36. Arevalo-Cortes, A; Mejia-Jaramillo, AM; Granada, Y; Coatsworth, H; Lowenberger, C; Triana-Chavez, O. (2020) The Midgut Microbiota of Colombian Aedes aegypti Populations with Different Levels of Resistance to the Insecticide Lambda-cyhalothrin.Insects 11 The Midgut Microbiota of Colombian Aedes aegypti Populations with Different Levels of Resistance to the Insecticide Lambda-cyhalothrin
Aedes aegypti; microbiome; lambda-cyhalothrin; insecticide resistance
Simple Summary Aedes aegypti is a mosquito capable of transmitting many viral diseases such as dengue, Zika, and chikungunya. Since no effective treatments are available for these viruses, eliminating the mosquito with insecticides is vital to combat these diseases. However, the mosquito can generate resistance to the insecticide by changing its genes or its physiology. It has been recognized that the type of bacteria that live inside the mosquito's gut can contribute to this resistance. In this study, we evaluated Ae. aegypti mosquitoes from six locations in Colombia to determine if they are resistant to lambda-cyhalothrin insecticide, and we analyze their gut microbiota. We observed resistance in five of the six areas. We compared the gut microbiota from susceptible and resistant mosquitoes and found specific bacteria in resistant mosquitoes that may play a role in insecticide resistance. Overall, our findings contribute to the understanding of insecticide resistance in Ae. aegypti that will generate alternatives for interventions to control this mosquito in Colombia. Insecticide resistance in Aedes aegypti populations is a problem that hinders vector control and dengue prevention programs. In this study, we determined the susceptibility of Ae. aegypti populations from six Colombian regions to the pyrethroid lambda-cyhalothrin and evaluated the presence of the V1016I mutation in the sodium channel gene, which has been broadly involved in the resistance to this insecticide. The diversity of the gut microbiota of these mosquito populations was also analyzed. Only mosquitoes from Bello were susceptible to lambda-cyhalothrin and presented a lower allelic frequency of the V1016I mutation. Remarkably, there was not an important change in allelic frequencies among populations with different resistance ratios, indicating that other factors or mechanisms contributed to the resistant phenotype. Treatment of mosquitoes with antibiotics led us to hypothesize that the intestinal microbiota could contribute to the resistance to lambda-cyhalothrin. Beta diversity analysis showed significant differences in the species of bacteria present between susceptible and resistant populations. We identified 14 OTUs of bacteria that were unique in resistant mosquitoes. We propose that kdr mutations are important in the development of resistance to lambda-cyhalothrin at low insecticide concentrations but insect symbionts could play an essential role in the metabolization of pyrethroid insecticides at higher concentrations, contributing to the resistant phenotype in Ae. aegypti. DOI PubMed
34. Caicedo, P.A., I.M. Serrato, S. Zim, G. Dimopoulos, C. Lowenberger, and C.B. Ocampo. (2019) Immune response-related genes associated to blocking midgut dengue virus infection in Aedes aegypti strains that differ in susceptibility to Dengue-2 Virus.Insect Science 26, 635–648 Immune response-related genes associated to blocking midgut dengue virus infection in Aedes aegypti strains that differ in susceptibility to Dengue-2 Virus.
Aedes aegypti; dengue virus; innate immune response; knockdown; microarray; vector competence
Aedes (Stegomyia) aegypti, the principal global vector of dengue viruses, has differences in its susceptibility to dengue virus infection. We compared the global expression of genes in the midguts of Colombian Ae. aegypti dengue-susceptible (Cali-S) and dengue-refractory (Cali-MIB) field derived strains after ingesting either a sugarmeal, a bloodmeal, or a bloodmeal containing dengue virus serotype 2 (DENV-2). Microarray-based transcriptome analysis among treatments indicated a total of 4725 transcripts with differential expression between the two strains. Eleven genes were selected from different functional groups based on their significant up or down expression levels as well as reports in the literature suggesting they are associated with dengue virus elimination. We measured mRNA abundance of these 11 genes at 0, 8, 24, and 36 h postinfection using quantitative real time PCR (qPCR) to confirm the microarray results and assess any temporal patterns. Four genes were selected (Gram-negative binding protein-GNBP [AAEL009176], Niemann Pick Type-C2-NPC2 [AAEL015136], Keratinocyte lectin [AAEL009842], and Cathepsin-b [AAEL007585]) for knockdown experiments using RNA interference (RNAi) methodology to determine the phenotype (DENV-2 susceptible or refractory). Silencing GNBP, Cathepsin-b and Keratinocyte lectin reduced the percentage of mosquitoes with disseminated virus in the Cali-S strain to 8%, 20%, and 12% respectively compared with 96% in the controls. Silencing of NPC2 increased the percentage of mosquitos with disseminated virus infections in Cali-MIB to 66% compared with 35% in the controls. This study provides insight into genes that may contribute to the Cali-S susceptible and Cali-MIB refractory phenotypes in Ae. aegypti. DOI PubMed
33. Gutiérrez-Cabrera, A.E., W.F. Zandberg, E.Zenteno, M.H. Rodríguez, B. Espinoza and C. Lowenberger. (2019) Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.Insect Science 26: 796-808 Glycosylation on proteins of the intestine and perimicrovillar membrane of Triatoma (Meccus) pallidipennis, under different feeding conditions.
feeding conditions; glycoproteins; lectins; perimicrovillar membrane; triatomine; vector-parasite interactions
Trypanosoma cruzi, the causative agent of Chagas disease, interacts with molecules in the midgut of its insect vector to multiply and reach the infective stage. Many studies suggest that the parasite binds to midgut-specific glycans. We identified several glycoproteins expressed in the intestine and perimicrovillar membrane (PMM) of Triatoma (Meccus) pallidipennis under different feeding conditions. In order to assess changes in protein-linked glycans, we performed lectin and immunoblot analyses on glycoprotein extracts from these intestinal tissues using well-characterized lectins, and an antibody, which collectively recognize a wide range of different glycans epitopes. We observed that the amount and composition of proteins and glycoproteins associated with different glycans structures changed over time in the intestines and PMM under different physiological conditions. PMM extracts contained a wide variety of glycoproteins with different sugar residues, including abundant high-mannose and complex sialylated glycans. We propose that these molecules could be involved in the process of parasite-vector interactions. DOI PubMed
32. Salcedo-Porras, N; Guarneri, A; Oliveira, PL; Lowenberger, C. (2019) Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteria.PLoS One 14 Rhodnius prolixus: Identification of missing components of the IMD immune signaling pathway and functional characterization of its role in eliminating bacteria
The innate immune system in insects is regulated by specific signalling pathways. Most immune related pathways were identified and characterized in holometabolous insects such as Drosophila melanogaster, and it was assumed they would be highly conserved in all insects. The hemimetabolous insect, Rhodnius prolixus, has served as a model to study basic insect physiology, but also is a major vector of the human parasite, Trypanosome cruzi, that causes 10,000 deaths annually. The publication of the R. prolixus genome revealed that one of the main immune pathways, the Immune-deficiency pathway (IMD), was incomplete and probably non-functional, an observation shared with other hemimetabolous insects including the pea aphid (Acyrthosiphon pisum) and the bedbug (Cimex lectularius). It was proposed that the IMD pathway is inactive in R. prolixus as an adaptation to prevent eliminating beneficial symbiont gut bacteria. We used bioinformatic analyses based on reciprocal BLAST and HMM-profile searches to find orthologs for most of the "missing" elements of the IMD pathway and provide data that these are regulated in response to infection with Gram-negative bacteria. We used RNAi strategies to demonstrate the role of the IMD pathway in regulating the expression of specific antimicrobial peptides (AMPs) in the fat body of R. prolixus. The data indicate that the IMD pathway is present and active in R. prolixus, which opens up new avenues of research on R. prolixus-T. cruzi interactions. DOI PubMed
31. Coatsworth, H; Caicedo, PA; Van Rossum, T; Ocampo, CB; Lowenberger, C. (2018) The Composition of Midgut Bacteria in Aedes aegypti (Diptera: Culicidae) That Are Naturally Susceptible or Refractory to Dengue Viruses.J Insect Sci. 18 The Composition of Midgut Bacteria in Aedes aegypti (Diptera: Culicidae) That Are Naturally Susceptible or Refractory to Dengue Viruses
vector competence; microbiome; midgut infection barrier
The composition, abundance, and diversity of midgut bacteria in mosquitoes can influence pathogen transmission. We used 16S rRNA microbiome profiling to survey midgut microbial diversity in pooled samples of laboratory colonized dengue-refractory, Cali-MIB, and dengue-susceptible, Cali-S Aedes aegypti (Linnaeus). The 16S rRNA sequences from the sugar-fed midguts of adult females clustered to 63 amplicon sequence variants (ASVs), primarily from Proteobacteria, Firmicutes, Flavobacteria, and Actinobacteria. An average of five ASVs dominated the midguts, and most ASVs were present in both Cali-MIB and Cali-S midguts. No differences in abundance were noted at any phylogenetic level (Phylum, Class, Order, Family, Genus) by analysis of composition of microbiome (w=0). No community diversity metrics were significantly different between refractory and susceptible mosquitoes. These data suggest that phenotypic differences in the susceptibility to dengue virus between Cali-MIB and Cali-S are not likely due to major differences in midgut bacterial communities. DOI PubMed
30. de Fuentes-Vicente, JA; Gutierrez-Cabrera, AE; Flores-Villegas, AL; Lowenberger, C; Benelli, G; Salazar-Schettino, PM; Cordoba-Aguilar, A. (2018) What makes an effective Chagas disease vector? Factors underlying Trypanosoma cruzi-triatomine interactions.Acta Trop. 183 What makes an effective Chagas disease vector? Factors underlying Trypanosoma cruzi-triatomine interactions
Chagas disease; Evolution; Host-parasite interaction; Meccus pallidipennis; Rhodnius prolixus; Triatoma barberi; Triatoma infestans; Triatoma dimidiata; Triatominae; Trypanosoma cruzi
The Chagas disease is caused by the parasite Trypanosoma cruzi, which infect blood-feeding triatomine bugs to finally reach mammal hosts. Chagas disease is endemic in Latin America, and is ranked among the 13 neglected tropical diseases worldwide. Currently, an estimate of 7 million people is infected by T. cruzi, leading to about 22000 deaths per year throughout the Americas. As occurs with other vectors, a major question towards control programs is what makes a susceptible bug. In this review, we focus on findings linked to insect gut structure and microbiota, immunity, genetics, blood sources, abiotic factors (with special reference to ambient temperature and altitude) to understand the interactions occurring between T. cruzi and triatomine bugs, under a co-evolutionary scenario. These factors lead to varying fitness benefits and costs for bugs, explaining why infection in the insect takes place and how it varies in time and space. Our analysis highlights that major factors are gut components and microbiota, blood sources and temperature. Although their close interaction has never been clarified, knowledge reviewed here may help to boost the success of triatomine control programs, reducing the use of insecticides. DOI PubMed
29. Serrato, IM; Caicedo, PA; Orobio, Y; Lowenberger, C; Ocampo, CB. (2017) Vector competence and innate immune responses to dengue virus infection in selected laboratory and field-collected Stegomyia aegypti (= Aedes aegypti).Med. Vet. Entomol. 31: 312-319 Vector competence and innate immune responses to dengue virus infection in selected laboratory and field-collected Stegomyia aegypti (= Aedes aegypti)
Apoptosis; caspases; dengue virus; innate immunity; vector competence
Control of dengue virus (DenV) transmission, primarily based on strategies to reduce populations of the principle vector Stegomya aegypti (= Aedes aegypti) (Diptera: Culicidae), is difficult to sustain over time. Other potential strategies aim to manipulate characteristics such as vector competence (VC), the innate capacity of the vector to transmit the virus. Previous studies have identified genetic factors, including differential expression of apoptosis-related genes, associated with the refractory and susceptible phenotypes in selected strains of S. aegypti from Cali, Colombia. The present study was designed to evaluate the variability of VC in selected strains against different DenV serotypes and to determine whether field-collected mosquitoes respond similarly to selected laboratory strains in terms of enhanced or reduced expression of apoptosis-related genes. Vector competence differed between strains, but did not differ in response to different DenV serotypes. Differences in VC were observed among mosquitoes collected from different localities in Cali. The overexpression of the pro-apoptosis genes, caspase 16 and Aedronc, was conserved in field-collected refractory mosquitoes and the selected laboratory refractory strain. The results suggest that the apoptosis response is conserved among all refractory mosquitoes to inhibit the development of all DenV serotypes. DOI
28. Gutierrez-Cabrera, AE; Cordoba-Aguilar, A; Zenteno, E; Lowenberger, C; Espinoza, B. (2016) Origin, evolution and function of the hemipteran perimicrovillar membrane with emphasis on Reduviidae that transmit Chagas disease.Bulletin of Entomological Research 106: 279-291 Origin, evolution and function of the hemipteran perimicrovillar membrane with emphasis on Reduviidae that transmit Chagas disease
evolution; perimicrovillar membrane; insects; triatomines
The peritrophic matrix is a chitin-protein structure that envelops the food bolus in the midgut of the majority of insects, but is absent in some groups which have, instead, an unusual extra-cellular lipoprotein membrane named the perimicrovillar membrane. The presence of the perimicrovillar membrane (PMM) allows these insects to exploit restricted ecological niches during all life stages. It is found only in some members of the superorder Paraneoptera and many of these species are of medical and economic importance. In this review we present an overview of the midgut and the digestive system of insects with an emphasis on the order Paraneoptera and differences found across phylogenetic groups. We discuss the importance of the PMM in Hemiptera and the apparent conservation of this structure among hemipteran groups, suggesting that the basic mechanism of PMM production is the same for different hemipteran species. We propose that the PMM is intimately involved in the interaction with parasites and as such should be a target for biological and chemical control of hemipteran insects of economic and medical importance. DOI
27. Nunes, RD; Ventura-Martins, G; Moretti, DM; Medeiros-Castro, P; Rocha-Santos, C; Daumas, CRD; Bittencourt-Cunha, PRB; Martins-Cardoso, K; Cudischevitch, CO; Menna-Barreto, RFS; Oliveira, JHM; Gusmao, DS; Lemos, FJA; Alviano, DS; Oliveira, PL; Lowenberger, C; Majerowicz, D; Oliveira, RM; Mesquita, RD; Atella, GC; Silva-Neto, MAC. (2016) Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan.PLoS Neglected Tropical Diseases 10 Polyphenol-Rich Diets Exacerbate AMPK-Mediated Autophagy, Decreasing Proliferation of Mosquito Midgut Microbiota, and Extending Vector Lifespan
Background Mosquitoes feed on plant-derived fluids such as nectar and sap and are exposed to bioactive molecules found in this dietary source. However, the role of such molecules on mosquito vectorial capacity is unknown. Weather has been recognized as a major determinant of the spread of dengue, and plants under abiotic stress increase their production of polyphenols. Results Here, we show that including polyphenols in mosquito meals promoted the activation of AMP-dependent protein kinase (AMPK). AMPK positively regulated midgut autophagy leading to a decrease in bacterial proliferation and an increase in vector lifespan. Suppression of AMPK activity resulted in a 6-fold increase in midgut microbiota. Similarly, inhibition of polyphenol-induced autophagy induced an 8-fold increase in bacterial proliferation. Mosquitoes maintained on the polyphenol diet were readily infected by dengue virus. Conclusion The present findings uncover a new direct route by which exacerbation of autophagy through activation of the AMPK pathway leads to a more efficient control of mosquito midgut microbiota and increases the average mosquito lifespan. Our results suggest for the first time that the polyphenol content and availability of the surrounding vegetation may increase the population of mosquitoes prone to infection with arboviruses. DOI
26. Marliere, NP; Latorre-Estivalis, JM; Lorenzo, MG; Carrasco, D; Alves-Silva, J; Rodrigues, JD; Ferreira, LD; Lara, LD; Lowenberger, C; Guarneri, AA. (2015) Trypanosomes Modify the Behavior of Their Insect Hosts: Effects on Locomotion and on the Expression of a Related Gene.PLoS Neglected Tropical Diseases 9 Trypanosomes Modify the Behavior of Their Insect Hosts: Effects on Locomotion and on the Expression of a Related Gene
Background As a result of evolution, the biology of triatomines must have been significantly adapted to accommodate trypanosome infection in a complex network of vector-vertebrate-parasite interactions. Arthropod-borne parasites have probably developed mechanisms, largely still unknown, to exploit the vector-vertebrate host interactions to ensure their transmission to suitable hosts. Triatomines exhibit a strong negative phototaxis and nocturnal activity, believed to be important for insect survival against its predators. Methodology/Principal Findings In this study we quantified phototaxis and locomotion in starved fifth instar nymphs of Rhodnius prolixus infected with Trypanosoma cruzi or Trypanosoma rangeli. T. cruzi infection did not alter insect phototaxis, but induced an overall 20% decrease in the number of bug locomotory events. Furthermore, the significant differences induced by this parasite were concentrated at the beginning of the scotophase. Conversely, T. rangeli modified both behaviors, as it significantly decreased bug negative phototaxis, while it induced a 23% increase in the number of locomotory events in infected bugs. In this case, the significant effects were observed during the photophase. We also investigated the expression of Rpfor, the triatomine ortholog of the foraging gene known to modulate locomotion in other insects, and found a 4.8 fold increase for T. rangeli infected insects. Conclusions/Significance We demonstrated for the first time that trypanosome infection modulates the locomotory activity of the invertebrate host. T. rangeli infection seems to be more broadly effective, as besides affecting the intensity of locomotion this parasite also diminished negative phototaxis and the expression of a behavior-associated gene in the triatomine vector. DOI
25. 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, JMC; Sorgine, MHF; 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, GRC; Calderon-Fernandez, G; Carareto, CMA; Christensen, MB; Costa, IR; Costa, SG; Dansa, M; Daumas, CRO; 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, DST; 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, ACA; Missirlis, F; Mota, T; Noriega, FG; Nouzova, M; Nunes, RD; Oliveira, RLL; 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, ES; Silva-Cardoso, L; Silva-Neto, MAC; Souza-Gomes, M; Sterkel, M; Taracena, ML; Tojo, M; Tu, ZJ; Tubio, JMC; Ursic-Bedoya, R; Venancio, TM; Walter-Nuno, AB; Wilson, D; Warren, WC; Wilson, RK; Huebner, E; Dotson, EM; Oliveira, PL. (2015) Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection.Proceedings of the National Academy of Sciences of the United States of America 112: 14936-14941 Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection
Rhodnius prolixus; genome; hematophagy; immunity; Chagas disease
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 (similar to 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. DOI
24. Taracena, ML; Oliveira, PL; Almendares, O; Umana, C; Lowenberger, C; Dotson, EM; Paiva-Silva, GO; Pennington, PM. (2015) Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi.PLoS Neglected Tropical Diseases 9 Genetically Modifying the Insect Gut Microbiota to Control Chagas Disease Vectors through Systemic RNAi
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 10(7) 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. DOI
23. Caicedo, PA; Baron, OL; Perez, M; Alexander, N; Lowenberger, C; Ocampo, CB. (2013) Selection of Aedes aegypti (Diptera: Culicidae) strains that are susceptible or refractory to Dengue-2 virus.Canadian Entomologist 145: 273-282 Selection of Aedes aegypti (Diptera: Culicidae) strains that are susceptible or refractory to Dengue-2 virus
QUANTITATIVE TRAIT LOCI; VECTOR COMPETENCE; GEOGRAPHIC STRAINS; ORAL INFECTION; TRANSMISSION; FECUNDITY; GENETICS
The vector competence (VC) of Aedes aegypti (Linnaeus) (Diptera: Culicidae) varies geographically and is affected by both genetic and environmental factors. Understanding the molecular mechanisms that influence VC may help develop novel control strategies. The selection of susceptible and refractory strains is the first step in this process. We collected immature A. aegypti in the field and established strains that were susceptible and refractory to Dengue-2 virus by isofamily selection through several generations. Infection was detected by immunofluorescence of head or midgut tissues to determine infection barriers and the % of VC by tissue. We selected three strains: Susceptible (Cali-S) (96.4% susceptible at F-19), Refractory with a midgut escape barrier (Cali-MEB) (44.1% refractory at F-15), and Refractory with a midgut infection barrier (Cali-MIB) (40% refractory at F-16). The effects of the infection were measured using Kaplan-Meier survival rates over the first seven generations. All selected strains showed a similar decrease in survival and in the number of eggs laid/female through the seven generations, suggesting that changes were a result of the selection process rather than the virus infection. The results of this study suggest that VC is associated with multiple genes, which have additive effects on susceptibility. DOI
22. Ocampo, CB; Caicedo, PA; Jaramillo, G; Bedoya, RU; Baron, O; Serrato, IM; Cooper, DM; Lowenberger, C. (2013) Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus.PLOS One 8(4): e61187. doi:10.1371/journal.pone.0061187 Differential Expression of Apoptosis Related Genes in Selected Strains of Aedes aegypti with Different Susceptibilities to Dengue Virus
YELLOW-FEVER MOSQUITO; EQUINE ENCEPHALOMYELITIS VIRUS; QUANTITATIVE TRAIT LOCI; VECTOR COMPETENCE; RNA INTERFERENCE; INNATE IMMUNITY; DROSOPHILA-MELANOGASTER; ORAL INFECTION; CULICIDAE; DIPTERA
Aedes aegypti is the principal vector of Dengue viruses worldwide. We identified field collected insects with differential susceptibility to Dengue-2 virus (DENv-2) and used isofemale selection to establish susceptible and refractory strains based on midgut infection barriers. Previous experiments had identified higher expression of apoptosis-related genes in the refractory strain. To identify potential molecular mechanisms associated with DENv susceptibility, we evaluated the differential expression of Caspase-16, Aedronc, Aedredd, Inhibitor of apoptosis (AeIAP1) and one member of the RNAi pathway, Argonaute-2 in the midguts and fat body tissues of the selected strains at specific times post blood feeding or infection with DENv-2. In the refractory strain there was significantly increased expression of caspases in midgut and fatbody tissues in the presence of DENv-2, compared to exposure to blood alone, and significantly higher caspase expression in the refractory strain compared with the susceptible strain at timepoints when DENv was establishing in these tissues. We used RNAi to knockdown gene expression; knockdown of AeIAP1 was lethal to the insects. In the refractory strain, knockdown of the pro-apoptotic gene Aedronc increased the susceptibility of refractory insects to DENv-2 from 53% to 78% suggesting a contributing role of this gene in the innate immune response of the refractory strain. DOI
21. Mitchell-Foster, K; Ma, BO; Warsame-Ali, S; Logan, C; Rau, ME; Lowenberger, C. (2012) The influence of larval density, food stress, and parasitism on the bionomics of the dengue vector Aedes aegypti (Diptera: Culicidae): implications for integrated vector management.Journal of Vector Ecology 37: 221-229 The influence of larval density, food stress, and parasitism on the bionomics of the dengue vector Aedes aegypti (Diptera: Culicidae): implications for integrated vector management
Aedes aegypti; Plagiorchis elegans; fitness consequences; biocontrol
New larval control strategies for integrated vector management of Aedes aegypti are in high demand, including the use of biological control agents. Exposure of Aedes aegypti to parasites, starvation, and overcrowded conditions during larval development reduces the probability of survival to eclosion, can directly affect fitness parameters such as adult size and fecundity, and can affect the size, provisioning, and viability of eggs produced by females. We compared these parameters after exposing larvae to 1) abundant food at low larval densities, 2) food deprivation and high larval density, and 2) infection with the endoparasite Plagiorchis elegans, an entomopathogenic digenean trematode. Female mosquitoes that eclosed from larval conditions of starvation and overcrowding were smaller and laid fewer and smaller eggs than controls. The proportion of females to complete an oviposition cycle was reduced in the P. elegans-infected treatment group. Parasite load was negatively correlated with wing length and egg size. Infection of Ae. aegypti with P. elegans has sublethal effects and may reduce population-level reproductive output, but one-time low-density P. elegans exposure does not have sufficient effect on Ae. aegypti fitness parameters to be considered a viable biocontrol option. DOI
20. Pacheco, MA; Reid, MJC; Schillaci, MA; Lowenberger, CA; Galdikas, BMF; Jones-Engel, L; Escalante, AA. (2012) The Origin of Malarial Parasites in Orangutans.PLOS One 7(4): e34990. Epub Apr 20 The Origin of Malarial Parasites in Orangutans
Background: Recent findings of Plasmodium in African apes have changed our perspectives on the evolution of malarial parasites in hominids. However, phylogenetic analyses of primate malarias are still missing information from Southeast Asian apes. In this study, we report molecular data for a malaria parasite lineage found in orangutans. Methodology/Principal Findings: We screened twenty-four blood samples from Pongo pygmaeus (Kalimantan, Indonesia) for Plasmodium parasites by PCR. For all the malaria positive orangutan samples, parasite mitochondrial genomes (mtDNA) and two antigens: merozoite surface protein 1 42 kDa (MSP-1(42)) and circumsporozoite protein gene (CSP) were amplified, cloned, and sequenced. Fifteen orangutans tested positive and yielded 5 distinct mitochondrial haplotypes not previously found. The haplotypes detected exhibited low genetic divergence among them, indicating that they belong to one species. We report phylogenetic analyses using mitochondrial genomes, MSP-1(42) and CSP. We found that the orangutan malaria parasite lineage was part of a monophyletic group that includes all the known non-human primate malaria parasites found in Southeast Asia; specifically, it shares a recent common ancestor with P. inui (a macaque parasite) and P. hylobati (a gibbon parasite) suggesting that this lineage originated as a result of a host switch. The genetic diversity of MSP-1(42) in orangutans seems to be under negative selection. This result is similar to previous findings in non-human primate malarias closely related to P. vivax. As has been previously observed in the other Plasmodium species found in non-human primates, the CSP shows high polymorphism in the number of repeats. However, it has clearly distinctive motifs from those previously found in other malarial parasites. Conclusion: The evidence available from Asian apes indicates that these parasites originated independently from those found in Africa, likely as the result of host switches from other non-human primates. DOI
19. Waliwitiya, R; Belton, P; Nicholson, RA; Lowenberger, CA. (2012) Plant Terpenoids: Acute Toxicities and Effects on Flight Motor Activity and Wing Beat Frequency in the Blow Fly Phaenicia sericata.Journal of Economic Entomology 105: 72-84 Plant Terpenoids: Acute Toxicities and Effects on Flight Motor Activity and Wing Beat Frequency in the Blow Fly Phaenicia sericata
flight muscles; eugenol; pulegone; alpha-terpineol; citronellal
We evaluated the acute toxicities and the physiological effects of plant monoterpenoids (eugenol, pulegone, citronellal and alpha-terpineol) and neuroactive insecticides (malathion, dieldrin and RH3421) on flight muscle impulses (FMI) and wing beat signals (WBS) of the blow fly (Phaenicia sericata). Topically-applied eugenol, pulegone, citronellal, and alpha-terpineol produced neurotoxic symptoms, but were less toxic than malathion, dieldrin, or RH3421. Topical application of eugenol, pulegone, and citronellal reduced spike amplitude in one of the two banks of blow fly dorsolongitudinal flight muscles within 6-8 min, but with citronellal, the amplitude of FMIs reverted to a normal pattern within 1 hr. In contrast to pulegone and citronellal, where impulse frequency remained relatively constant, eugenol caused a gradual increase, then a decline in the frequency of spikes in each muscle bank. Wing beating was blocked permanently within 6-7 min of administering pulegone or citronellal and within 16 mins with eugenol. alpha-Terpineol-treated blow flies could not beat their wings despite normal FMI patterns. The actions of these monoterpenoids on blow fly flight motor patterns are discussed and compared with those of dieldrin, malathion, RH3421, and a variety of other neuroactive substances we have previously investigated in this system. Eugenol, pulegone and citronellal readily penetrate blow fly cuticle and interfere with flight muscle and/or central nervous function. Although there were differences in the effects of these compounds, they mainly depressed flight-associated responses, and acted similarly to compounds that block sodium channels and facilitate GABA action. DOI
18. Waliwitiya, R; Nicholson, RA; Kennedy, CJ; Lowenberger, CA. (2012) The Synergistic Effects of Insecticidal Essential Oils and Piperonyl Butoxide on Biotransformational Enzyme Activities in Aedes aegypti (Diptera: Culicidae).Journal of Medical Entomology 49: 614-623 The Synergistic Effects of Insecticidal Essential Oils and Piperonyl Butoxide on Biotransformational Enzyme Activities in Aedes aegypti (Diptera: Culicidae)
ethoxyresorufin O-deethylase; glutathione S-transferase; esterase; essential oil; Ae. Aegypti
The biochemical mechanisms underlying the increased toxicity of several plant essential oils (thymol, eugenol, pulegone, terpineol, and citronellal) against fourth instar of Aedes aegypti L. when exposed simultaneously with piperonyl butoxide (PBO) were examined. Whole body biotransformational enzyme activities including cytochrome P450-mediated oxidation (ethoxyresorufin O-dethylase [EROD]), glutathione S-transferase (GST), and beta-esterase activity were measured in control, essential oil-exposed only (single chemical), and essential oil + PBO (10 mg/liter) exposed larvae. At high concentrations, thymol, eugenol, pulegone, and citronellal alone reduced EROD activity by 5-25% 16 h postexposure. Terpineol at 10 mg/liter increased EROD activity by 5 +/- 1.8% over controls. The essential oils alone reduced GST activity by 3-20% but PBO exposure alone did not significantly affect the activity of any of the measured enzymes. All essential oils in combination with PBO reduced EROD activity by 58-76% and reduced GST activity by 3-85% at 16 h postexposure. This study indicates a synergistic interaction between essential oils and PBO in inhibiting the cytochrome P450 and GST detoxification enzymes in Ae. aegypti. DOI
17. Ursic-Bedoya, R; Buchhop, J; Joy, JB; Durvasula, R; Lowenberger, C. (2011) Prolixicin: a novel antimicrobial peptide isolated from Rhodnius prolixus with differential activity against bacteria and Trypanosoma cruzi.Insect Molecular Biology 20: 775-786 Prolixicin: a novel antimicrobial peptide isolated from Rhodnius prolixus with differential activity against bacteria and Trypanosoma cruzi
hemiptera; innate immunity; antimicrobial peptide; attacin; diptericin; Trypanosoma cruzi
We identified and characterized the activity of prolixicin, a novel antimicrobial peptide (AMP) isolated from the hemipteran insect, Rhodnius prolixus. Sequence analysis reveals one region of prolixicin that may be related to the diptericin/attacin family of AMPs. Prolixicin is an 11-kDa peptide containing a putative 21 amino acid signal peptide, two putative phosphorylation sites and no glycosylation sites. It is produced by both adult fat body and midgut tissues in response to bacterial infection of the haemolymph or the midgut. Unlike most insect antibacterial peptides, the prolixicin gene does not seem to be regulated by NF-kappa B binding sites, but its promoter region contains several GATA sites. Recombinant prolixicin has strong activity against the Gram-negative bacterium Escherichia coli and differential activity against several Gramnegative and Gram-positive bacteria. No significant toxicity was demonstrated against Trypanosoma cruzi, the human parasite transmitted by R. prolixus. DOI
16. Baron, OL; Ursic-Bedoya, RJ; Lowenberger, CA; Ocampo, CB. (2010) Differential gene expression from midguts of refractory and susceptible lines of the mosquito, Aedes aegypti, infected with Dengue-2 virus.Journal of Insect Science 10: 41 Differential gene expression from midguts of refractory and susceptible lines of the mosquito, Aedes aegypti, infected with Dengue-2 virus
suppressive subtractive hybridization
Suppressive subtractive hybridization was used to evaluate the differential expression of midgut genes of feral populations of Aedes aegypti (Diptera: Culicidae) from Colombia that are naturally refractory or susceptible to Dengue-2 virus infection. A total of 165 differentially expressed sequence tags (ESTs) were identified in the subtracted libraries. The analysis showed a higher number of differentially expressed genes in the susceptible Ae. aegypti individuals than the refractory mosquitoes. The functional annotation of ESTs revealed a broad response in the susceptible library that included immune molecules, metabolic molecules and transcription factors. In the refractory strain, there was the presence of a trypsin inhibitor gene, which could play a role in the infection. These results serve as a template for more detailed studies aiming to characterize the genetic components of refractoriness, which in turn can be used to devise new approaches to combat transmission of dengue fever.Website DOI
15. Waliwitiya, R; Belton, P; Nicholson, RA; Lowenberger, CA. (2010) Effects of the essential oil constituent thymol and other neuroactive chemicals on flight motor activity and wing beat frequency in the blowfly Phaenicia sericata.Pest Management Science 66: 277-289 Effects of the essential oil constituent thymol and other neuroactive chemicals on flight motor activity and wing beat frequency in the blowfly Phaenicia sericata
flight motor activity; wing beat; thymol; GABA
BACKGROUND: The effects were evaluated of the plant terpenoid thymol and eight other neuroactive compounds on flight muscle impulses (FMIs) and wing beat frequency (WBF) of tethered blowflies (Phaenicia sericata Meig.). RESULTS: The electrical activity of the dorsolongitudinal flight muscles was closely linked to the WBF of control insects. Topically applied thymol inhibited WBF within 15-30 min and reduced FMI frequency. Octopamine and chlordimeform caused a similar, early-onset bursting pattern that decreased in amplitude with time. Desmethylchlordimeform blocked wing beating within 60 min and generated a profile of continuous but lower-frequency FMIs. Fipronil suppressed wing beating and induced a pattern of continuous, variable-frequency spiking that diminished gradually over 6 h. Cypermethrin- and rotenone-treated flies had initial strong FMIs that declined with time. In flies injected with GABA, the FMIs were generally unidirectional and frequency was reduced, as was seen with thymol. CONCLUSIONS: Thymol readily penetrates the cuticle and interferes with flight muscle and central nervous function in the blowfly. The similarity of the action of thymol and GABA suggests that this terpenoid acts centrally in blowflies by mimicking or facilitating GABA action. (C) 2009 Society of Chemical Industry DOI
14. Cooper, DM; Chamberlain, CM; Lowenberger, C. (2009) Aedes FADD: A novel death domain-containing protein required for antibacterial immunity in the yellow fever mosquito, Aedes aegypti.Insect Biochemistry and Molecular Biology 39: 47-54 Aedes FADD: A novel death domain-containing protein required for antibacterial immunity in the yellow fever mosquito, Aedes aegypti
KAPPA-B KINASE; DROSOPHILA-MELANOGASTER; SIGNALING PATHWAYS; ANOPHELES-GAMBIAE; INSECT IMMUNITY; FACTOR RELISH; EXPRESSION; CASPASE; INFECTION; APOPTOSIS
Microbial infections in insects activate a series of immune responses that culminate in the production of antimicrobial peptides (AMPS). In Drosophila, two signaling pathways, govern the challenge-dependent expression of AMPS; the Toll and IMD pathways. While AMPS have been the subject of much research in mosquitoes, the regulation of the pathways required for AMP expression remains largely unknown. We report here the identification of Aedes FADD (AeFADD), a death domain protein in Aedes aegypti. AeFadd is expressed in all immune-competent tissues and all developmental stages examined. At the transcriptional level, AeFadd transcripts increased when challenged with Escherichia coli but not Micrococcus luteus. In both cases, we observed the induction of two AMP genes; cecropin and defensin. Loss of AeFadd function by dsRNA interference impaired the inducible expression of both AMPS, and rendered adult mosquitoes susceptible to both types of bacteria. Identifying molecules that regulate mosquito immunity may help elucidate the factors that contribute to the vectorial capacity and provide insights into general mechanisms that regulate innate immunity. (C) 2008 Elsevier Ltd. All rights reserved. DOI
13. Cooper, DM; Granville, DJ; Lowenberger, C. (2009) The insect caspases.Apoptosis 14: 247-256 The insect caspases
PROGRAMMED CELL-DEATH; YELLOW-FEVER MOSQUITO; DROSOPHILA CASPASE; AEDES-AEGYPTI; COMPENSATORY PROLIFERATION; BACULOVIRUS INHIBITOR; DOMAIN SUPERFAMILY; MOLECULAR-CLONING; IAP PROTEINS; IN-VIVO
Developmental and tissue homeostasis is a delicate balance between cell proliferation and cell death. The activation of caspases, a conserved family of cysteine proteases, is a main event in the initiation and execution of programmed cell death. While caspases have been characterized from many organisms, comparatively little is known about insect caspases. In Drosophila melanogaster, seven caspases have been characterized; three initiators and four effectors. In mosquitoes, several putative caspases have been identified in the genomes of Aedes aegypti and Anopheles gambiae. A small number of caspases have been identified in the Lepidoptera, the flour beetle, Tribolium castaneum, and the pea aphid, Acyrthosiphon pisum. The availability of new insect genome sequences will provide a unique opportunity to examine the caspase family across an evolutionarily diverse phylum and will provide valuable insights into their function and regulation. DOI
12. Ericsson, JD; Janmaat, AF; Lowenberger, C; Myers, JH. (2009) Is decreased generalized immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni?Journal of Invertebrate Pathology 100: 61-67 Is decreased generalized immunity a cost of Bt resistance in cabbage loopers Trichoplusia ni?
INSECTICIDE BACILLUS-THURINGIENSIS; PROPHENOLOXIDASE-ACTIVATING SYSTEM; MOTH EPHESTIA-KUEHNIELLA; CRY1AB TOXIN; PEPTIDOGLYCAN RECOGNITION; ANTIMICROBIAL PEPTIDES; DIAMONDBACK MOTH; DELTA-ENDOTOXIN; AEDES-AEGYPTI; BACTERIA
We studied the immune response to Bacillus thuringiensis kurstaki (Btk) in Susceptible (Bt-RS) and resistant (Bt-R) Trichoplusia ni after exposure to low doses of Btk and injection with Escherichia coli. We measured the levels of resistance, the expression profiles of hemolymph proteins, the phenoloxidase (PO) activity, and the differential number of circulating hemocytes in resistant and susceptible individuals. Individuals from the Bt-RS line became more resistant following a previous exposure to sub lethal concentrations of Btk, but the resistance to Btk of the Bt-R line did not change significantly, Similarly the Bt-R strain showed no significant changes in any of the potential immune responses, hemolymph protein levels or PO activity. The number of circulating hemocytes was significantly lower in the Bt-R strain than in the Bt-RS strain. Exposure to Btk decreased the hemocyte Counts and reduced PO activity of Bt-RS larvae. Hemolymph protein concentrations also declined significantly in the susceptible larvae continually exposed to Btk. Seven peptides with antibacterial activity were identified in the hemolymph of Bt-RS larvae after exposure to Btk and five Were found in the Bt-R larvae. When exposed to a low level Bt challenge the susceptible strain increases in tolerance and there are concomitant reductions in hemolymph protein concentrations, PO activity and the number of circulating hemocytes. (C) 2008 Elsevier Inc. All rights reserved. DOI
11. Plunkett, RM; Murray, SI; Lowenberger, CA. (2009) Generation and characterization of the antibacterial activity of a novel hybrid antimicrobial peptide comprising functional domains from different insect cecropins.Canadian Journal of Microbiology 55: 520-528 Generation and characterization of the antibacterial activity of a novel hybrid antimicrobial peptide comprising functional domains from different insect cecropins
ESCHERICHIA-COLI; GENE-REGULATION; AEDES-AEGYPTI; EXPRESSION; DROSOPHILA; CLONING; MOSQUITO; IMMUNITY; PURIFICATION; HEMOLYMPH
The search for new antimicrobial compounds involves finding novel sources of chemotherapeutic compounds or manipulating and combining structures from existing molecules. Small antimicrobial peptides (AMPs) are components of innate immune defenses characterized in greatest detail in insect-derived AMPs. We have generated hybrid AMPs (hAMPs) by combining functional motifs from different insect AMPs as a proof of principle that we can generate molecules with lower minimum inhibitory concentrations, and with different activity and target specificity than either parent molecule. A two-helix, cecropin-like hAMP was created by linking the N-terminal a helix of cecropin A from Aedes aegypti to the C-terminal a helix of cecropin A1 from Drosophila melanogaster. This molecule exhibits antibacterial activity at sub-micromolar concentrations with a target specificity that differs from either parent molecule. Antibacterial activity of the hybrid molecule was found to be greater against Gram-negative than Gram-positive bacteria. No hemolysis was observed in sheep red blood cells exposed to concentrations up to 50 mu mol/L, suggesting the peptide is not detrimental to eukaryotic cells. DOI
10. Ursic-Bedoya, R; Buchhop, J; Lowenberger, C. (2009) Cloning and characterization of Dorsal homologues in the hemipteran Rhodnius prolixus.Insect Molecular Biology 18: 681-689 Cloning and characterization of Dorsal homologues in the hemipteran Rhodnius prolixus
NF-KAPPA-B; MOSQUITO AEDES-AEGYPTI; IMMUNE-RESPONSE; TRANSCRIPTION FACTOR; ANOPHELES-GAMBIAE; CHAGAS-DISEASE; FACTOR RELISH; DROSOPHILA; GENE; DEFENSE
Rhodnius prolixus is an ancient haematophagous hemipteran insect capable of mounting a powerful immune response. This response is transcriptionally regulated in part by transcription factors of the Rel/Nuclear Factor kappa B (Rel/NF-kappa B) family. We have cloned and characterized three members of this transcription factor family in this insect. Dorsal 1A is primarily expressed in early developmental stages. In contrast, dorsal 1B and 1C, both differentially spliced products of dorsal 1A, are expressed primarily in the adult fat body in response to septic injury, suggesting their exclusive role in immunity. Additionally, we identified putative kappa B binding sites in the 5' upstream regions of target genes known to be involved in the innate immune response of insects. DOI
9. Waliwitiya, R., Kennedy, C., and Lowenberger, C. (2009) Larvicidal and antiovipositional activity of monoterpenoids and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae).Pest Management Science 65: 241-248. Larvicidal and antiovipositional activity of monoterpenoids and rosemary oil to the yellow fever mosquito Aedes aegypti (Diptera: Culicidae)
Aedes aegypti, acute toxicity, essential oils, larvicides, monoterpenoids, oviposition, piperonyl butoxide
Aedes aegypti L. is the major vector of dengue fever and dengue hemorrhagic fever. In an effort to find effective tools for control programs to reduce mosquito populations, the authors assessed the acute toxicities of 14 monoterpenoids, trans-anithole and the essential oil of rosemary against different larval stages of Ae. aegypti. The potential for piperonyl butoxide (PBO) to act as a synergist for these compounds to increase larvicidal activity was also examined, and the oviposition response of gravid Ae. aegypti females to substrates containing these compounds was evaluated in behavioral bioassays.
RESULTS: Pulegone, thymol, eugenol, trans-anithole, rosemary oil and citronellal showed high larvicidal activity against all larval stages of Ae. aegypti (LC50 values 10.3-40.8 mg L-1). The addition of PBO significantly increased the larvicidal activity of all test compounds (3-250-fold). Eugenol, citronellal, thymol, pulegone, rosemary oil and cymene showed oviposition deterrent and/or repellent activities, while the presence of borneol, camphor and -pinene increased the number of eggs laid in test containers.
CONCLUSIONS: This study quantified the lethal and sublethal effects of several phytochemical compounds against all larval stages of Aedes aegypti, providing information that ultimately may have potential in mosquito control programs through acute toxicity and/or the ability to alter reproductive behaviorsWebsite DOI
8. Lardeux, F; Tejerina, R; Aliaga, C; Ursic-Bedoya, R; Lowenberger, C; Chavez, T. (2008) Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies.Transactions of the Royal Society of Tropical Medicine and Hygiene 102: 485-492 Optimization of a semi-nested multiplex PCR to identify Plasmodium parasites in wild-caught Anopheles in Bolivia, and its application to field epidemiological studies
PCR; Anopheles; Plasmodium; prevalence; entomology; Bolivia
Without an adequate DNA extraction protocol, the identification of Plasmodium species in whole mosquitoes by PCR is difficult because of the presence of reaction inhibitors from the insects. In this study, eight DNA extraction protocols were tested, from which a chelex-based protocol was selected. Then a semi-nested multiplex PCR technique that detects and distinguishes among the four human Plasmodium species in single mosquitoes and in pools of up to 100 mosquitoes was optimized. The technique was used to detect P. vivax in wild-caught Anopheles pseudopunctipennis from a village in the Andean valleys of Bolivia in May 2003. The prevalence of infection was 0.9%. This is the first direct evidence of P vivax transmission by this vector in this country. The extraction and PCR technique presented here can be useful to: (1) estimate Plasmodium prevalence in Anopheles populations in low prevalence areas where large numbers of individual mosquitoes would need to be processed to obtain a reliable estimate; (2) incriminate Anopheles species as malaria vectors; (3) identify all the circulating Plasmodium species in vectors from an area; (4) detect mixed infections in mosquitoes; and (5) detect mosquitoes with low-level parasite infections. (C) 2008 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved. DOI
7. Leighton, BJ; Roitberg, BD; Belton, P; Lowenberger, CA. (2008) Host antibodies in mosquito bloodmeals: A potential tool to detect and monitor infectious diseases in wildlife.Journal of Medical Entomology 45: 470-475 Host antibodies in mosquito bloodmeals: A potential tool to detect and monitor infectious diseases in wildlife
mosquito; enzyme-linked immunosorbent assay; bloodmeal; antibody; wildlife disease
When a female mosquito bites, it carries away a blood sample containing specific antibodies that can provide a history of the immune responses of its vertebrate host. This research examines the limits and reliability of a technique to detect antibodies in blood-fed mosquitoes in the laboratory. Mosquitoes were fed on blood containing a specific antibody, and then they were assayed using an enzyme-linked immunosorbent assay to determine the limits of detection of antibody over time, at different temperatures and initial antibody concentrations. The antibody, at an initial concentration of 1 mu g/ml, could be detected in mosquitoes for 24-48 h after feeding. Blind tests simulating the assay of feral mosquitoes were used to test the reliability of the method and detected positive mosquitoes with few false negatives and no false positives. Specific antibodies also could be detected in mosquitoes that had been air-dried or preserved in ethanol. This research indicates that, in theory, the collection and immunological assay of blood-fed mosquitoes could be developed to detect and monitor infectious disease in wildlife.
6. Ursic-Bedoya, RJ; Nazzari, H; Cooper, D; Triana, O; Wolff, M; Lowenberger, C. (2008) Identification and characterization of two novel lysozymes from Rhodnius prolixus, a vector of Chagas disease.Journal of Insect Physiology 54: 593-603 Identification and characterization of two novel lysozymes from Rhodnius prolixus, a vector of Chagas disease
Rhodnius prolixus; lysozyme; antimicrobial peptides; Chagas disease; innate immunity; Trypanosoma cruzi
Lysozymes have been described in invertebrates as digestive or immune molecules. We report here the characterization of two novel c-type lysozymes, RpLys-A (EU250274) and RpLys-B (EU250275), isolated from the fat body and digestive tract of immune stimulated Rhodnius prolixus, a major vector of Chagas disease. Transcriptional profiles indicate that the temporal and spatial expression patterns of these two peptides are very different. RpLys-A is expressed predominantly in the midgut after ingestion of Trypanosoma cruzi in a bloodmeal, or after injection of bacteria into the hemocoel. RpLys-B is expressed primarily in the fat body after bacterial injection. Phylogenetic alignments indicate that RpLys-A aligns best with molecules from other hemipterans whose major expression is found in the intestinal tract whereas RpLys-B aligns best with mosquito and tick molecules whose expression is found principally in hemocytes and fat body and whose role has been described as immune-related. These data suggest a differential compartmentalized role of two closely related molecules; one for immunity in the hemocoel and the other for digestion in the midgut. (c) 2007 Elsevier Ltd. All rights reserved. DOI
5. Cooper, DM; Pio, F; Thi, EP; Theilmann, D; Lowenberger, C. (2007) Characterization of Aedes Dredd: A novel initiator caspase from the yellow fever mosquito, Aedes aegypti.Insect Biochem. Mol. Biol. 37: 559-569 Characterization of Aedes Dredd: A novel initiator caspase from the yellow fever mosquito, Aedes aegypti
Caspases play an essential role during programmed cell death in all metazoans. These enzymes are cysteine proteases and comprise a multi-gene family with more than a dozen mammalian family members. Although caspases have been characterized in many animals, including Drosophila melanogaster, little is known about the laspases that exist in mosquitoes. Here we describe the identification and characterization of Aedes Dredd (AeDredd), a novel caspase in the yellow fever mosquito, Aedes aegypti. AeDredd contains two N-terminal death effector domains and the well conserved caspase catalytic domain. Multiple sequence alignments and functional substrate assays of recombinant protein Suggest that AcDredd is an orthologue of Drosophila Dredd and human caspase-8, both central effectors of the death receptor-inediated apoptotic pathway. AeDredd exhibits substrate specificity most similar to human caspase-8. AeDredd transcripts were found in all developmental stages with highest expression in early pupae. Within adults, AeDredd was found in all the tissues examined, with the highest transcript levels detected in fat body tissues. This is the first functional characterization of a death domain-containing caspase in in insect vector of human disease, and will initiate Studies on the role of apoptosis in the innate immune response of vectors towards intracellular parasites Such as Viruses. (c) 2007 Elsevier Ltd. All rights reserved. DOI PubMed
4. Cooper, DM; Thi, EP; Chamberlain, CM; Pio, F; Lowenberger, C. (2007) Aedes Dronc: a novel ecdysone-inducible caspase in the yellow fever mosquito, Aedes aegypti.Insect Mol. Biol. 16: 563-572 Aedes Dronc: a novel ecdysone-inducible caspase in the yellow fever mosquito, Aedes aegypti
apoptosis; caspase; Aedes aegypti; ecdysone; development
Caspases are cysteinyl-aspartate-specific proteases known for their role in apoptosis. Here, we describe the characterization of Aedes Dronc, a novel caspase in the yellow fever mosquito, Aedes aegypti. Aedes Dronc is predicted to contain an N-terminal caspase recruitment domain and is a homologue of Drosophila Dronc and human caspase-9. An increase in transcripts and caspase activity coincides with developmental changes in the mosquito, suggesting that Aedes Dronc plays a role in developmental apoptosis. Exposure of third instar larvae to ecdysone resulted in a significant increase in both transcript levels and caspase activity. We present here a functional characterization of the first caspase recruitment domain-containing caspase in mosquitoes, and will initiate studies on the role of apoptosis in the innate immune response of vectors. DOI PubMed
3. Ursic-Bedoya, RJ; Lowenberger, CA. (2007) Rhodnius prolixus: Identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization.Developmental and Comparative Immunology 31: 109-120 Rhodnius prolixus: Identification of immune-related genes up-regulated in response to pathogens and parasites using suppressive subtractive hybridization
Rhodnius prolixus; parasitic infection; innate immunity; suppressive subtractive hybridization; expressed sequence tags
We report the identification of immune-related molecules from the fat body, and intestine of Rhodnius prolixus, an important vector of Chagas disease. Insects were challenged by introducing pathogens or Trypanosoma cruzi, the parasite that causes Chagas disease, into the hemocoel. RNA from intestines, or fat body were isolated 24 h after stimulation. We used suppressive subtractive hybridization to identify immune-related genes, generated three subtracted libraries, sequenced the clones and assembled the sequences. The functional annotation revealed expressed sequence tags (ESTs) generated in response to various stimuli in all tissues, and included pathogen recognition molecules, regulatory molecules, and effector molecules. (c) 2006 Elsevier Ltd. All rights reserved.
2. Boulanger, N; Bulet, P; Lowenberger, C. (2006) Antimicrobial peptides in the interactions between insects and flagellate parasites.Trends Parasitol 22: 262-268 Antimicrobial peptides in the interactions between insects and flagellate parasites
Innate immunity has a key role in the control of microbial infections in both vertebrates and invertebrates. In insects, including vectors that transmit parasites that cause major human and animal diseases, antimicrobial peptides (AMPs) are important components of innate immunity. AMPs are induced upon parasitic infections and can participate in regulating parasite development in the digestive tract and in the hemolymph. This review presents our current knowledge of a field that is in its infancy: the role of innate immunity in different models of insects infected with flagellate parasites, and in particular the potential role of AMPs in regulating these parasitic infections.
1. Reid, MJC; Ursic, R; Cooper, D; Nazzari, H; Griffiths, M; Galdikas, BM; Garriga, RM; Skinner, M; Lowenberger, C. (2006) Transmission of human and macaque Plasmodium spp. to ex-captive Orangutans in Kalimantan, Indonesia.Emerg Infect Dis 12: 1902-1908 Transmission of human and macaque Plasmodium spp. to ex-captive Orangutans in Kalimantan, Indonesia
Data are lacking on the specific diseases to which great apes are susceptible and the transmission dynamics and overall impact of these diseases. We examined the prevalence of Plasmodium spp. infections in semicaptive orangutans housed at the Orangutan Care Center and Quarantine, Central Kalimantan, Indonesia, by using a combination of microscopic and DNA molecular techniques to identify the Plasmodium spp. in each animal. Previous studies indicated 2 orangutan-specific Plasmodium spp., but our data show 4 Plasmodium spp. These findings provide evidence for P vivax transmission between humans and orangutans and for P cynomolgi transmission between macaques and orangutans. These data have potential implications for the conservation of orangutans and also for the bidirectional transmission of parasites between orangutans and humans visiting or living in the region.
