victor; Sharakhov, Igor V.; Tu, Zhijian Jake (Genetics Society of America, 2020-10-01)
Chromosome level assemblies are accumulating in various taxonomic groups including mosquitoes. However, even in the few reference-quality mosquito assemblies, a significant portion of the heterochromatic regions including telomeres remain unresolved. Here we produce a de novo assembly of the New World malaria mosquito, Anopheles albimanus by integrating Oxford Nanopore sequencing, Illumina, Hi-C and optical mapping. This 172.6 Mbps female assembly, which we call AalbS3, is obtained by scaffolding polished large contigs (contig N50 = 13.7 Mbps) into three chromosomes. All chromosome arms end with telomeric repeats, which is the first in mosquito assemblies and represents a significant step toward the completion of a genome assembly. These telomeres consist of tandem repeats of a novel 30-32 bp Telomeric Repeat Unit (TRU) and are confirmed by analyzing the termini of long reads and through both chromosomal in situ hybridization and a Bal31 sensitivity assay. The AalbS3 assembly included previously uncharacterized centromeric and rDNA clusters and more than doubled the content of transposable elements and other repetitive sequences. This telomere-to-telomere assembly, although still containing gaps, represents a significant step toward resolving biologically important but previously hidden genomic components. The comparison of different scaffolding methods will also inform future efforts to obtain reference-quality genomes for other mosquito species.
Chromosome-level genome assemblies of the malaria vectors Anopheles coluzzii and Anopheles arabiensis
Zamyatin, Anton; Avdeyev, Pavel; Liang, Jiangtao; Sharma, Atashi; Chen, Chujia; Lukyanchikova, Varvara; Alexeev, Nikita; Tu, Zhijian Jake; Alekseyev, Max A.; Sharakhov, Igor V. (Oxford University Press, 2021-03-01)
Background: Anopheles coluzzii and Anopheles arabiensis belong to the Anopheles gambiae complex and are among the major malaria vectors in sub-Saharan Africa. However, chromosome-level reference genome assemblies are still lacking for these medically important mosquito species. Findings: In this study, we produced de novo chromosome-level genome assemblies for A. coluzzii and A. arabiensis using the long-read Oxford Nanopore sequencing technology and the Hi-C scaffolding approach. We obtained 273.4 and 256.8 Mb of the total assemblies for A. coluzzii and A. arabiensis, respectively. Each assembly consists of 3 chromosome-scale scaffolds (X, 2, 3), complete mitochondrion, and unordered contigs identified as autosomal pericentromeric DNA, X pericentromeric DNA, and Y sequences. Comparison of these assemblies with the existing assemblies for these species demonstrated that we obtained improved reference-quality genomes. The new assemblies allowed us to identify genomic coordinates for the breakpoint regions of fixed and polymorphic chromosomal inversions in A. coluzzii and A. arabiensis. Conclusion: The new chromosome-level assemblies will facilitate functional and population genomic studies in A. coluzzii and A. arabiensis. The presented assembly pipeline will accelerate progress toward creating high-quality genome references for other disease vectors.
A Gene-Based Method for Cytogenetic Mapping of Repeat-Rich Mosquito Genomes
Masri, Reem A.; Karagodin, Dmitriy A.; Sharma, Atashi; Sharakhova, Maria V. (MDPI, 2021-02-06)
Long-read sequencing technologies have opened up new avenues of research on the mosquito genome biology, enabling scientists to better understand the remarkable abilities of vectors for transmitting pathogens. Although new genome mapping technologies such as Hi-C scaffolding and optical mapping may significantly improve the quality of genomes, only cytogenetic mapping, with the help of fluorescence in situ hybridization (FISH), connects genomic scaffolds to a particular chromosome and chromosome band. This mapping approach is important for creating and validating chromosome-scale genome assemblies for mosquitoes with repeat-rich genomes, which can potentially be misassembled. In this study, we describe a new gene-based physical mapping approach that was optimized using the newly assembled Aedes albopictus genome, which is enriched with transposable elements. To avoid amplification of the repetitive DNA, 15 protein-coding gene transcripts were used for the probe design. Instead of using genomic DNA, complementary DNA was utilized as a template for development of the PCR-amplified probes for FISH. All probes were successfully amplified and mapped to specific chromosome bands. The genome-unique probes allowed to perform unambiguous mapping of genomic scaffolds to chromosome regions. The method described in detail here can be used for physical genome mapping in other insects.
Genome Landscape and Evolutionary Plasticity of Chromosomes in Malaria Mosquitoes
Xia, Ai; Sharakhova, Maria V.; Leman, Scotland C.; Tu, Zhijian Jake; Bailey, Jeffrey A.; Smith, Christopher D.; Sharakhov, Igor V. (PLOS, 2010-05-12)
Background: Nonrandom distribution of rearrangements is a common feature of eukaryotic chromosomes that is not well understood in terms of genome organization and evolution. In the major African malaria vector Anopheles gambiae, polymorphic inversions are highly nonuniformly distributed among five chromosomal arms and are associated with epidemiologically important adaptations. However, it is not clear whether the genomic content of the chromosomal arms is associated with inversion polymorphism and fixation rates. Methodology/Principal Findings: To better understand the evolutionary dynamics of chromosomal inversions, we created a physical map for an Asian malaria mosquito, Anopheles stephensi, and compared it with the genome of An. gambiae. We also developed and deployed novel Bayesian statistical models to analyze genome landscapes in individual chromosomal arms An. gambiae. Here, we demonstrate that, despite the paucity of inversion polymorphisms on the X chromosome, this chromosome has the fastest rate of inversion fixation and the highest density of transposable elements, simple DNA repeats, and GC content. The highly polymorphic and rapidly evolving autosomal 2R arm had overrepresentation of genes involved in cellular response to stress supporting the role of natural selection in maintaining adaptive polymorphic inversions. In addition, the 2R arm had the highest density of regions involved in segmental duplications that clustered in the breakpoint-rich zone of the arm. In contrast, the slower evolving 2L, 3R, and 3L, arms were enriched with matrixattachment regions that potentially contribute to chromosome stability in the cell nucleus. Conclusions/Significance: These results highlight fundamental differences in evolutionary dynamics of the sex chromosome and autosomes and revealed the strong association between characteristics of the genome landscape and rates of chromosomal evolution. We conclude that a unique combination of various classes of genes and repetitive DNA in each arm, rather than a single type of repetitive element, is likely responsible for arm-specific rates of rearrangements.
Genomic composition and evolution of Aedes aegypti chromosomes revealed by the analysis of physically mapped supercontigs
Timoshevskiy, Vladimir A.; Kinney, Nicholas A.; deBruyn, Becky S.; Mao, Chunhong; Tu, Zhijian Jake; Severson, D. W.; Sharakhov, Igor V.; Sharakhova, Maria V. (Biomed Central, 2014-04-14)
Background An initial comparative genomic study of the malaria vector Anopheles gambiae and the yellow fever mosquito Aedes aegypti revealed striking differences in the genome assembly size and in the abundance of transposable elements between the two species. However, the chromosome arms homology between An. gambiae and Ae. aegypti, as well as the distribution of genes and repetitive elements in chromosomes of Ae. aegypti, remained largely unexplored because of the lack of a detailed physical genome map for the yellow fever mosquito. Results Using a molecular landmark-guided fluorescent in situ hybridization approach, we mapped 624-Mb of the Ae. aegypti genome to mitotic chromosomes. We used this map to analyze the distribution of genes, tandem repeats and transposable elements along the chromosomes and to explore the patterns of chromosome homology and rearrangements between Ae. aegypti and An. gambiae. The study demonstrated that the q arm of the sex-determining chromosome 1 had the lowest gene content and the highest density of minisatellites. A comparative genomic analysis with An. gambiae determined that the previously proposed whole-arm synteny is not fully preserved; a number of pericentric inversions have occurred between the two species. The sex-determining chromosome 1 had a higher rate of genome rearrangements than observed in autosomes 2 and 3 of Ae. aegypti. Conclusions The study developed a physical map of 45% of the Ae. aegypti genome and provided new insights into genomic composition and evolution of Ae. aegypti chromosomes. Our data suggest that minisatellites rather than transposable elements played a major role in rapid evolution of chromosome 1 in the Aedes lineage. The research tools and information generated by this study contribute to a more complete understanding of the genome organization and evolution in mosquitoes.
Improved reference genome of Aedes aegypti informs arbovirus vector control
Matthews, Benjamin J.; Dudchenko, Olga; Kingan, Sarah B.; Koren, Sergey; Antoshechkin, Igor; Crawford, Jacob E.; Glassford, William J.; Herre, Margaret; Redmond, Seth N.; Rose, Noah H.; Weedall, Gareth D.; Wu, Yang; Batra, Sanjit S.; Brito-Sierra, Carlos A.; Buckingham, Steven D.; Campbell, Corey L.; Chan, Saki; Cox, Eric; Evans, Benjamin R.; Fansiri, Thanyalak; Filipovic, Igor; Fontaine, Albin; Gloria-Soria, Andrea; Hall, Richard; Joardar, Vinita S.; Jones, Andrew K.; Kay, Raissa G. G.; Kodali, Vamsi K.; Lee, Joyce; Lycett, Gareth J.; Mitchell, Sara N.; Muehling, Jill; Murphy, Michael R.; Omer, Arina D.; Partridge, Frederick A.; Peluso, Paul; Aiden, Aviva Presser; Ramasamy, Vidya; Rasic, Gordana; Roy, Sourav; Saavedra-Rodriguez, Karla; Sharan, Shruti; Sharma, Atashi; Smith, Melissa Laird; Turner, Joe; Weakley, Allison M.; Zhao, Zhilei; Akbari, Omar S.; Black, William C.; Cao, Han; Darby, Alistair C.; Hill, Catherine A.; Johnston, J. Spencer; Murphy, Terence D.; Raikhel, Alexander S.; Sattelle, David B.; Sharakhov, Igor V.; White, Bradley J.; Zhao, Li; Aiden, Erez Lieberman; Mann, Richard S.; Lambrechts, Louis; Powell, Jeffrey R.; Sharakhova, Maria V.; Tu, Zhijian Jake; Robertson, Hugh M.; McBride, Carolyn S.; Hastic, Alex R.; Korlach, Jonas; Neafsey, Daniel E.; Phillippy, Adam M.; Vosshall, Leslie B. (2018-11-22)
Female Aedes aegypti mosquitoes infect more than 400 million people each year with dangerous viral pathogens including dengue, yellow fever, Zika and chikungunya. Progress in understanding the biology of mosquitoes and developing the tools to fight them has been slowed by the lack of a high-quality genome assembly. Here we combine diverse technologies to produce the markedly improved, fully re-annotated AaegL5 genome assembly, and demonstrate how it accelerates mosquito science. We anchored physical and cytogenetic maps, doubled the number of known chemosensory ionotropic receptors that guide mosquitoes to human hosts and egg-laying sites, provided further insight into the size and composition of the sex-determining M locus, and revealed copy-number variation among glutathione S-transferase genes that are important for insecticide resistance. Using high-resolution quantitative trait locus and population genomic analyses, we mapped new candidates for dengue vector competence and insecticide resistance. AaegL5 will catalyse new biological insights and intervention strategies to fight this deadly disease vector.
Insights into the Preservation of the Homomorphic Sex-Determining Chromosome of Aedes aegypti from the Discovery of a Male-Biased Gene Tightly Linked to the M-Locus
Hall, Andrew Brantley; Timoshevskiy, Vladimir A.; Sharakhova, Maria V.; Jiang, Xiaofang; Basu, Sanjay; Anderson, Michelle A. E.; Hu, Wanqi; Sharakhov, Igor V.; Adelman, Zach N.; Tu, Zhijian Jake (Oxford University Press, 2014-01-01)
The preservation of a homomorphic sex-determining chromosome in some organisms without transformation into a heteromorphic sex chromosome is a long-standing enigma in evolutionary biology. A dominant sex-determining locus (or M-locus) in an undifferentiated homomorphic chromosome confers the male phenotype in the yellow fever mosquito Aedes aegypti. Genetic evidence suggests that the M-locus is in a nonrecombining region. However, the molecular nature of the M-locus has not been characterized. Using a recently developed approach based on Illumina sequencing of male and female genomic DNA, we identified a novel gene, myo-sex, that is present almost exclusively in the male genome but can sporadically be found in the female genome due to recombination. For simplicity, we define sequences that are primarily found in the male genome as male-biased. Fluorescence in situ hybridization (FISH) on A. aegypti chromosomes demonstrated that the myo-sex probe localized to region 1q21, the established location of theM-locus.Myo-sex is a duplicated myosin heavy chain gene that is highly expressed in the pupa and adult male.Myo-sex shares 83% nucleotide identity and 97% amino acid identity with its closest autosomal paralog, consistent with ancient duplication followed by strong purifying selection. Compared with males, myo-sex is expressed at very low levels in the females that acquired it, indicating that myo-sexmay be sexually antagonistic. This study establishes a framework to discover male-biased sequences within a homomorphic sex-determining chromosome and offers new insights into the evolutionary forces that have impeded the expansion of the nonrecombining M-locus in A. aegypti.
Integrating transcriptomic and proteomic data for accurate assembly and annotation of genomes
Prasad, T. S. Keshava; Mohanty, Ajeet Kumar; Kumar, Manish; Sreenivasamurthy, Sreelakshmi K.; Dey, Gourav; Nirujogi, Raja Sekhar; Pinto, Sneha M.; Madugundu, Anil K.; Pati, Arun H.; Advani, Jayshree; Manda, Srikanth S.; Gupta, Manoj Kumar; Dwivedi, Sutopa B.; Kelkar, Dhanashree S.; Hall, Brantley; Jiang, Xiaofang; Peery, Ashley; Rajagopalan, Pavithra; Yelamanchi, Soujanya D.; Solanki, Hitendra S.; Raja, Remya; Sathe, Gajanan J.; Chavan, Sandip; Verma, Renu; Patel, Krishna M.; Jain, Ankit P.; Syed, Nazia; Datta, Keshava K.; Khan, Aafaque Ahmed; Dammalli, Manjunath; Jayaram, Savita; Radhakrishnan, Aneesha; Mitchell, Christopher J.; Na, Chan-Hyun; Kumar, Nirbhay; Sinnis, Photini; Sharakhov, Igor V.; Wang, Charles; Gowda, Harsha; Tu, Zhijian Jake; Kumar, Ashwani; Pandey, Akhilesh (2017-01)
Complementing genome sequence with deep transcriptome and proteome data could enable more accurate assembly and annotation of newly sequenced genomes. Here, we provide a proof-of-concept of an integrated approach for analysis of the genome and proteome of Anopheles stephensi, which is one of the most important vectors of the malaria parasite. To achieve broad coverage of genes, we carried out transcriptome sequencing and deep proteome profiling of multiple anatomically distinct sites. Based on transcriptomic data alone, we identified and corrected 535 events of incomplete genome assembly involving 1196 scaffolds and 868 protein-coding gene models. This proteogenomic approach enabled us to add 365 genes that were missed during genome annotation and identify 917 gene correction events through discovery of 151 novel exons, 297 protein extensions, 231 exon extensions,192 novel protein start sites,19 novel translational frames, 28 events of joining of exons, and 76 events of joining of adjacent genes as a single gene. Incorporation of proteomic evidence allowed us to change the designation of more than 87 predicted "noncoding RNAs" to conventional mRNAs coded by protein-coding genes. Importantly, extension of the newly corrected genome assemblies and gene models to 15 other newly assembled Anopheline genomes led to the discovery of a large number of apparent discrepancies in assembly and annotation of these genomes. Our data provide a framework for how future genome sequencing efforts should incorporate transcriptomic and proteomic analysis in combination with simultaneous manual curation to achieve near complete assembly and accurate annotation of genomes.
Molecular action of pyriproxyfen: Role of the Methoprene-tolerant protein in the pyriproxyfen-induced sterilization of adult female mosquitoes
Ahmed, Tahmina Hossain; Saunders, T. Randolph; Mullins, Donald E.; Rahman, Mohammad Zillur; Zhu, Jinsong (2020-08)
Exposure of adult mosquitoes to pyriproxyfen (PPF), an analog of insect juvenile hormone (JH), has shown promise to effectively sterilize female mosquitoes. However, the underlying mechanisms of the PPF-induced decrease in mosquito fecundity are largely unknown. We performed a comprehensive study to dissect the mode of PPF action inAedes aegyptimosquitoes. Exposure to PPF prompted the overgrowth of primary follicles in sugar-fedAe.aegyptifemales but blocked the development of primary follicles at Christopher's Stage III after blood feeding. Secondary follicles were precociously activated in PPF-treated mosquitoes. Moreover, PPF substantially altered the expression of many genes that are essential for mosquito physiology and oocyte development in the fat body and ovary. In particular, many metabolic genes were differentially expressed in response to PPF treatment, thereby affecting the mobilization and utilization of energy reserves. Furthermore, PPF treatment on the previtellogenic female adults considerably modified mosquito responses to JH and 20-hydroxyecdysone (20E), two major hormones that govern mosquito reproduction. Kruppel homolog 1, a JH-inducible transcriptional regulator, showed consistently elevated expression after PPF exposure. Conversely, PPF upregulated the expression of several key players of the 20E regulatory cascades, includingHR3andE75A, in the previtellogenic stage. After blood-feeding, the expression of these 20E response genes was significantly weaker in PPF-treated mosquitoes than the solvent-treated control groups. RNAi-mediated knockdown of the Methoprene-tolerant (Met) protein, the JH receptor, partially rescued the impaired follicular development after PPF exposure and substantially increased the hatching of the eggs produced by PPF-treated female mosquitoes. Thus, the results suggested that PPF relied on Met to exert its sterilizing effects on female mosquitoes. In summary, this study finds that PPF exposure disturbs normal hormonal responses and metabolism inAe.aegypti, shedding light on the molecular targets and the downstream signaling pathways activated by PPF. Author summary Aedes aegyptimosquitoes are responsible for the transmission of dengue, yellow fever, chikungunya, and Zika fever. Insecticides are widely used as the primary tool in the prevention and control of these infectious diseases. In light of the rapid increase of insecticide resistance in mosquito populations, there is an urgent need to find new classes of insecticides with a different mode of action. Here we found that pyriproxyfen, an analog of insect juvenile hormone (JH), had a large impact on the oocyte development, both before and after blood feeding, in female mosquitoes. Pyriproxyfen disturbed normal hormonal responses and caused metabolic shifting in female adults. These actions appear to collectively impair oocyte development and substantially reduce viable progenies of female mosquitoes. Besides, we demonstrated the involvement of the JH receptor Met in pyriproxyfen-induced female sterilization. This study significantly advances our understanding of mosquito reproductive biology and the molecular basis of pyriproxyfen action, which are invaluable for the development of new mosquito control strategies.
Multigene Phylogenetics Reveals Temporal Diversification of Major African Malaria Vectors
Kamali, M.; Marek, Paul E.; Peery, A.; Antonio-Nkondjio, Christophe; Ndo, C.; Tu, Zhijian Jake; Simard, F.; Sharakhov, Igor V. (PLOS, 2014-04-04)
The major vectors of malaria in sub-Saharan Africa belong to subgenus Cellia. Yet, phylogenetic relationships and temporal diversification among African mosquito species have not been unambiguously determined. Knowledge about vector evolutionary history is crucial for correct interpretation of genetic changes identified through comparative genomics analyses. In this study, we estimated a molecular phylogeny using 49 gene sequences for the African malaria vectors An. gambiae, An. funestus, An. nili, the Asian malaria mosquito An. stephensi, and the outgroup species Culex quinquefasciatus and Aedes aegypti. To infer the phylogeny, we identified orthologous sequences uniformly distributed approximately every 5 Mb in the five chromosomal arms. The sequences were aligned and the phylogenetic trees were inferred using maximum likelihood and neighbor-joining methods. Bayesian molecular dating using a relaxed log normal model was used to infer divergence times. Trees from individual genes agreed with each other, placing An. nili as a basal clade that diversified from the studied malaria mosquito species 47.6 million years ago (mya). Other African malaria vectors originated more recently, and independently acquired traits related to vectorial capacity. The lineage leading to An. gambiae diverged 30.4 mya, while the African vector An. funestus and the Asian vector An. stephensi were the most closely related sister taxa that split 20.8 mya. These results were supported by consistently high bootstrap values in concatenated phylogenetic trees generated individually for each chromosomal arm. Genome-wide multigene phylogenetic analysis is a useful approach for discerning historic relationships among malaria vectors, providing a framework for the correct interpretation of genomic changes across species, and comprehending the evolutionary origins of this ubiquitous and deadly insect-borne disease.
A New Chromosomal Phylogeny Supports the Repeated Origin of Vectorial Capacity in Malaria Mosquitoes of the Anopheles gambiae Complex
Kamali, Maryam; Xia, Ai; Tu, Zhijian Jake; Sharakhov, Igor V. (PLOS, 2012-10-01)
Understanding phylogenetic relationships within species complexes of disease vectors is crucial for identifying genomic changes associated with the evolution of epidemiologically important traits. However, the high degree of genetic similarity among sibling species confounds the ability to determine phylogenetic relationships using molecular markers. The goal of this study was to infer the ancestral–descendant relationships among malaria vectors and nonvectors of the Anopheles gambiae species complex by analyzing breakpoints of fixed chromosomal inversions in ingroup and several outgroup species. We identified genes at breakpoints of fixed overlapping chromosomal inversions 2Ro and 2Rp of An. merus using fluorescence in situ hybridization, a whole-genome mate-paired sequencing, and clone sequencing. We also mapped breakpoints of a chromosomal inversion 2La (common to An. merus, An. gambiae, and An. arabiensis) in outgroup species using a bioinformatics approach. We demonstrated that the ‘‘standard’’ 2R+p arrangement and ‘‘inverted’’ 2Ro and 2La arrangements are present in outgroup species Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The data indicate that the ancestral species of the An. gambiae complex had the 2Ro, 2R+p, and 2La chromosomal arrangements. The ‘‘inverted’’ 2Ro arrangement uniquely characterizes a malaria vector An. merus as the basal species in the complex. The rooted chromosomal phylogeny implies that An. merus acquired the 2Rp inversion and that its sister species An. gambiae acquired the 2R+o inversion from the ancestral species. The karyotype of nonvectors An. quadriannulatus A and B was derived from the karyotype of the major malaria vector An. gambiae. We conclude that the ability to effectively transmit human malaria had originated repeatedly in the complex. Our findings also suggest that saltwater tolerance originated first in An. merus and then independently in An. melas. The new chromosomal phylogeny will facilitate identifying the association of evolutionary genomic changes with epidemiologically important phenotypes.
Next-Generation Sequencing Reveals Recent Horizontal Transfer of a DNA Transposon between Divergent Mosquitoes
Diao, Yupu; Qi, Yumin; Ma, Yajun; Xia, Ai; Sharakhov, Igor V.; Chen, Xiaoguang; Biedler, James K.; Ling, Erjun; Tu, Zhijian Jake (PLOS, 2011-02-10)
Horizontal transfer of genetic material between complex organisms often involves transposable elements (TEs). For example, a DNA transposon mariner has been shown to undergo horizontal transfer between different orders of insects and between different phyla of animals. Here we report the discovery and characterization of an ITmD37D transposon, MJ1, in Anopheles sinensis. We show that some MJ1 elements in Aedes aegypti and An. sinensis contain intact open reading frames and share nearly 99% nucleotide identity over the entire transposon, which is unexpectedly high given that these two genera had diverged 145–200 million years ago. Chromosomal hybridization and TE-display showed that MJ1 copy number is low in An. sinensis. Among 24 mosquito species surveyed, MJ1 is only found in Ae. aegypti and the hyrcanus group of anopheline mosquitoes to which An. sinensis belongs. Phylogenetic analysis is consistent with horizontal transfer and provides the basis for inference of its timing and direction. Although report of horizontal transfer of DNA transposons between higher eukaryotes is accumulating, our analysis is one of a small number of cases in which horizontal transfer of nearly identical TEs among highly divergent species has been thoroughly investigated and strongly supported. Horizontal transfer involving mosquitoes is of particular interest because there are ongoing investigations of the possibility of spreading pathogen-resistant genes into mosquito populations to control malaria and other infectious diseases. The initial indication of horizontal transfer of MJ1 came from comparisons between a 0.4x coverage An. sinensis 454 sequence database and available TEs in mosquito genomes. Therefore we have shown that it is feasible to use low coverage sequencing to systematically uncover horizontal transfer events. Expanding such efforts across a wide range of species will generate novel insights into the relative frequency of horizontal transfer of different TEs and provide the evolutionary context of these lateral transfer events.
De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug
Lancaster, Jason; Khrimian, Ashot; Young, Sharon; Lehner, Bryan; Luck, Katrin; Wallingford, Anna K.; Ghosh, Saikat Kumar B.; Zerbe, Philipp; Muchlinski, Andrew; Marek, Paul E.; Sparks, Michael E.; Tokuhisa, James G.; Tittiger, Claus; Köllner, Tobias G.; Weber, Donald C.; Gundersen-Rindal, Dawn E.; Kuhar, Thomas P.; Tholl, Dorothea (2018-09-11)
Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (15,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.
The Physical Genome Mapping of Anopheles albimanus Corrected Scaffold Misassemblies and Identified Interarm Rearrangements in Genus Anopheles
Artemov, Gleb N.; Peery, Ashley N.; Jiang, Xiaofang; Tu, Zhijian Jake; Stegniy, Vladimir N.; Sharakhova, Maria V.; Sharakhov, Igor V. (Genetics Society of America, 2017-01-01)
The genome of the Neotropical malaria vector Anopheles albimanus was sequenced as part of the 16 Anopheles Genomes Project published in 2015. The draft assembly of this species consisted of 204 scaffolds with an N50 scaffold size of 18.1 Mb and a total assembly size of 170.5 Mb. It was among the smallest genomes with the longest scaffolds in the 16 Anopheles species cluster, making An. albimanus the logical choice for anchoring the genome assembly to chromosomes. In this study, we developed a high-resolution cytogenetic photomap with completely straightened polytene chromosomes from the salivary glands of the mosquito larvae. Based on this photomap, we constructed a chromosome-based genome assembly using fluorescent in situ hybridization of PCR-amplified DNA probes. Our physical mapping, assisted by an ortholog-based bioinformatics approach, identified and corrected nine misassemblies in five large genomic scaffolds.Misassemblies mostly occurred in junctions between contigs. Our comparative analysis of scaffolds with the An. gambiae genome detected multiple genetic exchanges between pericentromeric regions of chromosomal arms caused by partial-arm translocations. The finalmap consists of 40 ordered genomic scaffolds and corrected fragments ofmisassembled scaffolds. The An. albimanus physical map comprises 98.2% of the total genome assembly and represents the most complete genome map among mosquito species. This study demonstrates that physical mapping is a powerful tool for correcting errors in draft genome assemblies and for creating chromosome-anchored reference genomes.
Six novel Y chromosome genes in Anopheles mosquitoes discovered by independently sequencing males and females
Hall, Andrew Brantley; Qi, Yumin; Timoshevskiy, Vladimir A.; Sharakhova, Maria V.; Sharakhov, Igor V.; Tu, Zhijian Jake (Biomed Central, 2013-04-23)
Background Y chromosomes are responsible for the initiation of male development, male fertility, and other male-related functions in diverse species. However, Y genes are rarely characterized outside a few model species due to the arduous nature of studying the repeat-rich Y. Results The chromosome quotient (CQ) is a novel approach to systematically discover Y chromosome genes. In the CQ method, genomic DNA from males and females is sequenced independently and aligned to candidate reference sequences. The female to male ratio of the number of alignments to a reference sequence, a parameter called the chromosome quotient (CQ), is used to determine whether the sequence is Y-linked. Using the CQ method, we successfully identified known Y sequences from Homo sapiens and Drosophila melanogaster. The CQ method facilitated the discovery of Y chromosome sequences from the malaria mosquitoes Anopheles stephensi and An. gambiae. Comparisons to transcriptome sequence data with blastn led to the discovery of six Anopheles Y genes, three from each species. All six genes are expressed in the early embryo. Two of the three An. stephensi Y genes were recently acquired from the autosomes or the X. Although An. stephensi and An. gambiae belong to the same subgenus, we found no evidence of Y genes shared between the species. Conclusions The CQ method can reliably identify Y chromosome sequences using the ratio of alignments from male and female sequence data. The CQ method is widely applicable to species with fragmented genome assemblies produced from next-generation sequencing data. Analysis of the six Y genes characterized in this study indicates rapid Y chromosome evolution between An. stephensi and An. gambiae. The Anopheles Y genes discovered by the CQ method provide unique markers for population and phylogenetic analysis, and opportunities for novel mosquito control measures through the manipulation of sexual dimorphism and fertility.

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