VTechWorks Repository :: Browsing by Author "Tu, Zhijian Jake"

Browsing by Author "Tu, Zhijian Jake"

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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.
Anopheles mosquitoes reveal new principles of 3D genome organization in insects
Lukyanchikova, Varvara; Nuriddinov, Miroslav; Belokopytova, Polina; Taskina, Alena; Liang, Jiangtao; Reijnders, Maarten J. M. F.; Ruzzante, Livio; Feron, Romain; Waterhouse, Robert M.; Wu, Yang; Mao, Chunhong; Tu, Zhijian Jake; Sharakhov, Igor V.; Fishman, Veniamin (Nature Portfolio, 2022-04-12)
Chromosomes are hierarchically folded within cell nuclei into territories, domains and subdomains, but the functional importance and evolutionary dynamics of these hierarchies are poorly defined. Here, we comprehensively profile genome organizations of five Anopheles mosquito species and show how different levels of chromatin architecture influence each other. Patterns observed on Hi-C maps are associated with known cytological structures, epigenetic profiles, and gene expression levels. Evolutionary analysis reveals conservation of chromatin architecture within synteny blocks for tens of millions of years and enrichment of synteny breakpoints in regions with increased genomic insulation. However, in-depth analysis shows a confounding effect of gene density on both insulation and distribution of synteny breakpoints, suggesting limited causal relationship between breakpoints and regions with increased genomic insulation. At the level of individual loci, we identify specific, extremely long-ranged looping interactions, conserved for similar to 100 million years. We demonstrate that the mechanisms underlying these looping contacts differ from previously described Polycomb-dependent interactions and clustering of active chromatin.
Application of Chromosome Mapping to Understanding Evolutionary History of Anopheles Species
Kamali, Maryam (Virginia Tech, 2013-06-13)
Malaria is the main cause of approximately one million deaths every year that mostly affect children in south of Sub-Saharan Africa. The Anopheles gambiae complex consists of seven morphologically indistinguishable sibling species. However, their behavior, ecological adaptations, vectorial capacity, and geographical distribution differ. Studying the phylogenetic relationships among the members of the complex is crucial to understanding the genomic changes that underlie evolving traits. These evolutionary changes can be related to the gain or loss of human blood choice or to other epidemiologically important traits. In order to understand the phylogenetic relationships and evolutionary history of the members of the An. gambiae complex, breakpoints of the 2Ro and 2Rp inversions in An. merus and their homologous sequence in the outgroup species were analyzed using fluorescent in situ hybridization (FISH), library screening, whole-genome mate-paired sequencing and bioinformatics analysis. Molecular phylogenies of breakpoint genes were constructed afterwards. In addition, multigene phylogenetic analyses of African malaria vectors were performed. Our findings revised the chromosomal phylogeny, and demonstrated the ancestry of 2Ro, 2R+p and 2La arrangements. Our new chromosomal phylogeny strongly suggests that vectorial capacity evolved repeatedly in members of the An. gambiae complex, and the most important vector of malaria in the world, An. gambiae, is more closely related to ancestral species than was previously thought. Our molecular phylogeny data were in agreement with chromosomal phylogeny, indicating that the position of the genetic markers with respect to chromosomal inversion is important for interpretation of the phylogenetic trees. Multigene phylogenetic analysis revealed that a malaria mosquito from humid savannah and degraded rainforest areas, An. nili, belongs to the basal clade and is more distantly related to other major African malaria vectors than was assumed previously. Finally, for the first time a physical map of 12 microsatellite markers for the Asian malaria vector An. stephensi was developed. Knowledge about the chromosomal position of microsatellites was shown to be important for a proper estimation of population genetic parameters. In conclusion, our study improved understanding of genetics and evolution of some of the major malaria vectors in Africa and Asia.
Assembly of Iron-Sulfur Clusters In Vivo
O'Carroll, Ina Puleri (Virginia Tech, 2009-02-03)
Iron-sulfur [Fe-S] clusters are protein cofactors that facilitate various life-sustaining biological processes. Their in vivo assembly is accomplished by three different systems known to date. These are: the NIF system which provides [Fe-S] clusters for nitrogenase and other nitrogen-fixing proteins, the SUF system which is induced during conditions of oxidative stress and iron starvation in E. coli, and the ISC system which serves as the housekeeping assembly apparatus. The latter is the focus of this dissertation and includes the proteins IscR, IscS, IscU, IscA, HscB, HscA, Fdx, and IscX. IscU is purified in its cluster-less (apo) form, but can serve as a scaffold to assemble [Fe-S] clusters in vitro in the presence of excess iron and sulfide. To test the scaffold hypothesis and gain insight into the events that occur during [Fe-S] cluster assembly and delivery, we developed two methods that allow the isolation of IscU and other ISC proteins in vivo. In the first method, Azotobacter vinelandii IscU is isolated from its native host, whereas in the second, it is isolated recombinantly from E. coli using a vector that allows expression of the entire isc operon. We found that IscU exists in vivo in two forms: apo-IscU and [2Fe-2S]2+ cluster-loaded IscU which are believed to be conformationally distinct. Both transient and stable IscU-IscS complexes were identified, indicating that the two proteins interact in vivo in a manner that involves their association and dissociation. The [2Fe-2S]2+-IscU species was present as a single entity, whereas significant amounts of apo-IscU were found associated with IscS, suggesting that IscU-IscS dissociation is triggered by the completion of [2Fe-2S] clusters. Both apo and [2Fe-2S]2+-IscU were predominantly monomeric whereas IscU-IscS complexes were determined to have an Î±2Î²2 composition. IscU was purified in the absence of the chaperones HscA and HscB and was also shown to accommodate a [2Fe-2S]2+ cluster similar to the one bound to IscU isolated from wild type cells. The findings suggest that [2Fe-2S]2+-IscU exists in one conformation in vivo and that any conformational changes on IscU are exerted after [2Fe-2S] cluster formation. In silico studies showed that a flexible loop containing the conserved LPPVK motif, which is responsible for interactions with HscA, may facilitate cluster exposure to either mediate its delivery to acceptor proteins or participation in the construction of [4Fe-4S] clusters. Experiments with NfuA, a protein similar to the C-terminal domain of NifU, demonstrated that NfuA and similar proteins might serve as [Fe-S] cluster carriers to accomplish the efficient delivery of nascent cofactors to the various recipient proteins.
The "atypical" protein kinase, SsoPK5, an archaeal member of the piD261/Bud32 subfamily
Haile, January Dendi (Virginia Tech, 2009-08-12)
Open reading frame (ORF) sso0433 from the archaeon Sulfolobus solfataricus encodes a protein kinase, SsoPK5 that exhibits 33% sequence identity to p53 related protein kinase (PRPK) from Homo sapiens and 26% sequence identity to piD261/Bud32 from Saccharomyces cerevisiae. Given this high degree of similarity, the objectives of this thesis were to (a) clone and purify recombinant SsoPK5, (b) examine its commonalities and differences with its eukaryotic homologues, and (c) determine if it was regulated by nucleotides or related compounds. Substantial progress was achieved on each objective. After successful cloning of ORF sso0433 and purification of its protein product, SsoPK5, it was determined that SsoPK5 was cold labile and incubation at 4ºC for an extended period of time rendered SsoPK5 incapable of phosphotransferase activity. When stored at room temperature, SsoPK5 was capable of transferring the γ-phosphate from ATP to casein, reduced carboxyamidomethylated and maleylated (RCM) lysozyme,and p53. SsoPK5 phosphotransferase activity required a divalent metal cofactor; like pid261/Bud32, SsoPK5 preferred Mn²⁺ over the more commonly preferred Mg²⁺. SsoPK5 was shown to phosphorylate itself on threonine and serine residues; one of the specific amino acid residues modified is threonine-151. Recombinant SsoPK5 is activated by ADP-ribose and 5′-AMP. Activation was observed when SsoPK5 was stabilized by ATP or a nonhydrolytic analogue, such as β,γ- methylene adenosine 5′-triphosphate (AMP-PCP). Activation was not a result of phosphoryl transfer nor hydrolytic breakdown of ATP or 5′-AMP. This was deduced by the lack of ³²P radioactivity incorporated into SsoPK5 during pre-incubation with [γ-³²P] ATP for 60 min at 65ºC, and activation by adenosine 5′-O-thiomonophosphate (AMPS), a hydrolysis-resistant analog of AMP. These results may indicate that ADP-ribose acts as a pseudochaperone for SsoPK5 thereby facilitating maximal activity.
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.
Biochemical Characterization of Arabidopsis Enzymes Involved in Inositol Pyrophosphate Biosynthesis
Adepoju, Olusegun Adeboye (Virginia Tech, 2019-09-05)
To compensate for the sessile nature of plants, thousands of years of evolution have led to the development of many sophisticated signaling pathways that help plants sense and respond appropriately to different environmental cues. One such signaling pathway is called inositol phosphate signaling. This research dissertation focuses on the inositol phosphate signaling pathway in plants, with emphasis on elucidating how a new class of signaling molecules collectively referred to inositol pyrophosphates are synthesized. Inositol pyrophosphates are an emerging class of "high-energy" intracellular signaling molecules containing one or two diphosphate groups attached to an inositol ring, with suggested roles in bioenergetic homeostasis and inorganic phosphate sensing. Information regarding the biosynthesis of this unique class of signaling molecules in plants is scarce, however the enzymes responsible for their biosynthesis in other eukaryotes have been well described. This work aims to characterize the biochemical activity of the kinase domain (KD) of the Arabidopsis plant diphosphoinositol pentakisphosphate kinase enzymes (AtVIP1 and AtVIP2), and elucidate the biosynthesis pathway of inositol pyrophosphates in plants. Our data indicate that AtVIP1-KD and AtVIP2-KD function primarily as diphosphoinositol pentakisphosphate 5 kinases that phosphorylate this substrate at the 1-position. We also discovered a previously unreported inositol hexakisphosphate kinase activity for the Arabidopsis inositol(1,3,4) triphosphate 5/6kinase enzymes, that can convert InsP6 to InsP7. Together, these enzymes can function in plants to produce inositol pyrophosphates, which have been implicated in signal transduction and phosphate sensing pathways. The significance and potential application of these findings in terms of reduced phytate content and phosphate pollution, improved plant fitness, and improved nutrient use efficiency are discussed. The future outlook of inositol phosphate signaling research is also discussed.
Biochemical studies of enzymes in insect cuticle hardening
Liu, Pingyang (Virginia Tech, 2013-03-28)
In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially contributing to the biosynthesis of taurine and/or beta-alanine in mammalian species. Finally, the metabolic engineering work of L-3, 4-dihydroxyphenylalanine decarboxylase (DDC) and 3, 4-dihydroxylphenylacetaldehyde (DHPAA) synthase has revealed that the reactions of these enzymes could be determined by a few conserved residues at their active site. As both enzymes have been implicated in the biosynthesis of sclerotizing reagents, it is of great scientific and practical importance to understand the similarity and difference in their reaction mechanisms. The results of this dissertation provide valuable biochemical information of ADC, DDC, DHPAA synthase, and GADL1, all of which are PLP-dependent decarboxylases. ADC, DDC, DHPAA synthase are important enzymes in insect cuticle hardening by contributing to the biosynthesis of sclerotizing reagents. Knowledge towards understanding of these enzymes will promote the comprehension of insect cuticle hardening and help scientists to search for ideal insecticide targets. The characterization of GADL1 lays groundwork for future research of its potential role in taurine and beta-alanine metabolism.
Biogeography and biosystematics of plum curculio, Conotrachelus nenuphar (Herbst)/Wolbachia interactions
Zhang, Xing (Virginia Tech, 2006-11-28)
This research focused on the reproductive incompatibility and genetic differences between the two strains of plum curculio, Conotrachelus nenuphar (Herbst). Two molecular markers served as the basis for the strain distribution analysis of plum curculio and Wolbachia symbiont. One marker is the partial mitochondrial cytochrome oxidase gene subunit I (mtCOI) of plum curculio. Another marker is the Wolbachia Surface Protein (wsp) gene of Wolbachia associated with plum curculio. First, the reproductive compatibility of cross-populations mating in plum curculio was studied during the summers of 2004 and 2006. The results confirmed the reproductive incompatibility among plum curculio geographic populations. A unidirectional incompatibility was revealed in an approximate north and south transect of the range of plum curculio (4 x 4 two factorial design: NY, VA, FL, and WV): there was a significant low fertility in WV males mated with NY (40%) and VA (29%) females. The Florida population showed a different pattern: FL males have a significantly lower fertility with VA (46%) and WV (37%) females while FL females were compatible with all males from the four populations. The results of experiment 2 indicated that within the northern geographic area populations (3 x 3 two factorial design: NY, MA, and NJ) were compatible with each other. An opposite unidirectional reproductive incompatibility was revealed in the combination of NJ males with FL females, which showed a significant low fertility (47%). A bi-directional incompatibility occurred between FL and WV reciprocal cross mating. FL males mated with WV females (26%) and WV males mated with FL females (21%) both have the significant low fertility compared to fertility of within their population matings. The genetic diversity among plum curculio populations from different geographic locations was investigated using the partial mtCOI gene. A total of 50 samples from 10 populations were sequenced. PCR products were 863 bp in length. A total of 23 unique sequence haplotypes were found in the 50 samples tested. Haplotype G (n = 5), L (n = 12) and T (n = 13) comprised 60% of 50 samples. The nucleotide distances between those haplotypes ranged from 0.12% to 4.87%. Genetic distances between northern and southern group plum curculios range from 4.17% to 4.87%. Two distinct major clades were found, using three different phylogenetic analyses: 1) neighbor joining (NJ), 2) maximum-parsimony (MP), and 3) maximum-likelihood (ML). 100% bootstraps support the northern clade and the southern clade was strongly supported (100/100/86, NJ/MP/ML) as well. The mid-southern subclade within the southern clade was also strongly supported (70/82/71, NJ/MP/ML) and the far-southern subclade was supported in NJ tree (81%) but was not resovled in MP and ML trees. The mid-southern subclade included haplotypes from two NJ, Washington, VA (Ra), Blacksburg, VA (BL) and 50% of WV populations and the far-southern subclade included haplotypes from FL, GA, Whitethorne, VA (Ke), Troutville, VA (Bo) and another 50% of WV populations. The results suggested that the northern and the southern clade could correspond with the northern and southern strains, respectively, of plum curculio. In this study, the mtCOI sequence was highly informative as a molecular marker in that it was useful to distinguish C. nenuphar from northern and from southern geographic locations in the eastern United States. However, the number of generations per year of several geographic populations within the southern clade still needs to be determined. The distribution of Wolbachia infection associated with plum curculio strains was investigated. 91 of 93 samples were infected by Wolbachia. Three unique Wolbachia strains were identified. The strains wCne1 and wCne2 (593 bp) were 97% identical, and their sequences were both 84% identical with wCne3 (590 bp). The wsp sequence of wCne1 was 99% identical to Wolbachia sequenced from the neotropical beetle, Chelymorpha alternans Boheman (Keller et al. 2004). The wCne2 sequence was 98.5% identical to the flower bug, Orius nagaii Yasunaga (Miura and Tagami, unpublished). The wCne3 sequence was 100% identical to Wolbachia sequenced from the tephritid fruit fly, Dacus destillatoria (Jamnongluk et al. 2000) and the ant, Formica exsecta (Reuter and Keller 2003). PCR - Restriction Fragment Length Polymorphism (RFLP) was used for superinfection detection. Of 93 samples, 15 (16.1%), 21 (22.6%), 19 (20.4%), 36 (38.7%) samples were infected by wCne1, wCne2, wCne1 plus wCne2, and wCne3, respectively. Only two (2.2%) samples had no infection. The wCne3 strain was always present as a single infection. Therefore, current results suggest that Wolbachia strains approximate the distribution of plum curculio strains: the northern strain is infected with wCne1 and wCne2 strains in supergroup B, the southern strain is infected with wCne3 strain in supergroup A and the mid-Atlantic region is the convergence area. Compared with the haplotype distribution of plum curculio mtCOI gene, there was a closer relation of the mid-southern PC clade to the far-southern clade than to the northern clade. However, Wolbachia symbionts in mid-southern PC are more closely related to those in northern PC than to those in far-southern PC. The relationship of Wolabchia infection with reproductive incompatibility between plum curculio populations is also discussed.
A Broad Analysis of Tandemly Arrayed Genes in the Genomes of Human, Mouse, and Rat
Shoja, Valia (Virginia Tech, 2006-11-10)
Tandemly arrayed genes (TAG) play an important functional and physiological role in the genome. Most previous studies have focused on individual TAG families in a few species, yet a broad characterization of TAGs is not available. We identified all the TAGs in the genomes of human, chimp, mouse, and rat and performed a comprehensive analysis of TAG distribution, TAG sizes, TAG gene orientations and intergenic distances, and TAG gene functions. TAGs account for about 14-17% of all the genomic genes and nearly one third of all the duplicated genes in the four genomes, highlighting the predominant role that tandem duplication plays in gene duplication. For all species, TAG distribution is highly heterogeneous along chromosomes and some chromosomes are enriched with TAG forests while others are enriched with TAG deserts. The majority of TAGs are of size two for all genomes, similar to the previous findings in C. elegans, A. thaliana, and O. sativa, suggesting that it is a rather general phenomenon in eukaryotes. The comparison with the genome patterns shows that TAG members have a significantly higher proportion of parallel gene orientation in all species, corroborating Graham's claim that parallel orientation is the preferred form of orientation in TAGs. Moreover, TAG members with parallel orientation tend to be closer to each other than all neighboring genes with parallel orientation in the genome. The analysis of GO function indicate that genes with receptor or binding activities are significantly over-represented by TAGs. Simulation reveals that random gene rearrangements have little effect on the statistics of TAGs for all genomes. It is noteworthy to mention that gene family sizes are significantly correlated with the extent of tandem duplication, suggesting that tandem duplication is a preferred form of duplication, especially in large families. There has not been any systematic study of TAG genes' expression patterns in the genome. Taking advantage of recent large-scale microarray data, we were able to study expression divergence of some of the TAGs of size two in human and mouse for which the expression data is available and examine the effect of sequence divergence, gene orientation, and physical proximity on the divergence of gene expression patterns. Our results show that there is a weak negative correlation between sequence divergence and expression similarity between the two members of a TAG, and also a weak negative correlation between physical proximity of two genes and their expression similarity. No significant relationship was detected between gene orientation and expression similarity. Moreover, we compared the expression breadth of upstream and downstream duplicate copies and found that downstream duplicate does not show significantly narrower expression breadth. We also compared TAG gene pairs with their neighboring non-TAG pairs for both physical proximity and expression similarity. Our results show that TAG gene pairs do not show any distinct differences in the two aspects from their neighboring gene pairs, suggesting that sufficient divergence has occurred to these duplicated genes during evolution and their original similarity conferred by duplication has decayed to a level that is comparable to their surrounding regions.
Brown marmorated stink bug, Halyomorpha halys (Stål), genome: putative underpinnings of polyphagy, insecticide resistance potential and biology of a top worldwide pest
Sparks, Michael E.; Bansal, Raman; Benoit, Joshua B.; Blackburn, Michael B.; Chao, Hsu; Chen, Mengyao; Cheng, Sammy; Childers, Christopher; Dinh, Huyen; Doddapaneni, Harsha V.; Dugan, Shannon; Elpidina, Elena N.; Farrow, David W.; Friedrich, Markus; Gibbs, Richard A.; Hall, Brantley; Han, Yi; Hardy, Richard W.; Holmes, Christopher J.; Hughes, Daniel S. T.; Ioannidis, Panagiotis; Cheatle Jarvela, Alys M.; Johnston, J. Spencer; Jones, Jeffery W.; Kronmiller, Brent A.; Kung, Faith; Lee, Sandra L.; Martynov, Alexander G.; Masterson, Patrick; Maumus, Florian; Munoz-Torres, Monica; Murali, Shwetha C.; Murphy, Terence D.; Muzny, Donna M.; Nelson, David R.; Oppert, Brenda; Panfilio, Kristen A.; Paula, Débora P.; Pick, Leslie; Poelchau, Monica F.; Qu, Jiaxin; Reding, Katie; Rhoades, Joshua H.; Rhodes, Adelaide; Richards, Stephen; Richter, Rose; Robertson, Hugh M.; Rosendale, Andrew J.; Tu, Zhijian Jake; Velamuri, Arun S.; Waterhouse, Robert M.; Weirauch, Matthew T.; Wells, Jackson T.; Werren, John H.; Worley, Kim C.; Zdobnov, Evgeny M.; Gundersen-Rindal, Dawn E. (2020-03-14)
Background Halyomorpha halys (Stål), the brown marmorated stink bug, is a highly invasive insect species due in part to its exceptionally high levels of polyphagy. This species is also a nuisance due to overwintering in human-made structures. It has caused significant agricultural losses in recent years along the Atlantic seaboard of North America and in continental Europe. Genomic resources will assist with determining the molecular basis for this species’ feeding and habitat traits, defining potential targets for pest management strategies. Results Analysis of the 1.15-Gb draft genome assembly has identified a wide variety of genetic elements underpinning the biological characteristics of this formidable pest species, encompassing the roles of sensory functions, digestion, immunity, detoxification and development, all of which likely support H. halys’ capacity for invasiveness. Many of the genes identified herein have potential for biomolecular pesticide applications. Conclusions Availability of the H. halys genome sequence will be useful for the development of environmentally friendly biomolecular pesticides to be applied in concert with more traditional, synthetic chemical-based controls.
Candidate Gene Expression and SNP Analyses of Toxin-Induced Dilated Cardiomyopathy in the Turkey(Meleagris gallopavo)
Lin, Kuan-chin (Virginia Tech, 2006-03-28)
Dilated cardiomyopathy (DCM), a heart disease, affects many vertebrates including humans and poultry. The disease can be either idiopathic (IDCM) or toxin-induced. Idiopathic DCM often occurs without a consensus cause. Though genetic and other studies of IDCM are extensive, the specific etiology of toxin-induced is still unknown. Here, our objective was to compare the level of mRNA expression of two candidate genes including troponin T (cTnT) and phospholamban (PLN) using quantitative reverse transcription polymerase chain reaction (RT-PCR) in toxin-induced DCM affected and unaffected turkeys. Cardiac TnT and PLN were chosen because their spontaneous expression has been reported to be associated with IDCM. We also scanned these genes for single nucleotide polymorphisms (SNPs) that could be useful in evaluating their functions in the incidence and severity of toxin-induced DCM in turkeys. There were no significant differences between affected and unaffected birds in the expression of both cTnT and PLN. A total of 12 SNPs were detected in cTnT and PLN DNA sequences. One of the seven haplotypes detected in cTnT was the most frequent. Linkage analysis showed that cTnT gene was unlinked on the current turkey genetic map. Resources developed here, including SNPs, haplotypes, cDNA sequences, and the PCR-RFLP genotype procedure will be used for future investigations involving cTnT and PLN and toxin-induced DCM.
Characterization of a female germline and early zygote promoter from the transcription factor bZip1 in the dengue mosquito Aedes aegypti
Kojin, Bianca B.; Biedler, James K.; Tu, Zhijian Jake; Adelman, Zach N. (2020-07-17)
Background The wide distribution of Aedes aegypti, the main vector of dengue and yellow fever viruses, currently puts three billion people in the world at risk of infection with these viruses. Continuous transmission of these and other viruses despite aggressive efforts to prevent this emphasizes the need to develop new control strategies. Proposals to control disease transmission based on vector engineering, including both population suppression and population replacement, rely on the development of transgenes under the control of regulatory elements able to drive molecules in a specific tissue, time and strength. Methods Here we report the characterization of a promoter active in both the female germline and early zygote, derived from the transcription factor bZip1 in the mosquito Ae. aegypti, using transposon-based methods and RT-qPCR. Results We generated seven transgenic lines carrying AabZip1-reporter constructs and observed expression in both the ovary and early embryo. RT-qPCR analysis was performed to evaluate transcript expression patterns for each line, confirming that transgenic expression from the AabZip1 promoter largely recapitulated the endogenous expression pattern, albeit the strength of maternal expression appeared to be strongly influenced by chromosomal position. Conclusions This study provides a new regulatory sequence that can be useful for generating transgenic lines that can become a tool in vector control strategies.
Chromosomal evolution in mosquitoes - vectors of diseases
Naumenko, Anastasia Nikolayevna (Virginia Tech, 2017-06-23)
The World Health Organization estimates that vector-borne diseases account for 17% of the global burden of all infectious diseases and has identified the mosquito as the most dangerous of all disease-transmitting insects, being responsible for several million deaths and hundreds of millions of cases each year. The study of mosquito genomics provides a deeper understanding of the molecular mechanisms involved in every aspect of vector biology, such as sex determination, host-parasite interaction, ecology, feeding behavior, immunity and evolutionary trends and can be used for the development of new strategies for vector control. We developed the first map of the mitotic chromosomes of the major vector for West Nile fever and lymphatic filariasis, Culex quinquefasciatus. The map was then successfully utilized for mapping of approximately 90% of available genetic markers to their precise positions on the chromosomes. Idiograms were integrated with 140 genetic supercontigs representing 26.5% of the genome. A linear regression analysis demonstrated good overall correlation between the positioning of markers on physical and genetic linkage maps. This will improve gene annotation and help in distinguishing potential haplotype scaffolds and regions of segmental duplications. It will also facilitate identification of epidemiologically important genes that can be used as targets for the vector control and provide a better framework for comparative genomics that will help understanding of the evolution of epidemiologically important traits. In another study, we confirmed the presence of the newly described species, Anopheles daciae, in regions of Russia using molecular data. Although sympatric with its sibling species, Anopheles messeae, five nucleotide substitutions in the internal transcribed spacer 2 of ribosomal DNA can be used to distinguish the morphologically similar species. Chromosome rearrangements have a significant impact on mosquito adaptation and speciation. Using sequencing data in combination with karyotyping, we demonstrated that significant differences in inversion frequencies distinguish An. messeae from An. daciae, suggesting that these inversions are actively involved in adaptation and speciation. It is essential to have reliable toolbox for correct identification of these species and to know their range for future possible malaria outbreaks prevention.
Chromosomal Evolution of Malaria Vectors
Peery, Ashley Nicole (Virginia Tech, 2016-07-01)
International malaria control initiatives such as the Roll Back Malaria Initiative (RBM) and the Medicines for Malaria Venture (MMV) mobilize resources and spur research aimed at vector control as well as the treatment and eventual eradication of the disease. These efforts have managed to reduce incidence of malaria by an estimated 37% worldwide since 2000. However, despite the promising success of control efforts such as these, the World Health Organization reports a staggering 438,000 deaths from malaria in 2015. The continuing high death toll of malaria as well as emerging insecticide and antimalarial drug resistance suggests that while encouraging, success in reducing malaria incidence may be tenuous. Current vector control strategies are often complicated by ecological and behavioral heterogeneity of vector mosquito populations. As an additional obstruction, mosquito genomes are highly plastic as evidenced by the wealth or chromosomal inversions that have occurred in this genus. Chromosomal inversions have been correlated with differences in adaptation to aridity, insecticide resistance, and differences in resting behavior. However, a good understanding of the molecular mechanisms for inversion generation is still lacking. One possible contributor to inversion formation in Anopheles mosquitoes includes repetitive DNA such as transposable elements (TEs), tandem repeats (TRs) and inverted repeats (IRs). This dissertation provides physical maps for two important malaria vectors, An. stephensi and An. albimanus (Ch.2 and Ch. 3) and then applies those maps to the identification of inversion breakpoints in malaria mosquitoes. Repeat content of each chromosomal arm and the molecular characterization of lineage specific breakpoints is also investigated (Ch. 2 and Ch.4). Our study reveals differences in patterns of chromosomal evolution of Anopheles mosquitoes vs. Drosophila. First, mosquito chromosomes tend to shuffle as intact elements via whole arm translocations and do not under fissions or fusions as seen in fruitflies. Second, the mosquito sex chromosome is changing at a much higher rate relative to the autosomes in malaria mosquitoes than in fruit flies. Third, our molecular characterization of inversion breakpoints indicates that TEs and TRs may participate in inversion genesis in an arm specific manner.
Chromosome and Genome Evolution in Culicinae Mosquitoes
Masri, Reem Abed (Virginia Tech, 2021-07-14)
The Culicinae is the most extensive subfamily among the Culicidae family of mosquitoes. Two genera, Culex and Aedes, from this subfamily have world-wide distribution and are responsible for transmitting of several deadly diseases including Zika, West Nile fevers, chikungunya, dengue, and Rift Valley fevers. Developing high-quality genome assembly for mosquitoes, studying their population structure, and evolution can help to facilitate the development of new strategies for vector control. Studies on Aedes albopitcus as well as on species from the Culex pipiens complex, which are widely spread in the United States, provide excellent models on these topics. Ae. albopictus is one of the most dangerous invasive mosquito species in the world that transmits more than 20 arboviruses. This species has highly repetitive genome that is the largest among mosquito genomes sequenced so far. Thus, sequencing and assembling of such genome is extremally challenging. As a result, the lack of high-quality Ae. albopictus genome assembly has delayed the progress in understanding its biology. To produce a high-quality genome assembly, it was important to anchor genomic scaffolds to the cytogenetic map creating a physical map of the genome assembly. We first developed a new gene-based approach for the physical mapping of repeat-rich mosquito genomes. The approach utilized PCR amplification of the DNA probes based on complementary DNA (cDNA) that does not include repetitive DNA sequences. This method was then used for the development of a physical map for Ae. albopictus based on the in situ hybridization of fifty cDNA fragments or gene exons from twenty-four scaffolds to the mitotic chromosomes from imaginal discs. This study resulted in the construction of a first physical map of the Ae. albopictus genome as well as mapping viral integration and polyphenol oxidase genes. Moreover, comparing our present Ae. albopictus physical map to the current Ae. aegypti assembly indicated the presence of multiple chromosomal inversions between them. To better understand population structure and chromosome evolution in Culicinae mosquitoes, especially in the Culex pipiens complex, we studied genomic and chromosomal differentiation between two subspecies Cx. pipiens pipiens and Cx. pipiens molestus. For the species responsible for the spread of human diseases, understanding the population dynamics and processes of taxa diversification is important for an effective mosquito control . Two vectors of West Nile virus, Cx. p. pipiens and Cx. p. molestus, exhibit epidemiologically important behavioral and physiological differences, but the whole-genome divergence between them was unexplored. The first goal of this study was to better understand the level of genomic differentiation and population structures of Cx. p. pipiens and Cx. p. molestus from different continents. We sequenced and compared whole genomes of 40 individual mosquitoes from two locations in Eurasia and two in North America. Principal Component, ADMIXTURE, and neighbor joining analyses of the nuclear genomes identified two major intercontinental, monophyletic clusters of Cx. p. pipiens and Cx. p. molestus. The level of genomic differentiation between the subspecies was uniform along chromosomes. The ADMIXTURE analysis determined signatures of admixture in Cx. p. pipens populations, but not in Cx. p. molestus populations. Thus, our study identified that Cx. p. molestus and Cx. p. pipiens represent different evolutionary units with monophyletic origin that have undergone incipient ecological speciation. The second goal was to study differences at the chromosome level between these two organisms. We first measured whole chromosome and chromosome arm length differences between Cx. p. molestus and Cx. p. pipiens as a basic cytogenetic approach. In addition, we used the novel Hi-C approach to detect chromosomal rearrangements between them since Hi-C was successful in detecting a known inversion in Cx. quinquefasciatus. Cx. p. molestus and Cx. p. pipiens embryos were used to perform the Hi-C technique. Analysis of the Hi-C data showed the presence of two different inversions in Cx. p. pipiens and Cx. p. molestus heatmap, which could explain their different physiology and adaptation in nature. Developing modern genomic and cytogenetic tools is important to enhance the quality of genome assemblies, improve gene annotation, and provide a better framework for comparative and population genomics of mosquitoes; also it is the foundation for the development of novel genome-based approaches for vector control.
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.
Cloning, characterization, and expression of microRNAs from the Asian malaria mosquito, Anopheles stephensi
Mead, Edward A.; Tu, Zhijian Jake (2008-05-23)
Background microRNAs (miRNAs) are non-coding RNAs that are now recognized as a major class of gene-regulating molecules widely distributed in metozoans and plants. miRNAs have been found to play important roles in apoptosis, cancer, development, differentiation, inflammation, longevity, and viral infection. There are a few reports describing miRNAs in the African malaria mosquito, Anopheles gambiae, on the basis of similarity to known miRNAs from other species. An. stephensi is the most important malaria vector in Asia and it is becoming a model Anopheline species for physiological and genetics studies. Results We report the cloning and characterization of 27 distinct miRNAs from 17-day old An. stephensi female mosquitoes. Seventeen of the 27 miRNAs matched previously predicted An. gambiae miRNAs, offering the first experimental verification of miRNAs from mosquito species. Ten of the 27 are miRNAs previously unknown to mosquitoes, four of which did not match any known miRNAs in any organism. Twenty-five of the 27 Anopheles miRNAs had conserved sequences in the genome of a divergent relative, the yellow fever mosquito Aedes aegypti. Two clusters of miRNAs were found within introns of orthologous genes in An. gambiae, Ae. aegypti, and Drosophila melanogaster. Mature miRNAs were detected in An. stephensi for all of the nine selected miRNAs, including the four novel miRNAs (miR-x1- miR-x4), either by northern blot or by Ribonuclease Protection Assay. Expression profile analysis of eight of these miRNAs revealed distinct expression patterns from early embryo to adult stages in An. stephensi. In both An. stephensi and Ae. aegypti, the expression of miR-x2 was restricted to adult females and predominantly in the ovaries. A significant reduction of miR-x2 level was observed 72 hrs after a blood meal. Thus miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. A mosquito homolog of miR-14, a regulator of longevity and apoptosis in D. melanogaster, represented 25% of all sequenced miRNA clones from 17-day old An. stephensi female mosquitoes. An. stephensi miR-14 displayed a relatively strong signal from late embryonic to adult stages. miR-14 expression is consistent during the adult lifespan regardless of age, sex, and blood feeding status. Thus miR-14 is likely important across all mosquito life stages. Conclusion This study provides experimental evidence for 23 conserved and four new microRNAs in An. stephensi mosquitoes. Comparisons between miRNA gene clusters in Anopheles and Aedes mosquitoes, and in D. melanogaster suggest the loss or significant change of two miRNA genes in Ae. aegypti. Expression profile analysis of eight miRNAs, including the four new miRNAs, revealed distinct patterns from early embryo to adult stages in An. stephensi. Further analysis showed that miR-x2 is likely involved in female reproduction and its function may be conserved among divergent mosquitoes. Consistent expression of miR-14 suggests that it is likely important across all mosquito life stages from embryos to aged adults. Understanding the functions of mosquito miRNAs will undoubtedly contribute to a better understanding of mosquito biology including longevity, reproduction, and mosquito-pathogen interactions, which are important to disease transmission.
Comparative analysis of Anopheles gambiae L-tyrosine decarboxylase and L-DOPA decarboxylase
Aljabri, Hareb Mohammed (Virginia Tech, 2010-08-16)
A major pathway of tyramine and dopamine synthesis in insects is through the decarboxylation of tyrosine and DOPA, respectively. Although tyrosine decarboxylase (TDC) has been mentioned in some reports, it has never been critically analyzed. The high sequence identity shared by tyrosine decarboxylase and DOPA decarboxylase in insects, and the similar structures of the substrates, tyrosine and DOPA, raise the possibility that both tyrosine decarboxylase and DOPA decarboxylase (DDC) have activities to tyrosine and DOPA. In this study, after tyrosine decarboxylase and DOPA decarboxylase enzymes of Anopheles gambiae were expressed, their substrate specificities and biochemical properties were critically analyzed. My results provide clear biochemical evidence establishing that the mosquito tyrosine decarboxylase functions primarily on the production of tyramine with low activity to DOPA. In contrast, mosquito DOPA decarboxylase is highly specific to DOPA with essentially no activity to tyrosine.
Comparative Analysis of Heterochromatin in the Anopheles gambiae Complex
Sharma, Atashi (Virginia Tech, 2016-05-10)
Mosquito borne diseases continue to be a big threat to human health worldwide. Despite using various vector control methods, we lose a great number of lives to this malicious disease in tropical and subtropical countries each year. Not surprisingly, mosquito is considered as the deadliest animal on the earth, because mortality rates from mosquito-vectored infections only lag behind other major diseases such as HIV and tuberculosis. Current approaches of vector control are mostly limited to using insecticidal bed nets, thus novel techniques are required to prevent a staggering loss to human health and quality of life. Advances in the genome sequencing in the past decade have helped to uncover numerous secrets of diverse genomes. The genome of malaria mosquito Anopheles gambiae was first sequenced in 2002 and since then has been updated to include additional scaffolds, their orientations and correction of mis-assemblies. Yet, the greatest challenge remains in assembling the heterochromatin regions, that are repeat rich part and contain relatively low-gene density. Although previously neglected by scientific studies due to its characteristic paucity of genes, heterochromatin is now recognized to be crucial for several processes such as cell viability, chromosome pairing, meiosis, longevity etc. It is therefore not surprising that heterochromatin comprises of a significant portion of the genome in many species. The efforts to analyze the genome of malaria mosquito in order to identify potential new leads for vector control warrant a better understanding of the heterochromatin. Mosquitoes diploid chromosome number equal 6. While autosomes 2 and 3 are submetacentric and present in both sexes, females are homogametic with XX and males are heterogametic with XY sex chromosomes. To achieve a better understanding of the Anopheles heterochromatin, we investigated heterochromatic region of the X chromosome. Despite one arm of the X chromosome being completely heterochromatic, few studies have investigated the molecular content of this region. Protocols were developed for performing fluorescent in situ hybridization (FISH) on mitotic X chromosomes in An. gambiae. Using cytogenetics and molecular biology techniques, we characterized the X chromosome heterochromatin in members of the An. gambiae complex. Specific satellite DNA and 18S ribosomal DNA probes (major components of heterochromatin) were mapped to X chromosomes enabling their differentiation and characterization in the An. gambiae complex. Microarray studies have highlighted the importance of X chromosome during investigation of nascent species An. gambiae and An. coluzzii. Here for the first time qualitative differences in heterochromatin in between nascent species are described. Cytogenetic idiograms are developed as to include the molecular and qualitative differences between the species of the An. gambiae complex. These idiograms are expected to provide a better resolution of the X chromosome heterochromatin for comparison in major malaria vectors, closing some of the gaps present due to poor sequencing of unassembled repeat rich regions in An. gambiae complex. The current understanding of Y chromosome for transgenic manipulation is poor and limited to very few genes. Due to its near total heterochromatic composition, it is the hardest part of the genome to assemble. In collaboration with other researchers, the Y chromosome content was characterized among sibling species of the An. gambiae complex. Our data revealed the swift changes the Y chromosome has undergone in a relatively short evolutionary time period. These include a rapid rate of turnover not only in heterochromatin but also in euchromatin. In addition to previously described repeats, a novel highly repetitive element called Zanzibar was discovered and mapped to the males of various Anopheles sibling species. Our data can form the basis for evolutionary studies in heterochromatin for male mosquitoes within the An. gambiae complex while also help identify novel targets to create successful transgenic male populations. Along with the X chromosome heterochromatin, to our knowledge this is the most extensive contribution to improve the understanding of mitotic chromosome heterochromatin in malaria mosquitoes. This study also investigated if epigenetics play role in mosquito development, fecundity and heterochromatin formation. DNA methylation, histone modifications and small noncoding RNAs are among the epigenetic mechanisms scrutinized in mammals. However, knowledge about epigenetic mechanisms and their effects is sparse in mosquitoes. A protocol for testing the various effects of epigenetics on different stages of malaria mosquito was developed. An epigenetic drug was utilized to probe the effects on immature and adult malaria mosquitoes. Different concentrations of DZNep, a histone methyltransferase inhibitor, were administered to An. coluzzii larvae. Total survivorship and pupation were compared for treated and untreated groups. The drug was also administered to adult blood feeding females to determine any effects on fecundity and egg morphology, revealing a negative association with an increase in drug concentration. A dose dependent decrease in SAH hydrolase concentration in An. coluzzii was also noticed. These results suggest epigenetics plays a critical role in mosquito pupation and ovarian development. Our work lays the groundwork for future investigations into the field of epigenetics in mosquitoes by revealing its effect on several important developmental stages in malaria mosquitoes. Although genomics and next-gen sequencing technology have come a long way in the last decade since the first Anopheles genome was sequenced, considerable gaps still exist in case characterization of heterochromatin function in an organism. Through our work, we have endeavored to elucidate a few of the major roles that heterochromatin may play in organization, evolution and adaptation of the malaria mosquitoes.

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