Browsing by Subject "Mosquito"
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Item Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae(JOVE, 2015-03-25) Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W.; Zhu, Kun Yan; Duman-Scheel, Molly; Department of Medical and Molecular Genetics, IU School of MedicineVector mosquitoes inflict more human suffering than any other organism-and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.Item Examination of the genetic basis for sexual dimorphism in the Aedes aegypti (dengue vector mosquito) pupal brain(BioMed Central, 2014-10-21) Tomchaney, Michael; Mysore, Keshava; Sun, Longhua; Li, Ping; Emrich, Scott J.; Severson, David W.; Duman-Scheel, Molly; Department of Medical and Molecular Genetics, IU School of MedicineBACKGROUND: Most animal species exhibit sexually dimorphic behaviors, many of which are linked to reproduction. A number of these behaviors, including blood feeding in female mosquitoes, contribute to the global spread of vector-borne illnesses. However, knowledge concerning the genetic basis of sexually dimorphic traits is limited in any organism, including mosquitoes, especially with respect to differences in the developing nervous system. METHODS: Custom microarrays were used to examine global differences in female vs. male gene expression in the developing pupal head of the dengue vector mosquito, Aedes aegypti. The spatial expression patterns of a subset of differentially expressed transcripts were examined in the developing female vs. male pupal brain through in situ hybridization experiments. Small interfering RNA (siRNA)-mediated knockdown studies were used to assess the putative role of Doublesex, a terminal component of the sex determination pathway, in the regulation of sex-specific gene expression observed in the developing pupal brain. RESULTS: Transcripts (2,527), many of which were linked to proteolysis, the proteasome, metabolism, catabolic, and biosynthetic processes, ion transport, cell growth, and proliferation, were found to be differentially expressed in A. aegypti female vs. male pupal heads. Analysis of the spatial expression patterns for a subset of dimorphically expressed genes in the pupal brain validated the data set and also facilitated the identification of brain regions with dimorphic gene expression. In many cases, dimorphic gene expression localized to the optic lobe. Sex-specific differences in gene expression were also detected in the antennal lobe and mushroom body. siRNA-mediated gene targeting experiments demonstrated that Doublesex, a transcription factor with consensus binding sites located adjacent to many dimorphically expressed transcripts that function in neural development, is required for regulation of sex-specific gene expression in the developing A. aegypti brain. CONCLUSIONS: These studies revealed sex-specific gene expression profiles in the developing A. aegypti pupal head and identified Doublesex as a key regulator of sexually dimorphic gene expression during mosquito neural development.Item Identification of Aedes aegypti cis-regulatory elements that promote gene expression in olfactory receptor neurons of distantly related dipteran insects(BMC, 2018-07-11) Mysore, Keshava; Li, Ping; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineBACKGROUND: Sophisticated tools for manipulation of gene expression in select neurons, including neurons that regulate sexually dimorphic behaviors, are increasingly available for analysis of genetic model organisms. However, we lack comparable genetic tools for analysis of non-model organisms, including Aedes aegypti, a vector mosquito which displays sexually dimorphic behaviors that contribute to pathogen transmission. Formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq) recently facilitated genome-wide discovery of putative A. aegypti cis-regulatory elements (CREs), many of which could be used to manipulate gene expression in mosquito neurons and other tissues. The goal of this investigation was to identify FAIRE DNA elements that promote gene expression in the olfactory system, a tissue of vector importance. RESULTS: Eight A. aegypti CREs that promote gene expression in antennal olfactory receptor neurons (ORNs) were identified in a Drosophila melanogaster transgenic reporter screen. Four CREs identified in the screen were cloned upstream of GAL4 in a transgenic construct that is compatible with transformation of a variety of insect species. These constructs, which contained FAIRE DNA elements associated with the A. aegypti odorant coreceptor (orco), odorant receptor 1 (Or1), odorant receptor 8 (Or8) and fruitless (fru) genes, were used for transformation of A. aegypti. Six A. aegypti strains, including strains displaying transgene expression in all ORNs, subsets of these neurons, or in a sex-specific fashion, were isolated. The CREs drove transgene expression in A. aegypti that corresponded to endogenous gene expression patterns of the orco, Or1, Or8 and fru genes in the mosquito antenna. CRE activity in A. aegypti was found to be comparable to that observed in D. melanogaster reporter assays. CONCLUSIONS: These results provide further evidence that FAIRE-seq, which can be paired with D. melanogaster reporter screening to test FAIRE DNA element activity in select tissues, is a useful method for identification of mosquito cis-regulatory elements. These findings expand the genetic toolkit available for the study of Aedes neurobiology. Moreover, given that the CREs drive comparable olfactory neural expression in both A. aegypti and D. melanogaster, it is likely that they may function similarly in multiple dipteran insects, including other disease vector mosquito species.Item Larvicidal Activity of Carbon Black against the Yellow Fever Mosquito Aedes aegypti(MDPI, 2022-03-20) Martínez Rodríguez, Erick J.; Evans, Parker; Kalsi, Megha; Rosenblatt, Noah; Stanley, Morgan; Piermarini, Peter M.; Surgery, School of MedicineThe yellow fever mosquito Aedes aegypti is one of the deadliest animals on the planet because it transmits several medically important arboviruses, including Zika, chikungunya, dengue, and yellow fever. Carbon-based nanoparticles (CNPs) derived from natural sources have previously been shown to have toxic effects on mosquito larvae and offer a potential alternative to chemical insecticides such as pyrethroids, for which mosquitoes have evolved resistance. However, CNPs derived from industrial sources, such as carbon black, have not previously been evaluated as larvicides. Here, we evaluate the effects of a commercially-available carbon black, EMPEROR® 1800 (E1800), on mortality and development of pyrethroid-susceptible (PS) and pyrethroid-resistant (PR) strains of Ae. aegypti. We found that E1800 exhibited concentration-dependent mortality against 1st instar larvae of both strains within the first 120 h after exposure, but after this period, surviving larvae did not show delays in their development to adults. Physical characterization of E1800 suspensions suggests that they form primary particles of ~30 nm in diameter that fuse into fundamental aggregates of ~170 nm in diameter. Notably, larvae treated with E1800 showed internal accumulation of E1800 in the gut and external accumulation on the respiratory siphon, anal papillae, and setae, suggesting a physical mode of toxic action. Taken together, our results suggest that E1800 has potential use as a larvicide with a novel mode of action for controlling PS and PR mosquitoes.Item Requirement for commissureless2 function during dipteran insect nerve cord development(Wiley, 2013-12) Sarro, Joseph; Andrews, Emily; Sun, Longhua; Behura, Susanta K.; Tan, John C.; Zeng, Erliang; Severson, David W.; Duman-Scheel, Molly; Medical & Molecular Genetics, School of MedicineBACKGROUND: In Drosophila melanogaster, commissureless (comm) function is required for proper nerve cord development. Although comm orthologs have not been identified outside of Drosophila species, some insects possess orthologs of Drosophila comm2, which may also regulate embryonic nerve cord development. Here, this hypothesis is explored through characterization of comm2 genes in two disease vector mosquitoes. RESULTS: Culex quinquefasciatus (West Nile and lymphatic filiariasis vector) has three comm2 genes that are expressed in the developing nerve cord. Aedes aegypti (dengue and yellow fever vector) has a single comm2 gene that is expressed in commissural neurons projecting axons toward the midline. Loss of comm2 function in both A. aegypti and D. melanogaster was found to result in loss of commissure defects that phenocopy the frazzled (fra) loss of function phenotypes observed in both species. Loss of fra function in either insect was found to result in decreased comm2 transcript levels during nerve cord development. CONCLUSIONS: The results of this investigation suggest that Fra down-regulates repulsion in precrossing commissural axons by regulating comm2 levels in both A. aegypti and D. melanogaster, both of which require Comm2 function for proper nerve cord development.Item Saccharomyces cerevisiae (Baker's Yeast) as an Interfering RNA Expression and Delivery System(Bentham Science Publishers, 2019) Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineThe broad application of RNA interference for disease prevention is dependent upon the production of dsRNA in an economically feasible, scalable, and sustainable fashion, as well as the identification of safe and effective methods for RNA delivery. Current research has sparked interest in the use of Saccharomyces cerevisiae for these applications. This review examines the potential for commercial development of yeast interfering RNA expression and delivery systems. S. cerevisiae is a genetic model organism that lacks a functional RNA interference system, which may make it an ideal system for expression and accumulation of high levels of recombinant interfering RNA. Moreover, recent studies in a variety of eukaryotic species suggest that this microbe may be an excellent and safe system for interfering RNA delivery. Key areas for further research and development include optimization of interfering RNA expression in S. cerevisiae, industrial-sized scaling of recombinant yeast cultures in which interfering RNA molecules are expressed, the development of methods for largescale drying of yeast that preserve interfering RNA integrity, and identification of encapsulating agents that promote yeast stability in various environmental conditions. The genetic tractability of S. cerevisiae and a long history of using this microbe in both the food and pharmaceutical industry will facilitate further development of this promising new technology, which has many potential applications of medical importance.Item Seroprevalence of West Nile Virus among Healthy Blood Donors from Different National Populations Residing in Qatar(Elsevier, 2021) Dargham, Soha R.; Al-Sadeq, Duaa W.; Yassine, Hadi M.; Ahmed, Muna; Kunhipurayil, Hasna; Humphrey, John M.; Abu-Raddad, Laith J.; Nasrallah, Gheyath K.; Medicine, School of MedicineObjective: To estimate the age- and nationality-specific West Nile virus (WNV) seroprevalence in select Middle East and North Africa (MENA) populations residing in Qatar. Methods: Sera were collected from male blood donors attending Hamad Medical Corporation. A total of 1,948 sera were tested for anti-WNV antibodies using Serion ELISA classic IgG and IgM kits. Results: Overall, seroprevalence estimates of WNV-specific IgG and IgM antibodies were 10.4% and 3.3%, respectively. Country-specific WNV-specific IgG seroprevalence was estimated to be 37.0% (34/92) in Sudanese, 33.0% in Egyptians (66/200), 13.0% (26/200) in Indians, 10.6% (11/104) in Iranians, 10.2% (14/137) in Yemenis, 9.2% (18/195) in Pakistanis, 7.0% (14/199) in Jordanians, 5.4% (6/111) in Filipinos, 2.5% (5/200) in Palestinians, 2.5% (5/200) in Syrians, 1.5% (3/200) in Qataris, and 0.9% (1/110) in Lebanese. Seroprevalence of WNV-specific IgM was lowest in Iranians (0/77), Lebanese (0/108), and Filipinos (0/107) at 0.0%, and was highest in Sudanese at 10.0% (8/80). While there seemed to be apparent trends in the prevalence of WNV-IgM and WNV-IgG antibodies, none of these trends were found to be statistically significant. Conclusion: The findings support the circulation of WNV in human populations in different countries of the MENA region. Seroprevalence was highest in Sudanese and Egyptians and lowest in Qataris and nationals of the Levant. The findings call for further animal, vector, and human studies, such as studying the actual prevalence of the viral RNA in blood donors to assess the risk of viral transmission through blood donation and for a better characterization of the epidemiology of this infection in this part of the world.Item Yeast interfering RNA larvicides targeting neural genes induce high rates of Anopheles larval mortality(BioMed Central, 2017-11-13) Mysore, Keshava; Hapairai, Limb K.; Sun, Longhua; Harper, Elizabeth I.; Chen, Yingying; Eggleson, Kathleen K.; Realey, Jacob S.; Scheel, Nicholas D.; Severson, David W.; Wei, Na; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineBackground Although larviciding can reduce the number of outdoor biting malaria vector mosquitoes, which may help to prevent residual malaria transmission, the current larvicide repertoire is faced with great challenges to sustainability. The identification of new effective, economical, and biorational larvicides could facilitate maintenance and expansion of the practice of larviciding in integrated malaria vector mosquito control programmes. Interfering RNA molecules represent a novel class of larvicides with untapped potential for sustainable mosquito control. This investigation tested the hypothesis that short interfering RNA molecules can be used as mosquito larvicides. Results A small interfering RNA (siRNA) screen for larval lethal genes identified siRNAs corresponding to the Anopheles gambiae suppressor of actin (Sac1), leukocyte receptor complex member (lrc), and offtrack (otk) genes. Saccharomyces cerevisiae (baker’s yeast) was engineered to produce short hairpin RNAs (shRNAs) for silencing of these genes. Feeding larvae with the engineered yeasts resulted in silenced target gene expression, a severe loss of neural synapses in the larval brain, and high levels of larval mortality. The larvicidal activities of yeast interfering RNA larvicides were retained following heat inactivation and drying of the yeast into user-friendly tablet formulations that induced up to 100% larval mortality in laboratory trials. Conclusions Ready-to-use dried inactivated yeast interfering RNA larvicide tablets may someday be an effective and inexpensive addition to malaria mosquito control programmes and a valuable, biorational tool for addressing residual malaria transmission. Electronic supplementary material The online version of this article (10.1186/s12936-017-2112-5) contains supplementary material, which is available to authorized users.Item A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide(MDPI, 2021-11-02) Mysore, Keshava; Sun, Longhua; Hapairai, Limb K.; Wang, Chien-Wei; Igiede, Jessica; Roethele, Joseph B.; Scheel, Nicholas D.; Scheel, Max P.; Li, Ping; Wei, Na; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineConcerns for widespread insecticide resistance and the unintended impacts of insecticides on nontarget organisms have generated a pressing need for mosquito control innovations. A yeast RNAi-based insecticide that targets a conserved site in mosquito Irx family genes, but which has not yet been identified in the genomes of nontarget organisms, was developed and characterized. Saccharomyces cerevisiae constructed to express short hairpin RNA (shRNA) matching the target site induced significant Aedes aegypti larval death in both lab trials and outdoor semi-field evaluations. The yeast also induced high levels of mortality in adult females, which readily consumed yeast incorporated into an attractive targeted sugar bait (ATSB) during simulated field trials. A conserved requirement for Irx function as a regulator of proneural gene expression was observed in the mosquito brain, suggesting a possible mode of action. The larvicidal and adulticidal properties of the yeast were also verified in Aedes albopictus, Anopheles gambiae, and Culexquinquefasciatus mosquitoes, but the yeast larvicide was not toxic to other nontarget arthropods. These results indicate that further development and evaluation of this technology as an ecofriendly control intervention is warranted, and that ATSBs, an emerging mosquito control paradigm, could potentially be enriched through the use of yeast-based RNAi technology.