Browsing by Author "Luckhart, Shirley"

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    Biochemical Separation of Geographical Strains of Plum Curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), and Evaluation of Olfactory Attractants in Virginia Orchards
    McClanan, Michelle Erin Garlic (Virginia Tech, 2002-04-18)
    Plum curculio, Conotrachelus nenuphar (Herbst), is an endemic pest of stone and pome crops of the eastern United States. Two morphologically identical strains of plum curculio have been described and documented in Virgina: a univoltine strain and a multivoltine strain. Because of the cryptic coloring and behaviours of the plum curculio adults, monitoring in orchards is difficult and often ineffective. RAPD-PCR assay was an effective method for separation of the geographical strains. Of the tested primers four, OPE 01, OPE 03, OPE 04, and OPE 07, gave 21 amplimers that are useful for distinguishing individuals from the univoltine and multivoltine populations. Gene targeted PCR revealed the presence of Wolbachia in both populations. Analysis of the wsp gene sequence showed the univoltine population of plum curculio is associated with a strain of Wolbachia in supergroup B, most closely related to a strain identified from Perithemis tenera (Say) (Odonata). The multivoltine populations of plum curculio are associated with strains of Wolbachia which are in supergroup A, and most closely related to Wolbachia strains associated with Dacus destillatoria, Bactrocera sp., and Callosobruchus chinensis Linn. Three different trap designs baited with grandisoic acid, plant volatiles, and a combination of pheromone and plant volatiles were tested. In 1999, significantly more plum curculios were captured with Tedders traps baited with grandisoic acid and unbaited control in traps baited with limonene, plum essence or ethyl isovalerate. In 2000, Circle traps baited with plum essence, sour cherry essence and grandisoic acid yielded no results. In 2001, branch mimic traps yielded no significant differences among three release rates of a blend of benzaldehyde, ethyl isovalerate, trans-2-hexenal, and limonene; although, there was a significant interaction between the pheromone and the host plant volatiles. Overall however, all three of the traps were not effective.
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    Bionomics of Ochlerotatus triseriatus Say (Diptera: Culicidae) and Aedes albopictus Skuse (Diptera: Culicidae) in emerging La Crosse virus foci in Virginia
    Barker, Christopher M. (Virginia Tech, 2001-07-10)
    Recently, the number of human cases of La Crosse encephalitis (LACE), an illness caused by mosquito-borne La Crosse (LAC) virus, has increased in southwestern Virginia, resulting in a need for better understanding of the virus cycle and the biology of its vectors in the region. This project examined the spatial and temporal distributions of the primary vector of LAC virus, Ochlerotatus triseriatus, and a potential secondary vector, Aedes albopictus. Ovitrapping surveys were conducted in 1998 and 1999 to determine distributions and oviposition habitat preferences of the two species in southwestern Virginia. For virus assay, adult mosquitoes were collected at a tire dump and a human case site during 1998 and 1999, and ovitrap samples were taken from a human case site in 2000. In a separate study, a landcover map of Wise County was created by supervised classification of Landsat Enhanced Thematic Mapper imagery, and maps indicating posterior probabilities of high mosquito abundance were created by combining ovitrap survey-derived, landcover-based prior and conditional probabilities for high and low mosquito abundance using remote sensing techniques and Bayesian decision-making rules. Both Oc. triseriatus and Ae. albopictus were collected from all ovitrap sites surveyed in Wise, Scott, and Lee Counties during 1998. Numbers of Oc. triseriatus remained high from late June through late August, while Ae. albopictus numbers increased gradually through June and July, reaching a peak in late August and declining thereafter. Overall, Oc. triseriatus accounted for 90.1% of eggs collected during this period, and Ae. albopictus made up the remaining 9.9%. Abundance of the two species differed among the sites, and in Wise County, relative Ae. albopictus abundance was highest in sites with traps placed in open residential areas. Lowest numbers of both species were found in densely forested areas. Ovitrapping at a human LACE case site during 1998 and 1999 revealed that Aedes albopictus was well-established and overwintering in the area. An oviposition comparison between yard and adjacent forest at the Duncan Gap human LACE case site in 1999 showed that Ae. albopictus preferentially oviposited in the yard surrounding the home over adjacent forested areas, but Oc. triseriatus showed no preference. LAC virus was isolated from 1 larval and 1 adult collection of Oc. triseriatus females from the Duncan Gap human case site, indicating the occurrence of transovarial transmission at this site. The supervised landcover classification for Wise County yielded a landcover map with an overall accuracy of 98% based on comparison of output classification with user-defined ground truth data. Posterior probability maps for Oc. triseriatus and Ae. albopictus abundance reflected seasonal and spatial fluctuations in mosquito abundance with an accuracy of 55-79% for Oc. triseriatus (Kappa=0.00-0.53) and 70-94% for Ae. albopictus (Kappa=0.00-0.49) when model output was compared with results of an ovitrapping survey. Other accuracy measures were also considered, and suggestions were offered for improvement of the model.
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    The Biosynthesis and Function of Nitrogenase Metalloclusters
    Dos Santos, Patricia C. (Virginia Tech, 2004-11-29)
    Nitrogenase catalyzes the biological reduction of N2 to ammonia (nitrogen fixation). The metalloclusters associated with the nitrogenase components include the [4Fe-4S] cluster of the Fe protein, and the P-cluster [8Fe7S] and FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], both contained within the MoFe protein. These metal-complexes play a vital role in enzyme activity during electron transport and substrate reduction. It is known that the FeMo-cofactor provides the site of substrate reduction, but the exact site of substrate binding remains a topic of intense debate. Some models for the substrate binding location favor the molybdenum atom, while other models favor one or more iron atoms within FeMo-cofactor. We have shown that the a-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site: a-70 approaches one 4Fe-4S face of the FeMo-cofactor. Substitutions at this position alter enzyme specificity for reduction of alternative alkyne substrates. These altered MoFe proteins and alternative alkyne substrates, such as propargyl alcohol, were used to trap an intermediate during substrate reduction. Further studies involving the effect of pH on substrate reduction of these altered MoFe proteins pinpointed the location of the bound substrate-derived intermediate on the FeMo-cofactor to a specific Fe atom, designated Fe6. In addition to understanding how substrates are bound and reduced at the active site, understanding how these clusters are biologically assembled is a second point of interest. Inactivation of NifU or NifS has been shown to affect the activity of both nitrogenase components. NifS is a cysteine desulfurase that provides the sulfur for cluster formation and NifU serves as a molecular scaffold during [Fe-S] cluster assembly. Genetic and biochemical experiments involving amino acid substitutions within the N-terminal and C-terminal domains of NifU indicate that both domains can separately participate in nitrogenase-specific [Fe-S] cluster formation. Furthermore, the NifU and NifS protein appear to have specialized functions in the maturation of metalloclusters of nitrogenase and cannot functionally replace the isc [Fe-S] cluster system used for the maturation of other [Fe-S] proteins. These results indicate that, in certain cases, [Fe-S] cluster biosynthetic machineries have evolved to perform only specialized functions.
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    Combined Roles of Glandular-haired Alfalfa and Natural Enemies in Alfalfa Pest Managment in Virginia
    Dellinger, Theresa Ann (Virginia Tech, 2003-09-19)
    Both alfalfa weevil, Hypera postica (Gyllenhal), (Coleoptera: Curculionidae), and potato leafhopper, Empoasca fabae (Harris), (Homoptera: Cicadellidae), remain key pests of alfalfa in Virginia. Commercial varieties of potato leafhopper-resistant (or glandular-haired) alfalfa were released in the mid-1990s, but the impact of alfalfa weevil on these varieties has not been well documented. In 1999, two large-scale field experiments were initiated to compare the performance of a glandular-haired alfalfa variety against a standard, non-glandular-haired variety under both alfalfa weevil and potato leafhopper pest pressures in the southwestern and Piedmont regions of Virginia over a 3 year period. Results indicated that alfalfa weevil must be managed in potato leafhopper-resistant alfalfa to limit crop loss. Surprisingly, similar densities of potato leafhoppers were found in both the glandular-haired and standard varieties. Both varieties frequently had similar yields and forage quality. In general, the glandular-haired variety did not outperform the standard variety. Results also indicated that insecticide application did not always provide the expected benefits of higher yields and forage quality, despite reducing pest densities for 2-3 weeks after application. These data suggest that the economic thresholds for one or both of these pests in Virginia may require adjustment. The potential impact of glandular-haired alfalfa on the natural enemies of alfalfa weevil was examined as well. Bathyplectes anurus (Thompson) (Hymenoptera: Ichneumonidae) was the dominant parasitoid attacking weevil larvae at both locations. Parasitization of weevil larvae by Bathyplectes spp. did not appear to be adversely affected by the presence of glandular trichomes on the potato leafhopper-resistant variety. Glandular trichomes had little impact on the infection of weevil larvae by the fungus Zoophthora phytonomi as well, but this was not unexpected. The genetic variation of B. anurus was surveyed at both study sites using RAPD-PCR to establish or eliminate the possibility that differences in parasitization levels between the Piedmont and southwestern regions could be attributed to the presence of different parasitoid strains. Most of the detected phenotypic variation was attributed to within population variation, with very little variation occurring between the two populations. However, the between population variation was statistically significant in 2000, but not in 2001.
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    Genome analysis of a major urban malaria vector mosquito, Anopheles stephensi
    Jiang, X.; Peery, A.; Hall, B.; Sharma, A.; Chen, X.-G.; Waterhouse, R. M.; Komissarov, A.; Riehle, M. M.; Shouche, Y.; Sharakhova, Maria V.; Lawson, D.; Pakpour, Nazzy; Arensburger, Peter; Davidson, V. L. M.; Eiglmeier, K.; Emrich, S.; George, P.; Kennedy, R. C.; Mane, S. P.; Maslen, G.; Oringanje, C.; Qi, Y.; Settlage, Robert E.; Tojo, M.; Tubio, J. M. C.; Unger, Maria F.; Wang, B.; Vernick, K. D.; Ribeiro, J. C.; James, A. A.; Michel, K.; Riehle, M. A.; Luckhart, Shirley; Sharakhov, Igor V.; Tu, Zhijian Jake (Biomed Central, 2014-01-01)
    Background: Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range. Results: Here, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism. Conclusions: The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.
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    Genomic Analysis of Human and Mouse Guanine-7-Methyltransferase with Active Site Characterization
    Bautz, David James (Virginia Tech, 2001-05-10)
    The 5' end of eukaryotic and viral mRNAs contain a "cap" structure with the sequence m7G(5')pppN(5'). The methylation of the 7-position on the guanine cap is very important to proper mRNA processing and initiation of translation. The enzyme responsible for this methylation, RNA guanine-7-methyltransferase, has been cloned and studied from a number of different species, including human, X. laevis, yeast, and C. elegans. The sequences for mouse guanine-7-methyltransferase cDNA and protein have been deduced based upon identity of mouse ESTs to the cDNA of the human enzyme. The deduced mouse cDNA encodes an ORF of 465 amino acids and is 76.4% identical to the human enzyme, or 86.5% within the C-terminal domain. Active site characterization of mouse and human guanine-7-methyltransferase indicates a cysteine residue is important to proper enzyme activity. Enzyme activity was completely eliminated when N-ethylmaleimide (NEM) was added to the assay mixture. When the product of the reaction, S-adenosyl-L-homocysteine (SAH), was added at a concentration of 40uM the mouse enzyme retained 60% activity while enzyme isolated from Human Osteosarcoma (HOS) cells retained 100% of the original activity. SAH demonstrated no protective effects on the cloned human enzyme. Factors that affect binding of RNA to the active site were also investigated. UV-cross-linking of RNA to the active site of the mouse enzyme was inhibited 35% by NEM. Cap analog, GpppG, at a concentration of 1mM, inhibited cross-linking, but the similar nucleotide GMP, at a concentration of 1mM, did not inhibit cross-linking. These analyses have given a clearer understanding of this very important enzyme.
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    Plasmodium-Induced Nitrosative Stress in Anopheles stephensi: The Cost of Host Defense
    Peterson, Tina Marie Loane (Virginia Tech, 2005-05-23)
    Both vertebrates and anopheline mosquitoes inhibit Plasmodium spp. (malaria parasite) development via induction of nitric oxide (·NO) synthase. Expression of Anopheles stephensi ·NO synthase (AsNOS) is induced in the midgut epithelium beginning at 6 h following a Plasmodium berghei-infected blood meal. ·NO reacts readily with other biocompounds forming a variety of reactive nitrogen intermediates (RNIs) that may impose a nitrosative stress. These RNIs are proposed to be responsible for the AsNOS-dependent inhibition of Plasmodium development. In my studies, I identified several RNIs that are induced in the blood-filled midgut in response to Plasmodium infection. Stable end products of ·NO (NO₃⁻ and NO₂⁻), measured using a modified Griess assay, are elevated in infected midguts at 24 h post-blood meal (pBM). Further studies using chemical reduction-chemiluminescence with Hg displacement showed that infected midguts contained elevated levels of potentially toxic higher oxides of nitrogen (NOx), but S-nitrosothiol (SNO) and nitrite levels did not differ between infected and uninfected midguts at 12.5 and 24 h pBM. Thus, nitrates contributed to elevated NOx levels. SNO-biotin switch westerns indicated that S-nitrosated midgut proteins change over the course of blood meal digestion, but not in response to infection. Photolysis-chemiluminescence was used to release and detect bound ·NO from compounds in blood-filled midguts dissected from 0-33 h pBM. Results showed increased ·NO levels in Plasmodium-infected midgut lysates beginning at 8 h, with significant increases at 12.5-13.5 h and 24-25.5 h pBM and peak levels at 20-24 h. Photolyzed ·NO is derived from SNOs and metal nitrosyls. Since SNO concentrations did not change in response to infection, I proposed that metal nitrosyls, specifically Fe nitrosyl hemoglobin (nitrosylHb) based on the concentration of hemoglobin, were elevated in the infected midgut. At 12-24 h pBM, levels of midgut RNIs in infected mosquitoes were typical of levels measured during mammalian septic inflammation. The inverse relationship between AsNOS activity and parasite abundance indicates that nitrosative stress has a detrimental effect on parasite development. However, nitrosative stress may impact mosquito tissues as well in a manner analogous to mammalian tissue damage during inflammation. Elevated levels of nitrotyrosine (NTYR), a marker for nitrosative stress in many mammalian disease states, were detected in tissues of parasite-infected A. stephensi at 24 h pBM. Greater nitration of tyrosine residues was detected in the blood bolus, midgut epithelium, eggs and fat body. In the midgut, Hb remained in an oxygenated state for the duration of blood digestion. The reaction between ·NO and oxyhemoglobin (oxyHb) can result in the formation of nitrate and methemoglobin (metHb). Although nitrate levels were elevated in response to parasite infection, there was little to no metHb present in the mosquito midgut. The simultaneous presence of nitrates, nitrosylHb, oxyHb, and NTYR, together with a lack of elevated nitrites and metHb, suggested that alternative reaction mechanisms involving â ¢NO had occurred in the reducing environment of the midgut. In addition, I proposed that nitroxyl and peroxynitrite participated in reactions that yielded observed midgut RNIs. To cope with the parasite-induced nitrosative stress, cellular defenses in the mosquito may be induced to minimize self damage. I proposed that peroxiredoxins (Prx), enzymes that can detoxify peroxides and peroxynitrite, may protect A. stephensi from nitrosative stress. Six Prx genes were identified in the A. gambiae genome based on homology with known D. melanogaster Prxs. I identified one A. stephensi Prx, AsPrx, that shared 78% amino acid identity with a D. melanogaster 2-Cys Prx known to protect fly cells against various oxidative stresses. AsPrx was expressed in the midgut epithelium and is encoded by a single-copy, intronless gene. Quantitative RT-PCR analyses confirmed that induction of AsPrx expression in the midgut was correlated with malaria parasite infection and nitrosative stress. To determine whether AsPrx could protect against RNI- and ROS-mediated cell death, transient transfection protocols were established for AsPrx overexpression in D. melanogaster (S2) and A. stephensi (MSQ43) cells and for AsPrx gene silencing using RNA interference in MSQ43 cells. Viability assays in MSQ43 cells showed that AsPrx conferred protection against hydrogen peroxide, ·NO, nitroxyl and peroxynitrite. These data suggested that the ·NO-mediated defense response is toxic to both host and parasite. However, AsPrx may shift the balance in favor of the mosquito.
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    Immunological Crosstalk between Human Transforming Growth Factor-β1 and the Malaria Vector Anopheles stephensi
    Lieber, Matthew Joshua (Virginia Tech, 2005-05-23)
    The emergence of pesticide-resistant mosquitoes and drug-resistant parasites in the last twenty years has made control of malaria more difficult. One novel strategy to better control malaria is the development and release of transgenic mosquitoes whose enhanced immunity prevents transmission of the parasite to the mammalian host. One candidate effector gene is Anopheles stephensi nitric oxide synthase (AsNOS), whose inducible expression and subsequent synthesis of nitric oxide (NO) limits Plasmodium development in A. stephensi. In mammals, one of the most potent physiological regulators of NOS gene expression and catalytic activity is transforming growth factor-β (TGF-β). Moreover, human TGF-β can activate Drosophila melanogaster Smads, the proteins responsible for TGF-β signal transduction. We have determined that following a bloodmeal, active human TGF-β1 (hTGF-β1) persists in the midgut of A. stephensi for up to 48 hours. My data demonstrate that the midgut epithelium recognizes hTGF-β1 as an immunomodulatory cytokine. Specifically, induction of AsNOS by hTGF-β1 occurs in the midgut within minutes of bloodfeeding. Moreover, hTGF-β1 limits development of the human malaria parasite Plasmodium falciparum in the midgut. In other experiments, provision of the AsNOS catalytic inhibitor L-NAME partially reverses the effect of hTGF-β1 on Plasmodium development. These results suggest that AsNOS is a target of hTGF-β1 signaling and additional effectors that impact parasite development may be regulated by hTGF-β1 as well. The fact that hTGF-β1 signals mosquito cells to limit malaria parasite development suggests that there is an endogenous TGF-β signaling network in place. An analysis of the A. gambiae genome database revealed the presence of six TGF-β ligands, including gene duplication in the 60A gene, the first evidence of ligand gene duplication outside of chordates. In addition to five receptors, three Smads were identified in the A. gambiae genome predicted to support TGF-β/Activin- and BMP-like signaling. Midgut epithelial cells and an immunocompetent A. stephensi cell line express all three Smads, confirming that a signaling pathway is in place to support signaling by divergent exogenous and endogenous TGF-β superfamily proteins. The results presented here provide the first evidence of immunological crosstalk between divergent free living hosts of a single parasite. Further, these results imply that the interface between mammals and the mosquitoes that feed on them provide a unique opportunity for circulating molecules in the blood, including TGF-β and other cytokines, to alter the mosquito immune response.
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    Induction of Anopheles stephensi nitric oxide synthase by Plasmodium-derived factor(s)
    Lim, Junghwa (Virginia Tech, 2004-11-03)
    Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h post-infection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that limit parasite development. However, the Plasmodium-derived factors that can induce AsNOS expression and the signaling pathways responsible for transduction in A. stephensi have not been identified until completion of the work described herein. In my studies, I have determined that P. falciparum glycosylphosphatidylinositol (PfGPIs) can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Based on related work in mammals, I hypothesized that parasite-derived AsNOS-inducing factors signal through the insulin signaling pathway and the NF-kappaB-dependent Toll and Immune deficiency (Imd) signaling pathways. In support of this hypothesis, I have determined that signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi protein kinase B (Akt/PKB) and DSOR1 (mitogen activated protein kinase kinase, MEK)/Extracellular signal-regulated protein kinase (ERK), kinases which are associated with the insulin signaling pathway. However, signaling by P. falciparum and PfGPIs is distinctively different from signaling by insulin and these parasite signals are not insulin-mimetic to A. stephensi cells. In other studies, treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced AsNOS expression by P. falciparum merozoites in A. stephensi cells. This result suggested the involvement of Toll and Imd pathways in parasite signaling of mosquito cells. Knockout of Pelle, a proximal signaling protein in the Toll pathway, increased AsNOS expression following parasite stimulation, suggesting that the Toll pathway may negatively regulate signaling by Plasmodium-derived AsNOS-inducing factors. In contrast, knockout of TGF-beta-activated kinase 1 (Tak1), a proximal signaling protein in the Imd pathway, reduced AsNOS expression by 20% relative to the control, suggesting that the Imd pathway is required for signaling by Plasmodium-derived AsNOS-inducing factors. Despite the NO-rich environment of the midgut, Plasmodium development is not completely inhibited. This observation suggests that Plasmodium may have efficient detoxification systems during sexual development in A. stephensi. To identify Plasmodium defense genes that may defend parasites against nitrosative stress caused by AsNOS induction, expression of several antioxidant defense genes known to function in nitrosative stress defense in a variety of organisms were examined during sporogonic development. Notably, increased expression levels of P. falciparum peroxiredoxins containing 1 or 2 cysteines (1-cys or 2-cys PfPrx) were associated with periods of parasite development just prior to and during parasite penetration of midgut epithelium, an event associated with significant AsNOS induction in the midgut. The provision of N omega-L-arginine (L-NAME), a known inhibitor of NOS enzyme activity, to A. stephensi with Plasmodium culture by artificial bloodmeal significantly reduced expression of 1-cys and 2-cys PfPrx indicating that these gene products may function to protect parasites against nitrosative stress induced by AsNOS.
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    Ingested histamine and serotonin interact to alter Anopheles stephensi feeding and flight behavior and infection with Plasmodium parasites
    Coles, Taylor A.; Briggs, Anna M.; Hambly, Malayna G.; Cespedes, Nora; Fellows, Abigail M.; Kaylor, Hannah L.; Adams, Alexandria D.; Van Susteren, Grace; Bentil, Ronald E.; Robert, Michael A.; Riffell, Jeffrey A.; Lewis, Edwin E.; Luckhart, Shirley (Frontiers, 2023-07-24)
    Blood levels of histamine and serotonin (5-HT) are altered in human malaria, and, at these levels, we have shown they have broad, independent effects on Anopheles stephensi following ingestion by this invasive mosquito. Given that histamine and 5-HT are ingested together under natural conditions and that histaminergic and serotonergic signaling are networked in other organisms, we examined effects of combinations of these biogenic amines provisioned to A. stephensi at healthy human levels (high 5-HT, low histamine) or levels associated with severe malaria (low 5-HT, high histamine). Treatments were delivered in water (priming) before feeding A. stephensi on Plasmodium yoelii-infected mice or via artificial blood meal. Relative to effects of histamine and 5-HT alone, effects of biogenic amine combinations were complex. Biogenic amine treatments had the greatest impact on the first oviposition cycle, with high histamine moderating low 5-HT effects in combination. In contrast, clutch sizes were similar across combination and individual treatments. While high histamine alone increased uninfected A. stephensi weekly lifetime blood feeding, neither combination altered this tendency relative to controls. The tendency to re-feed 2 weeks after the first blood meal was altered by combination treatments, but this depended on mode of delivery. For blood delivery, malaria-associated treatments yielded higher percentages of fed females relative to healthy-associated treatments, but the converse was true for priming. Female mosquitoes treated with the malaria-associated combination exhibited enhanced flight behavior and object inspection relative to controls and healthy combination treatment. Mosquitoes primed with the malaria-associated combination exhibited higher mean oocysts and sporozoite infection prevalence relative to the healthy combination, with high histamine having a dominant effect on these patterns. Compared with uninfected A. stephensi, the tendency of infected mosquitoes to take a second blood meal revealed an interaction of biogenic amines with infection. We used a mathematical model to project the impacts of different levels of biogenic amines and associated changes on outbreaks in human populations. While not all outbreak parameters were impacted the same, the sum of effects suggests that histamine and 5-HT alter the likelihood of transmission by mosquitoes that feed on hosts with symptomatic malaria versus a healthy host.
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    A Mosquito DNA Transposon Agh1: Structure, Evolution and Evidence of Activity
    Seok, Hee young (Virginia Tech, 2004-08-30)
    Transposable elements (TEs) are mobile genetic elements. They are a significant component of many eukaryotic genomes. They are involved in chromosomal rearrangement by serving as substrates for homologous recombination, in creating new genes through a process of TE "domestication", and in modifying and shuffling existing genes by transducing neighboring sequences (Lander et al., 2001). Therefore, both active and inactive TEs are potentially potent agents for genomic change (Kidwell and Lisch, 2001, 2002; Rizzon et al., 2002; Petrov et al., 2003). In the meantime, active TEs are being explored as useful tools for genetic transformation and possible gene drive mechanisms to deliver genes in natural populations (Ashburner et al.,1998; Alphey et al.,2002; Handler and O'Brochta, 2004). My thesis project focuses on AGH1, a novel DNA-mediated TE in Anopheles gambiae and related mosquitoes. I have studied its genomic structure, insertion polymorphism, evolution, and transposition activity. As part of the sequence and structural characterization of AGH1 in the A. gambiae genome, the boundaries of AGH1were determined. The TA target site duplications flanking AGH1 were verified by comparing a genomic sequence that had an AGH1 insertion with the sequence of a corresponding empty site. AGH1 has relatively long, 350bp, TIRs (Terminal inverted repeats). In addition to the transposase ORF (ORF1) that contains a DD34E catalytic motif, it contains an unusual ORF2 with unknown function. Phylogenic analyses clearly suggest that unlike most DD34E transposons that are similar to the Tc1 family, AGH1 belongs to a different clade that is related to the previously characterized fungal TE Ant and protozoan TEC1 and TEC2. Truncated AGH1 and AGH1-related MITE (Miniature inverted-repeat TE) families were also identified. AGH1 insertion polymorphism was studied using 4 natural populations that belong to two molecular forms of A. gambiae, M and S. AGH1 insertions showed considerable differences between M and S forms and the insertions of AGH1 are highly variable in two populations of M. These results are potentially significant in light of the hypothesis that M forms are newly derived incipient species that are only found in West Africa. PCR and sequencing results showed more than 99% sequence identity between AGH1 sequences in A. gambiae, A. arabiensis, and A. melas, which may indicate either purifying selection or recent horizontal transfer. To assess whether AGH1 is currently active, inverse PCR was performed which provided evidence for extrachromosomal circular AGH1 that may be a product of imprecise excision. RT-PCR detected transcripts for both intact and truncated transposase. Preliminary TE display experiments using genomic DNA isolated from different passages of an A. gambiae Sua1B cell line showed possible new insertions and deletions of AGH1 related elements, which may have been mobilized by AGH1. In summary, the structural and genomic characteristics of AGH1 and the phylogenetic relationship between AGH1 and other known transposons in the IS630-Tc1-mariner superfamily have been determined. Significant divergence was shown between M and S forms of A. gambiae according to AGH1 insertion patterns. Observations of high level of insertion polymorphism and low insertion frequency per site in M populations are preliminary indications that AGH1 may be active in some populations. AGH1 has at least been recently transposing and there are also indications for its current activity in A. gambiae cell lines. If AGH1 is indeed active, it has the potential to be used as genetic tools to study mosquito biology and to spread refractory genes into the field populations to help control mosquito-borne diseases. Although a few active DNA transposons have been discovered in different insects and are being used as tools to transform mosquitoes, no DNA active transposons have been reported in mosquitoes. It is our hope that active endogenous DNA transposons may present new features that will help us overcome some of the deficiencies of current transformation tools developed based on exogenous transposons. In addition, the discovery of an active DNA transposon will help us understand how TEs spread in natural populations of mosquitoes, which is critical if we are to use TEs to drive refractory genes into mosquito populations to control vector-borne infectious diseases. The differential insertion patterns of AGH1 in M and S populations are consistent with the hypothesis that the M and S forms of A. gambiae are in the process of incipient speciation. AgH1 showed much higher levels of insertion polymorphisms in two west African populations of the M molecular form compared to two east African S populations. Similarly, the maximum level of chromosomal differentiation is observed in west African dry savannah areas, while a much lower degree of chromosomal polymorphism is observed in east Africa. Therefore our insertion data support the hypothesis that the speciation process is likely to be originated in west Africa, probably as the result of the need of ecological flexibility created by the greater ecological variability of this region. From a biomedical perspective, this type of analysis is critical because the genetic differences between M and S forms may directly impact the effectiveness of mosquito control measure and perhaps disease transmission.
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    A New Role for an Old Antimicrobial: Lysozyme c-1 Can Function to Protect Malaria Parasites in Anopheles Mosquitoes
    Kajla, Mayur K.; Shi, Lei; Li, Bin; Luckhart, Shirley; Li, Jianyong; Paskewitz, Susan M. (PLOS, 2011-05-06)
    Background Plasmodium requires an obligatory life stage in its mosquito host. The parasites encounter a number of insults while journeying through this host and have developed mechanisms to avoid host defenses. Lysozymes are a family of important antimicrobial immune effectors produced by mosquitoes in response to microbial challenge. Methodology/Principal Findings A mosquito lysozyme was identified as a protective agonist for Plasmodium. Immunohistochemical analyses demonstrated that Anopheles gambiae lysozyme c-1 binds to oocysts of Plasmodium berghei and Plasmodium falciparum at 2 and 5 days after infection. Similar results were observed with Anopheles stephensi and P. falciparum, suggesting wide occurrence of this phenomenon across parasite and vector species. Lysozyme c-1 did not bind to cultured ookinetes nor did recombinant lysozyme c-1 affect ookinete viability. dsRNA-mediated silencing of LYSC-1 in Anopheles gambiae significantly reduced the intensity and the prevalence of Plasmodium berghei infection. We conclude that this host antibacterial protein directly interacts with and facilitates development of Plasmodium oocysts within the mosquito. Conclusions/Significance This work identifies mosquito lysozyme c-1 as a positive mediator of Plasmodium development as its reduction reduces parasite load in the mosquito host. These findings improve our understanding of parasite development and provide a novel target to interrupt parasite transmission to human hosts.
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    Non-LTR Retrotransposons in Mosquitoes: Diversity, Evolution, and Analysis of Potentially Active Elements
    Biedler, James K. (Virginia Tech, 2005-07-14)
    This research focuses on non-Long Terminal Repeat (non-LTR) retrotransposons in the African malaria mosquito, Anopheles gambiae and other mosquito species. An unprecedented diversity of non-LTRs was discovered by genome analysis of the An. gambiae genome assembly. One hundred and four families were found by a reiterative and comprehensive search using the conserved reverse transcriptase domains of known non-LTRs from a number of organisms as the starting queries. These families range in copy number from a few to approximately 2000 and occupy at least 3% of the genome. An. gambiae non-LTRs represent 8 of the 15 previously defined clades, plus two novel clades, Loner and Outcast, raising the total number of known clades to 17. The first invertebrate L1 clade representatives were also found. All clades except one have families with sequence characteristics suggesting recent activity. Juan, a non-LTR of the Jockey clade originally discovered in the mosquito Culex pipiens quinquefasciatus (Mouches et al. 1991), has been implicated in horizontal transfer in three non-sibling species of the Aedes genus (Mouches, Bensaadi, and Salvado 1992). PCR was used to obtain sequences from 18 mosquito species of six genera. Phylogenetic analysis demonstrates predominant vertical inheritance of Juan elements among these species. There is strong evidence from sequence analysis supporting the recent activity of Juan in several divergent species. We hypothesize that the sustained activity (versus quick inactivation) of non-LTRs in mosquitoes may contribute to the diversity we observe in the An. gambiae genome today. Promoter and transcriptional analyses were performed for several families previously identified as potentially active elements based on sequence analysis. RT-PCR results indicate that transcripts are present in An. gambiae cell lines that contain sequences corresponding to 13 of 15 tested non-LTR families. The 5' UTRs of An. gambiae non-LTRs from the I, Jockey, and L1 clades support basal transcription in divergent mosquito cell lines from 3 species. The Jen-1 5'UTR did not support transcription in Ae. aegypti and had low activity in Ae. albopictus. In summary, this research shows that Non-LTRs have been highly successful genomic elements that have flourished in many divergent mosquito species.
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    A Physiological, Biochemical and Structural Analysis of Inositol Polyphosphate 5-Phosphatases from Arabidopsis thaliana and Humans
    Burnette, Ryan Nelson (Virginia Tech, 2004-11-22)
    The complete role of inositol signaling in plants and humans is still elusive. The plant Arabidopsis thaliana contains fifteen predicted inositol polyphosphate 5- phosphatases (5PTases, E.C. 3.1.3.36) that have the potential to remove a 5-phosphate from various inositol second messenger substrates. To examine the substrate specificity of one of these Arabidopsis thaliana 5PTases (At5PTases), recombinant At5PTase1 was obtained from a Drosophila melanogaster expression system and analyzed biochemically. This analysis revealed that At5PTase1 has the ability to catalyze the hydrolysis of four potential inositol second messenger substrates. To determine whether At5PTase1 can be used to alter the signal transduction pathway of the major drought-sensing hormone abscisic acid (ABA), plants ectopically expressing At5PTase1 under the control of a constitutive promoter were characterized. This characterization revealed that plants ectopically expressing At5PTase1 had an altered response to ABA. These plants have stomata that are insensitive to ABA, and have lower basal and ABA-induced inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] levels. In addition, At5PTase1 mRNA and protein levels are transiently regulated by ABA. These data strongly suggest that At5PTase1 can act as a signal terminator of ABA signal transduction. Like the Arabidopsis At5PTase1, a human 5PTase, Ocrl, has the ability to catalyze the hydrolysis of a 5-phosphate from several inositol-containing substrates. The loss of functional Ocrl protein results in a rare genetic disorder known as Lowe oculocerebrorenal syndrome. To gather information concerning the specificity determinants of the Ocrl protein, a structure-function analysis of Ocrl was conducted using a vibrational technique, difference Fourier transform infrared (FT-IR) spectroscopy. Upon the introduction of Ins(1,4,5)P₃ substrate, structural changes in carboxylic acid and histidine residues were observed. The net result of changes in these residues indicates that upon Ins(1,4,5)P₃ introduction, a carboxylic acid-containing residue is protonated, and a histidine residue is deprotonated. This interpretation supports the idea that the deprotonation of the histidine residue is concomitant with the coordination of a divalent cation upon Ins(1,4,5)P₃ introduction. This work allows for the proposal of a new model for the role of the active site histidine of OCRL.
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    Protein Kinase C-Dependent Signaling Controls the Midgut Epithelial Barrier to Malaria Parasite Infection in Anopheline Mosquitoes
    Pakpour, Nazzy; Camp, Lauren; Smithers, Hannah M.; Wang, Bo; Tu, Zhijian Jake; Nadler, Steven A.; Luckhart, Shirley (PLOS, 2013-10-11)
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    Protein O-Kinases in the Archaeon Sulfolobus solfataricus
    Lower, Brian H. (Virginia Tech, 2001-07-23)
    For many years, it has been understood that protein phosphorylation-dephosphorylation constitutes one of the most ubiquitous mechanisms for controlling the functional properties of proteins. Although originally believed to be a eukaryotic phenomenon, protein phosphorylation is now known to occur in all three domains of life Eukarya, Bacteria, and Archaea. Very little is known, however, concerning the origins and evolution of protein phosphorylation-dephosphorylation. Knowledge of the structure and properties of the protein kinases resident in the members of the Archaea represents a key piece of this puzzle. The extreme acidothermophilic archaeon, Sulfolobus solfataricus, exhibits a membrane-associated protein kinase activity. Solubilization of the kinase activity requires the presence of detergent such as Triton X-100 or octyl glucoside, indicating its activity reside in an integral membrane protein. This protein kinase utilizes purine nucleotides as phosphoryl donors in vitro with a requirement for a divalent metal ion cofactor, favoring Mn⁺². A preference for NTPs over NDPs and for adenyl nucleotides over the analogous guanyl nucleotides was observed. The enzyme appears to be a glycoprotein that displays catalytic activity on SDS-PAGE corresponding to a molecular mass of ≈67 kDa, as well as an apparent molecular mass of –125 kDa on a gel filtration column. Challenged with several exogenous substrates revealed the protein kinase to be relatively selective. Only casein, reduced carboxyamidomethylated and maleylated lysozyme (RCM lysozyme), histone H4 proved, and a peptide modeled after myosin light chains (KKRAARATSNVFA) were phosphorylated to appreciable levels in vitro. All of the aforementioned substrates were phosphorylated on threonine, while histone H4 was phosphorylated on serine as well. When the phosphoacceptor threonine in the MLC peptide was substituted with serine an appreciable decrease in phosphorylation was noted. The protein kinase underwent autophosphorylation on threonine and was relatively insensitive to several known "eukaryotic" protein kinase inhibitors. Primary sequence motifs based on known conserved subdomains of eukaryotic protein kinases were used to search the genome of S. solfataricus for eukaryotic-like protein kinase sequences. Six hypothetical proteins were identified from S. solfataricus whose primary sequence exhibited noticeable similarities to eukaryotic protein kinases. The hypothetical protein encoded by ORF sso0197 contained 7 putative subdomains, ORFs sso0433, sso2291, sso2387, and sso3207 contained 8 putative subdomains, and ORF sso3182, contained 9 putative subdomains of the 12 characteristically conserved subdomains found within eukaryotic protein kinases. ORF sso2387 was cloned and expressed in Escherichia coli. The expressed protein, SsPK2, was solubilized from inclusion bodies using 5 M urea. SsPK2 was able to phosphorylate casein, BSA, RCM lysozyme, and mixed histones in vitro. Phosphoamino acid analysis of casein, BSA, and mixed histones revealed that they were all phosphorylated on serine. SsPK2 underwent autophosphorylation on serine at elevated temperature using both purine nucleotide triphosphates as phosphoryl donors in vitro, but exhibited a noticeable preference for ATP. Autophosphorylate of SsPK2 also occurred at elevated temperature using a variety of divalent metals cofactors in order of Mn⁺² > Mg⁺² >> Ca²⁺ ≈ Zn⁺². Polycations such as polyLys stimulated the phosphorylation of exogenous substrates while polyanions such as poly(Glu:Tyr) were shown to inhibit the phosphorylation of exogenous substrates. Of the "eukaryotic" protein kinases inhibitors tested, only tamoxifen had any noticeable effect of the catalytic activity of SsPK2 towards itself and exogenous substrates. A truncated form of SsPK2 containing the perceived catalytic domain also exhibited protein kinase activity towards itself and exogenous substrates. The observed protein kinase activity for SsPK2trunk was similar to that observed for SsPK2. Proteins from the membrane fraction of S. solfataricus subject to phosphorylation in vitro on serine or threonine residues were identified using MALDI-MS / peptide fingerprinting techniques. Nine phosphoproteins were assigned a tentative identification using the ProFound protein search engine from Rockefeller University. The identity of two of nine phosphoproteins, a translational endoplasmic reticulum ATPase and an ≈ 42 kDa hypothetical protein, were determined with a relatively high degree of confidence. Collectively the results suggested MALDI-MS peptide mapping coupled with [³²P] labeling in vivo will have a tremendous potential for mapping out a major portion of the phosphoproteome of S. solfataricus.
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    Reproduction and development of the black vine weevil (Coleoptera: Curculionidae) in relation to environmental factors
    Son, Youngsoo (Virginia Tech, 2004-09-15)
    Otiorhynchus sulcatus (Fabricius) is a serious pest of various horticultural crops in the United States. Despite its economic importance, this pest remains very challenging to manage. Integrated Pest Management Program for O. sulcatus has not yet been implemented due to lack of accurate information on its ecology. Investigation of temperature-dependent development and reproduction of O. sulcatus was conducted to enable the prediction of stage emergence more accurately. Immature-development study at constant temperatures 11-30 oC showed that there was the difference in optimal temperature regime among immature stages, which suggests that temperature can play a role to adapt each stage to the different temperature condition of the season. By applying mathematical models, optimum temperature for the fastest development was estimated to be 27.5, 25.1, and 24.1 oC for eggs, larvae, and pupae, respectively. Temperature also significantly influenced the adult reproductive life history traits, including preovipositional period, ovipositional period, longevity, per capita egg production, and egg viability. At 27 oC or above, reproductive success of O. sulcatus was substantially impaired due to shortened longevity, delayed reproduction, and lowered egg viability. Endosymbiont bacteria Wolbachia infect many arthropod species and they may alter the reproduction of their hosts. I first documented that Wolbachia is highly prevalent in O. sulcatus populations in the United States. Given that two antibiotics (tetracycline and gentamicin) differing only in their efficacy against Wolbachia, results strongly support the hypothesis that Wolbachia may be required for normal egg development of O. sulcatus. Despite intensive insecticide application, O. sulcatus populations often remain at unacceptable levels. I ask whether sublethal dosages of newer insecticides, imidacloprid and thiamethoxam, influence offspring production of O. sulcatus adults and its progeny under laboratory conditions. Weevils that survived short-term exposure to sublethal dosages of imidacloprid and thiamethoxam produce viable offspring when they have access to insecticide-free leaves after the exposure. Toxicity test showed the potential of imidacloprid and thiamethoxam for the control of first-instars but not for the control of eggs.