Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-7 (of 7 Records) |
Query Trace: Taracena M [original query] |
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Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes.
Taracena M , Hunt C , Pennington P , Andrew D , Jacobs-Lorena M , Dotson E , Wells M . J Vis Exp 2022 (181) RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond. |
Delivery of Double-Stranded RNAs (dsRNAs) Produced by Escherichia coli HT115(DE3) for Nontransgenic RNAi-Based Insect Pest Management.
Taracena ML , GarciaCaffaro I , Paiva-Silva GO , Oliveira PL , Rendon PA , Dotson EM , Pennington PM . Methods Mol Biol 2022 2360 279-294 RNA interference (RNAi) is a powerful mechanism that can be exploited not only for physiology research but also for designing insect pest management approaches. Some insects cause harm by vectoring diseases dangerous to humans, livestock, or plants or by damaging crops. For at least a decade now, different insect control strategies that induce RNAi by delivering double stranded RNA (dsRNA) targeting essential genes have been proposed. Here, we focus on nontransgenic RNAi-based approaches that use oral delivery of dsRNA through feeding of inactivated bacteria to produce RNAi in disease vectors and in a crop pest. This potential pest management method could be easily adapted to target different genes or similar organisms. |
Diverse cellular morphologies during lumen maturation in Anopheles gambiae larval salivary glands.
Chiu M , Trigg B , Taracena M , Wells M . Insect Mol Biol 2020 30 (2) 210-230 Mosquitoes are the greatest animal threat to human health, causing hundreds of millions of infections and around one million deaths each year. All mosquito-borne pathogens must traverse the salivary glands (SGs) to be transmitted to the next host, making this organ an ideal target for interventions. The adult SG develops from precursor cells located in the larval SG duct bud. Characterization of the larval SG has been limited. We sought to better understand larval SG architecture, secretion, and gene expression. We developed an optimized method for larval SG staining and surveyed hundreds of larval stage 4 (L4) SGs using fluorescence confocal microscopy. Remarkable variation in SG cell and chromatin organization differed among individuals and across the L4 stage. Lumen formation occurred during L4 stage through secretion likely involving a coincident cellular apical lipid enrichment and extracellular vesicle-like structures. Meta-analysis of microarray data showed that larval SG gene expression is divergent from adult SGs, more similar to larval gastric cecae, but different from other larval gut compartments. This work highlights the variable cell architecture of larval Anopheles gambiae SGs and provides candidate targets for genetic strategies aiming to disrupt SGs and transmission of mosquito-borne pathogens. |
Characterization of horizontally acquired ribotoxin encoding genes and their transcripts in Aedes aegypti.
Lapadula WJ , Marcet PL , Taracena M , Lenhart A , Juri Ayub M . Gene 2020 754 144857 Ribosome Inactivating Proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of the 28S rRNA. The occurrence of RIP genes has been described in a wide range of plant taxa, as well as in several species of bacteria and fungi. A remarkable case is the presence of these genes in metazoans belonging to the Culicinae subfamily. We reported that these genes are derived from a single horizontal gene transfer event, most likely from a bacterial donor species. Moreover, we have shown evidence that mosquito RIP genes are evolving under purifying selection, suggesting that these toxins have acquired a functional role in these organisms. In the present work, we characterized the intra-specific sequence variability of Aedes aegypti RIP genes (RIPAe1, RIPAe2, and RIPAe3) and tested their expression at the mRNA level. Our results show that RIPAe2 and RIPAe3 are transcribed and polyadenylated, and their expression levels are modulated across the developmental stages. Varibility among genes was observed, including the existence of null alleles for RIPAe1 and RIPAe2, with variants showing partial deletions. These results further support the existence of a physiological function for these foreign genes in mosquitoes. The possible nature of this functionality is discussed. |
Downregulation of female doublesex expression by oral-mediated RNA interference reduces number and fitness of Anopheles gambiae adult females
Taracena ML , Hunt CM , Benedict MQ , Pennington PM , Dotson EM . Parasit Vectors 2019 12 (1) 170 BACKGROUND: Mosquito-borne diseases affect millions worldwide, with malaria alone killing over 400 thousand people per year and affecting hundreds of millions. To date, the best strategy to prevent the disease remains insecticide-based mosquito control. However, insecticide resistance as well as economic and social factors reduce the effectiveness of the current methodologies. Alternative control technologies are in development, including genetic control such as the sterile insect technique (SIT). The SIT is a pivotal tool in integrated agricultural pest management and could be used to improve malaria vector control. To apply the SIT and most other newer technologies against disease transmitting mosquitoes, it is essential that releases are composed of males with minimal female contamination. The removal of females is an essential requirement because released females can themselves contribute towards nuisance biting and disease transmission. Thus, females need to be eliminated from the cohorts prior to release. Manual separation of Anopheles gambiae pupae or adult mosquitoes based on morphology is time consuming, is not feasible on a large scale and has limited the implementation of the SIT technique. The doublesex (dsx) gene is one of the effector switches of sex determination in the process of sex differentiation in insects. Both males and females have specific splicing variants that are expressed across the different life stages. Using RNA interference (RNAi) to reduce expression of the female specific (dsxF) variant of this gene has proven to have detrimental effects to the females in other mosquito species, such as Aedes aegypti. We tested oral RNAi on dsx (AgdsxF) in An. gambiae. METHODS: We studied the expression pattern of the dsx gene in the An. gambiae G3 strain. We knocked down AgdsxF expression in larvae through oral delivery of double stranded RNA (dsRNA) produced by bacteria and observed its effects in adults. RESULTS: Our results show that feeding of AgdsxF dsRNA can effectively reduce (> 66%) the mRNA of female dsx transcript and that there is a concomitant reduction in the number of female larvae that achieve adulthood. Control groups produced 52% (+/- 3.9% SE) of adult males and 48% (+/- 4.0% SE) females, while AgdsxF dsRNA treated groups had 72.1% (+/- 4.0% SE) males vs 27.8% females (+/- 3.3% SE). In addition, the female adults produce fewer progeny, 37.1% (+/- 8.2% SE) less than the controls. The knockdown was sex-specific and had no impact on total numbers of viable male adults, in the male dsx transcripts or male fitness parameters such as longevity or body size. CONCLUSIONS: These findings indicate that RNAi could be used to improve novel mosquito control strategies that require efficient sex separation and male-only release of An. gambiae by targeting sex determination genes such as AgdsxF. The advantages of using RNAi in a controlled setting for mosquito rearing are numerous, as the dose and time of exposure are controlled, and the possibility of off-target effects and the waste of female production would be significantly reduced. |
Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.
Mesquita RD , Vionette-Amaral RJ , Lowenberger C , Rivera-Pomar R , Monteiro FA , Minx P , Spieth J , Carvalho AB , Panzera F , Lawson D , Torres AQ , Ribeiro JM , Sorgine MH , Waterhouse RM , Montague MJ , Abad-Franch F , Alves-Bezerra M , Amaral LR , Araujo HM , Araujo RN , Aravind L , Atella GC , Azambuja P , Berni M , Bittencourt-Cunha PR , Braz GR , Calderon-Fernandez G , Carareto CM , Christensen MB , Costa IR , Costa SG , Dansa M , Daumas-Filho CR , De-Paula IF , Dias FA , Dimopoulos G , Emrich SJ , Esponda-Behrens N , Fampa P , Fernandez-Medina RD , da Fonseca RN , Fontenele M , Fronick C , Fulton LA , Gandara AC , Garcia ES , Genta FA , Giraldo-Calderon GI , Gomes B , Gondim KC , Granzotto A , Guarneri AA , Guigo R , Harry M , Hughes DS , Jablonka W , Jacquin-Joly E , Juarez MP , Koerich LB , Latorre-Estivalis JM , Lavore A , Lawrence GG , Lazoski C , Lazzari CR , Lopes RR , Lorenzo MG , Lugon MD , Majerowicz D , Marcet PL , Mariotti M , Masuda H , Megy K , Melo AC , Missirlis F , Mota T , Noriega FG , Nouzova M , Nunes RD , Oliveira RL , Oliveira-Silveira G , Ons S , Pagola L , Paiva-Silva GO , Pascual A , Pavan MG , Pedrini N , Peixoto AA , Pereira MH , Pike A , Polycarpo C , Prosdocimi F , Ribeiro-Rodrigues R , Robertson HM , Salerno AP , Salmon D , Santesmasses D , Schama R , Seabra-Junior ES , Silva-Cardoso L , Silva-Neto MA , Souza-Gomes M , Sterkel M , Taracena ML , Tojo M , Tu ZJ , Tubio JM , Ursic-Bedoya R , Venancio TM , Walter-Nuno AB , Wilson D , Warren WC , Wilson RK , Huebner E , Dotson EM , Oliveira PL . Proc Natl Acad Sci U S A 2015 112 (48) 14936-14941 Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome ( approximately 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods. |
Genetically modifying the insect gut microbiota to control Chagas disease vectors through systemic RNAi.
Taracena ML , Oliveira PL , Almendares O , Umana C , Lowenberger C , Dotson EM , Paiva-Silva GO , Pennington PM . PLoS Negl Trop Dis 2015 9 (2) e0003358 Technologies based on RNA interference may be used for insect control. Sustainable strategies are needed to control vectors of Chagas disease such as Rhodnius prolixus. The insect microbiota can be modified to deliver molecules to the gut. Here, Escherichia coli HT115(DE3) expressing dsRNA for the Rhodnius heme-binding protein (RHBP) and for catalase (CAT) were fed to nymphs and adult triatomine stages. RHBP is an egg protein and CAT is an antioxidant enzyme expressed in all tissues by all developmental stages. The RNA interference effect was systemic and temporal. Concentrations of E. coli HT115(DE3) above 3.35 x 107 CFU/mL produced a significant RHBP and CAT gene knockdown in nymphs and adults. RHBP expression in the fat body was reduced by 99% three days after feeding, returning to normal levels 10 days after feeding. CAT expression was reduced by 99% and 96% in the ovary and the posterior midgut, respectively, five days after ingestion. Mortality rates increased by 24-30% in first instars fed RHBP and CAT bacteria. Molting rates were reduced by 100% in first instars and 80% in third instars fed bacteria producing RHBP or CAT dsRNA. Oviposition was reduced by 43% (RHBP) and 84% (CAT). Embryogenesis was arrested in 16% (RHBP) and 20% (CAT) of laid eggs. Feeding females 105 CFU/mL of the natural symbiont, Rhodococcus rhodnii, transformed to express RHBP-specific hairpin RNA reduced RHBP expression by 89% and reduced oviposition. Modifying the insect microbiota to induce systemic RNAi in R. prolixus may result in a paratransgenic strategy for sustainable vector control. |
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