76. Bitencourt, RDB; Salcedo-Porras, N; Umana-Diaz, C; Angelo, ID; Lowenberger, C. (2021) Antifungal immune responses in mosquitoes (Diptera: Culicidae): A review.J. Invertebr. Pathol. 178 Antifungal immune responses in mosquitoes (Diptera: Culicidae): A review
Fungal infection; Biocontrol; Innate immunity; Mosquitoes; Entomopathogenic fungi
Mosquitoes transmit many parasites and pathogens to humans that cause significant morbidity and mortality. As such, we are constantly looking for new methods to reduce mosquito populations, including the use of effective biological controls. Entomopathogenic fungi are excellent candidate biocontrol agents to control mosquitoes. Understanding the complex ecological, environmental, and molecular interactions between hosts and pathogens are essential to create novel, effective and safe biocontrol agents. Understanding how mosquitoes recognize and eliminate pathogens such as entomopathogenic fungi may allow us to create insect-order specific biocontrol agents to reduce pest populations. Here we summarize the current knowledge of fungal infection, colonization, development, and replication within mosquitoes and the innate immune responses of the mosquitoes towards the fungal pathogens, emphasizing those features required for an effective mosquito biocontrol agent. DOI PubMed
75. Coatsworth, H; Caicedo, PA; Winsor, G; Brinkman, F; Ocampo, CB; Lowenberger, C. (2021) Transcriptome comparison of dengue-susceptible and -resistant field derived strains of Colombian Aedes aegypti using RNA-sequencing.Mem. Inst. Oswaldo Cruz 116 Transcriptome comparison of dengue-susceptible and -resistant field derived strains of Colombian Aedes aegypti using RNA-sequencing
dengue; Aedes aegypti; yellow fever mosquito; RNA sequencing; refractory mechanisms; innate immunity
BACKGROUND Forty percent of the world's population live in areas where they are at risk from dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Dengue viruses are transmitted primarily by the mosquito Aedes aegypti. In Cali, Colombia, approximately 30% of field collected Ae. aegypti are naturally refractory to all four dengue serotypes. OBJECTIVES Use RNA-sequencing to identify those genes that determine refractoriness in feral mosquitoes to dengue. This information can be used in gene editing strategies to reduce dengue transmission. METHODS We employed a full factorial design, analyzing differential gene expression across time (24, 36 and 48 h post bloodmeal), feeding treatment (blood or blood + dengue-2) and strain (susceptible or refractory). Sequences were aligned to the reference Ae. aegypti genome for identification, assembled to visualize transcript structure, and analyzed for dynamic gene expression changes. A variety of clustering techniques was used to identify the differentially expressed genes. FINDINGS We identified a subset of genes that likely assist dengue entry and replication in susceptible mosquitoes and contribute to vector competence. MAIN CONCLUSIONS The differential expression of specific genes by refractory and susceptible mosquitoes could determine the phenotype, and may be used to in gene editing strategies to reduce dengue transmission. DOI PubMed
74. Meraj, S; Mohr, E; Ketabchi, N; Bogdanovic, A; Lowenberger, C; Gries, G. (2021) Time- and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection.J. Insect Physiol. 135 Time- and tissue-specific antimicrobial activity of the common bed bug in response to blood feeding and immune activation by bacterial injection
Bed bug; Aedes aegypti; Midgut; Antimicrobial peptide activity; Immune activation; Blood feeding
Unlike almost all hematophagous insects, common bed bugs, Cimex lectularius, are not known to transmit pathogens to humans. To help unravel the reasons for their lack of vector competence, we studied the time-and tissue-dependent expression of innate immune factors after blood feeding or immune activation through the intrathoracic injection of bacteria. We used minimum inhibitory concentration (MIC1) bioassays and the Kirby Bauer protocol to evaluate antimicrobial peptide (AMP(2)) activity in tissue extracts from the midguts or 'rest of body' (RoB(3)) tissues (containing hemolymph and fat body AMPs) against Gram-positive and Gram-negative bacteria. We compared AMP activity between blood-fed female bed bugs and yellow fever mosquitoes, Aedes aegypti and determined how female and male bed bugs respond to immune challenges, and how long AMP gene expression remains elevated in bed bugs following a blood meal. Blood meal-induced AMP activity is 4-fold stronger in female bed bugs than in female mosquitoes. Male bed bugs have elevated AMP activity within 8 h of a blood meal or an intrathoracic injection with bacteria, with the strongest activity expressed in RoB tissue 24 h after the immune challenge. Female bed bugs have a stronger immune response than males within 24 h of a blood meal. The effects of blood meal-induced elevated AMP activity lasts longer against the Gram-positive bacterium, Bacillus subtilis, than against the Gram-negative bacterium Escherichia coli. Unravelling the specific immune pathways that are activated in the bed bugs' immune responses and identifying the bed bug-unique AMPs might help determine why these insects are not vectors of human parasites. DOI PubMed
72. Reid, MJC; Switzer, WM; Alonso, SK; Lowenberger, CA; Schillaci, MA. (2021) Evolutionary history of orangutan plasmodia revealed by phylogenetic analysis of complete mtDNA genomes and new biogeographical divergence dating calibration models.American Journal of Primatology Jul 5:e23298. Epub 2021 Jul 5. Evolutionary history of orangutan plasmodia revealed by phylogenetic analysis of complete mtDNA genomes and new biogeographical divergence dating calibration models
Bayesian models; malaria; orangutan; parasites; Plasmodium
During the past 15 years, researchers have shown a renewed interest in the study of the Plasmodium parasites that infect orangutans. Most recently, studies examined the phylogenetic relationships and divergence dates of these parasites in orangutans using complete mitochondrial DNA genomes. Questions regarding the dating of these parasites, however, remain. In the present study, we provide a new calibration model for dating the origins of Plasmodium parasites in orangutans using a modified date range for the origin of macaques in Asia. Our Bayesian phylogenetic analyses of complete Plasmodium sp. mitochondrial DNA genomes inferred two clades of plasmodia in orangutans (Pongo 1 and Pongo 2), and that these clades likely represent the previously identified species Plasmodium pitheci and Plasmodium silvaticum. However, we cannot identify which Pongo clade is representative of the morphologically described species. The most recent common ancestor of both Pongo sp. plasmodia, Plasmodium. hylobati, and Plasmodium. inui dates to 3-3.16 million years ago (mya) (95% highest posterior density [HPD]: 2.09-4.08 mya). The Pongo 1 parasite diversified 0.33-0.36 mya (95% HPD: 0.12-0.63), while the Pongo 2 parasite diversified 1.15-1.22 mya (95% HPD: 0.63-1.82 mya). It now seems likely that the monkey Plasmodium (P. inui) is the result of a host switch event from the Pongo 2 parasite to sympatric monkeys, or P. hylobati. Our new estimates for the divergence of orangutan malaria parasites, and subsequent diversification, are all several hundred thousand years later than previous Bayesian estimates. DOI PubMed
69. 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
68. Baharmand, I; Coatsworth, H; Peach, DAH; Belton, P; Lowenberger, C. (2020) Molecular relationships of introduced Aedes japonicus (Diptera: Culicidae) populations in British Columbia, Canada using mitochondrial DNA.J. Vector Ecol. 45: 285-296 Molecular relationships of introduced Aedes japonicus (Diptera: Culicidae) populations in British Columbia, Canada using mitochondrial DNA
Aedes japonicus; mitochondrial DNA; barcoding; polymorphism; cytochrome c oxidase 1; invasive species
Aedes japonicus japonicus (Theobald) is a relatively recent immigrant to the Pacific Northwest, having been collected in Washington State in 2001 and in British Columbia (BC) since 2014. We applied a molecular barcoding approach to determine the phylogenetic relationship of Ae. j. japonicus populations in BC with those from around the world. We sequenced a 617 base-pair segment of the cytochrome c oxidase 1 gene and a 330 base-pair region of the NADH dehydrogenase 4 gene to find genetic variation and characterize phylogenetic and haplotypic relationships based on nucleotide divergences. Our results revealed low genetic diversity in the BC samples, suggesting that these populations arose from the same introduction event. However, our approach lacked the granularity to identify the exact country of origin of the Ae. j. japonicus collected in BC. Future efforts should focus on detecting and preventing new Ae. j. japonicus introductions, recognizing that current molecular techniques are unable to pin-point the precise source of an introduction. PubMed
66. Salcedo-Porras, N; Umana-Diaz, C; Bitencourt, RDB; Lowenberger, C. (2020) The Role of Bacterial Symbionts in Triatomines: An Evolutionary Perspective.Microorganisms 8 The Role of Bacterial Symbionts in Triatomines: An Evolutionary Perspective
microbiota; Chagas disease; triatomines; microbiome; symbiosis; Trypanosoma; Hemiptera
Insects have established mutualistic symbiotic interactions with microorganisms that are beneficial to both host and symbiont. Many insects have exploited these symbioses to diversify and expand their ecological ranges. In the Hemiptera (i.e., aphids, cicadas, and true bugs), symbioses have established and evolved with obligatory essential microorganisms (primary symbionts) and with facultative beneficial symbionts (secondary symbionts). Primary symbionts are usually intracellular microorganisms found in insects with specialized diets such as obligate hematophagy or phytophagy. Most Heteroptera (true bugs), however, have gastrointestinal (GI) tract extracellular symbionts with functions analogous to primary endosymbionts. The triatomines, are vectors of the human parasite, Trypanosoma cruzi. A description of their small GI tract microbiota richness was based on a few culturable microorganisms first described almost a century ago. A growing literature describes more complex interactions between triatomines and bacteria with properties characteristic of both primary and secondary symbionts. In this review, we provide an evolutionary perspective of beneficial symbioses in the Hemiptera, illustrating the context that may drive the evolution of symbioses in triatomines. We highlight the diversity of the triatomine microbiota, bacterial taxa with potential to be beneficial symbionts, the unique characteristics of triatomine-bacteria symbioses, and the interactions among trypanosomes, microbiota, and triatomines. DOI PubMed
64. 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
63. 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
62. 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
61. Salcedo-Porras, N; Lowenberger, C. (2019) The innate immune system of kissing bugs, vectors of chagas disease.Dev. Comp. Immunol. 98: 119-128 The innate immune system of kissing bugs, vectors of chagas disease
Rhodnius prolixus; Triatomine; Kissing bug; Innate immunity; AMPs; Trypanosoma cruzi; Trypanosoma rangeli; Microbiome
Kissing bugs have long served as models to study many aspects of insect physiology. They also serve as vectors for the parasite Trypanosoma cruzi that causes Chagas disease in humans. The overall success of insects is due, in part, to their ability to recognize parasites and pathogens as non-self and to eliminate them using their innate immune system. This immune system comprises physical barriers, cellular responses (phagocytosis, nodulation and encapsulation), and humoral factors (antimicrobial peptides and the prophenoloxidase cascade). Trypanosomn cruzi survives solely in the gastrointestinal (GI) tract of the vector; if it migrates to the hemocoel it is eliminated. Kissing bugs may not mount a vigorous immune response in the GI tract to avoid eliminating obligate symbiotic microbes on which they rely for survival. Here we describe the current knowledge of innate immunity in kissing bugs and new opportunities using genomic and transcriptomic approaches to study the complex triatomine-trypanosome-microbiome interactions. DOI PubMed
60. Scholefield, JA; Shikano, I; Lowenberger, CA; Cory, JS. (2019) The impact of baculovirus challenge on immunity: The effect of dose and time after infection.J. Invertebr. Pathol. 167 The impact of baculovirus challenge on immunity: The effect of dose and time after infection
Nucleopolyhedrovirus; Cabbage looper; Immunity; Haemocytes; Phenoloxidase; Glucose dehydrogenase; Dose-response
Understanding how hosts respond to pathogen attack is crucial to disease management. The response of a host can be particularly important if hosts have to defend against multiple pathogens which could either benefit from or be suppressed by prior pathogen exposure. Insect defence against viruses is less well understood than responses to other entomopathogens and much of the information available relates to in vitro studies and model systems. Baculoviruses are natural pathogens of insects, particularly Lepidoptera, and have been well-studied in terms of their ecology, pest control potential and molecular biology. In order to examine how an insect reacts to baculovirus challenge, we measured components of the cellular and humoral immune response of the cabbage looper Trichoplusia ni to Trichoplusia ni SNPV, a narrow-host range nucleopolyhedrovirus (NPV), over four doses and three times after pathogen challenge (18, 42 and 90 h). We found that total haemocyte numbers peaked at 42 h post-exposure at all doses, and declined linearly with increasing dose after the 18 h time point. Two immune-related enzymes, phenoloxidase (PO) and FAD-glucose dehydrogenase (GLD), showed very different responses. PO levels were lowest at the 42 h time point and were not influenced by virus dose when each time point was examined separately. GLD levels declined over time but they interacted with virus dose in a non-linear manner, such that there was an increase in levels at intermediate virus doses after 18 h, no effect at 42 h, and then declined as infection progressed at 90 h post-infection. These data suggest that baculoviruses can rapidly infect haemocytes (or cause a reduction in their numbers) in a dose-dependent manner once the infection is systemic, likely reducing the ability of the host to counter subsequent infections. However, the data do not support a direct role for PO in defence against baculoviruses. Whether GLD plays a role in virus defence is still unclear. DOI PubMed
59. 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
58. 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
57. Gill, C; Bahrndorff, S; Lowenberger, C. (2017) Campylobacter jejuni in Musca domestica: An examination of survival and transmission potential in light of the innate immune responses of the house flies.Insect Sci. 24: 584-598 Campylobacter jejuni in Musca domestica: An examination of survival and transmission potential in light of the innate immune responses of the house flies
antimicrobial peptides; Campylobacter spp.; Campylobacteriosis; house fly; innate immunity; Musca domestica; suppression subtractive hybridization (SSH); vector
The house fly, Musca domestica, has been implicated as a vector of Campylobacter spp., a major cause of human disease. Little is known whether house flies serve as biological amplifying hosts or mechanical vectors for Campylobacter jejuni. We investigated the period after C. jejuni had been ingested by house flies in which viable C. jejuni colonies could be isolated from whole bodies, the vomitus and the excreta of adult M. domestica and evaluated the activation of innate immune responses of house flies to ingested C. jejuni over time. C. jejuni could be cultured from infected houseflies soon after ingestion but no countable C. jejuni colonies were observed > 24 h postingestion. We detected viable C. jejuni in house fly vomitus and excreta up to 4 h after ingestion, but no viable bacteria were detected >= 8 h. Suppression subtractive hybridization identified pathogen-induced gene expression in the intestinal tracts of adult house flies 4-24 h after ingesting C. jejuni. We measured the expression of immune regulatory (thor, JNK, and spheroide) and effector (cecropin, diptericin, attacin, defensing, and lysozyme) genes in C. jejuni-infected and -uninfected house flies using quantitative real time PCR. Some house fly factor, or combination of factors, eliminates C. jejuni within 24 h postingestion. Because C. jejuni is not amplified within the body of the housefly, this insect likely serves as a mechanical vector rather than as a true biological, amplifying vector for C. jejuni, and adds to our understanding of insect-pathogen interactions. DOI
56. Reid, MJC; Switzer, WM; Schillaci, MA; Klegarth, AR; Campbell, E; Ragonnet-Cronin, M; Joanisse, I; Caminiti, K; Lowenberger, CA; Galdikas, BMF; Hollocher, H; Sandstrom, PA; Brooks, JI. (2017) Bayesian inference reveals ancient origin of simian foamy virus in orangutans.Infect. Genet. Evol. 51: 54-66 Bayesian inference reveals ancient origin of simian foamy virus in orangutans
Pongo; SFV; Co-evolution; Paleobiogeography; Paleovirology; Mt. Toba; Orangutans
Simian foamy viruses (SFVs) infect most nonhuman primate species and appears to co-evolve with its hosts. This co-evolutionary signal is particularly strong among great apes, including orangutans (genus Pongo). Previous studies have identified three distinct orangutan SFV clades. The first of these three clades is composed of SFV from P. abelii from Sumatra, the second consists of SW from P. pygmaeus from Borneo, while the third Glade is mixed, comprising an SW strain found in both species of orangutan. The existence of the mixed clade has been attributed to an expansion of P. pygmaeus into Sumatra following the Mount Toba super-volcanic eruption about 73,000 years ago. Divergence dating, however, has yet to be performed to establish a temporal association with the Toba eruption. Here, we use a Bayesian framework and a relaxed molecular clock model with fossil calibrations to test the Toba hypothesis and to gain a more complete understanding of the evolutionary history of orangutan SW. As with previous studies, our results show a similar three-Glade orangutan SW phylogeny, along with strong statistical support for SW-host co-evolution in orangutans. Using Bayesian inference, we date the origin of orangutan SW to >4.7 million years ago (mya), while the mixed species Glade dates to approximately 1.7 mya, >1.6 million years older than the Toba super-eruption. These results, combined with fossil and paleogeographic evidence, suggest that the origin of SW in Sumatran and Bornean orangutans, including the mixed species Glade, likely occurred on the mainland of Indo-China during the Late Pliocene and Calabrian stage of the Pleistocene, respectively. (C) 2017 Elsevier B.V. All rights reserved. DOI
55. 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
54. 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
53. 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, H; 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; Lange, AB; 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; Oliveir, RLL; Oliveira-Silveira, G; Ons, S; Orchard, I; 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, 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. (2016) Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection (vol 112, pg 14936, 2015).Proceedings of the National Academy of Sciences of the United States of America 113: E1415-E1416 Genome of Rhodnius prolixus, an insect vector of Chagas disease, reveals unique adaptations to hematophagy and parasite infection (vol 112, pg 14936, 2015)
DOI
52. 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
51. Reid, MJC; Switzer, WM; Schillaci, MA; Ragonnet-Cronin, M; Joanisse, I; Caminiti, K; Lowenberger, CA; Galdikas, BMF; Sandstrom, PA; Brooks, JI. (2016) Detailed phylogenetic analysis of primate T-lymphotropic virus type 1 (PTLV-1) sequences from orangutans (Pongo pygmaeus) reveals new insights into the evolutionary history of PTLV-1 in Asia.Infection Genetics and Evolution 43: 434-450 Detailed phylogenetic analysis of primate T-lymphotropic virus type 1 (PTLV-1) sequences from orangutans (Pongo pygmaeus) reveals new insights into the evolutionary history of PTLV-1 in Asia
Orangutans; Pongo; PTLV-1; STLV-1; HTLV-1; Australasia; Cross-species transmission; Paleovirology
While human T-lymphotropic virus type 1 (HTLV-1) originates fromancient cross-species transmission of simian T-lymphotropic virus type 1 (STLV-1) from infected nonhuman primates, much debate exists on whether the first HTLV-1 occurred in Africa, or in Asia during early human evolution and migration. This topic is complicated by a lack of representative Asian STLV-1 to infer PTLV-1 evolutionary histories. In this study we obtained new STLV-1 LTR and tax sequences from a wild-born Bornean orangutan (Pongo pygmaeus) and performed detailed phylogenetic analyses using both maximum likelihood and Bayesian inference of available Asian PTLV-1 and African STLV-1 sequences. Phylogenies, divergence dates and nucleotide substitution rates were co-inferred and compared using six different molecular clock calibrations in a Bayesian framework, including both archaeological and/or nucleotide substitution rate calibrations. We then combined our molecular results with paleobiogeographical and ecological data to infer the most likely evolutionary history of PTLV-1. Based on the preferred models our analyses robustly inferred an Asian source for PTLV-1 with cross-species transmission of STLV-1 likely from a macaque (Macaca sp.) to an orangutan about 37.9-48.9 kya, and to humans between 20.3-25.5 kya. An orangutan diversification of STLV-1 commenced approximately 6.4-7.3 kya. Our analyses also inferred that HTLV-1 was first introduced into Australia similar to 3.1-3.7 kya, corresponding to both genetic and archaeological changes occurring in Australia at that time. Finally, HTLV-1 appears in Melanesia at similar to 2.3-2.7 kya corresponding to the migration of the Lapita peoples into the region. Our results also provide an important future reference for calibrating information essential for PTLV evolutionary timescale inference. Longer sequence data, or full genomes from a greater representation of Asian primates, including gibbons, leaf monkeys, and Sumatran orangutans are needed to fully elucidate these evolutionary dates and relationships using the model criteria suggested herein. (C) 2016 Elsevier B.V. All rights reserved. DOI
50. 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
49. 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
48. 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
47. Bahrndorff, S; Gill, C; Lowenberger, C; Skovgard, H; Hald, B. (2014) The Effects of Temperature and Innate Immunity on Transmission of Campylobacter jejuni (Campylobacterales: Campylobacteraceae) Between Life Stages of Musca domestica (Diptera: Muscidae).Journal of Medical Entomology 51: 670-677 The Effects of Temperature and Innate Immunity on Transmission of Campylobacter jejuni (Campylobacterales: Campylobacteraceae) Between Life Stages of Musca domestica (Diptera: Muscidae)
antimicrobial peptide; infection; vector; campylobacteriosis; house fly
The house fly (Musca domestica L.) is a well-established vector of human pathogens, including Campylobacter spp., which can cause infection of broiler chicken flocks, and through contaminated broiler meat can cause outbreaks of campylobacteriosis in humans. We investigated whether Campylobacter jejuni (Jones) could be transferred between life stages of M. domestica (larvae-pupae-adults) and determined bacterial counts of C. jejuni at different time points after bacterial exposure. C. jejuni was transmitted from infected larvae to pupae, but not to the adult stage. Infected larvae maintained at 25 degrees C had mean bacterial numbers of 6.5 +/- 0.2 SE log(10) (colony forming units [CFU]/g) that subsequently dropped to 3.6 +/- 0.3 SE log(10) (CFU/g) 8 h after infection. Pupae originating from infected larvae contained mean bacterial numbers of 5.3 +/- 0.1 SE log(10) (CFU/g), and these numbers dropped to 4.8 +/- 0.1 SE log(10) (CFU/g) 24 h after pupation. The decline in C. jejuni numbers during pupal development coincided with increased expression of antimicrobial peptides, including cecropin, diptericin, attacin, and defensin, in the larva-pupa transition stage and a later second peak in older pupae (4 or 48 h). Conversely, there was a reduced expression of the digestive enzyme, lysozyme, in pupae and adults compared with larvae. DOI
46. 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
45. 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
44. 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
43. 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
42. 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
41. 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
40. 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
39. 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
38. Stuyt, M., R. Ursic-Bedoya, D.M. Cooper, N. Huitson, G. Anderson, and C. Lowenberger. (2010) IDENTIFICATION OF HOST MATERIAL FROM CROPS AND WHOLE BODIES OF Protophormia terraenovae (R-D) (DIPTERA) LARVAE, PUPAE, AND ADULTS, AND THE IMPLICATIONS FOR FORENSIC STUDIES.Canadian Society of Forensic Science Journal 43: 97- 107 IDENTIFICATION OF HOST MATERIAL FROM CROPS AND WHOLE BODIES OF Protophormia terraenovae (R-D) (DIPTERA) LARVAE, PUPAE, AND ADULTS, AND THE IMPLICATIONS FOR FORENSIC STUDIES
Larvae of Diptera are used routinely in forensic entomology to estimate accurately the post mortem interval. These applications require a detailed understanding of insect growth rates and insect succession profiles under different climactic conditions and geographical locations. There may be times when larvae may have been removed from a single food source or the corpse has been moved from one location to another, confounding the interpretation of the entomological data. We report here a study that evaluates the time period during which a food source can be detected in larvae of Protophormia terraenovae. We could detect the food source (pork or chicken liver) that larvae had ingested from early second instar to late third instar larvae. No detection was possible in pupae or adults. When larvae were transferred from one food source to another, the primary source was detectable for only 8-12 hours after transfer, whereas it was detectable for 12 hours in starved larvae. In most studies that use insects to estimate PMI, investigators collect larvae directly from the corpse. In cases where there have been significant disturbances, investigators must understand the changes in insect dieting and development that may take place.Website
37. 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
36. 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
35. 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
34. 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
33. 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
32. 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
31. 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
30. 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
29. 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.
28. 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
27. 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
26. 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
25. 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.
24. 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.
23. 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.
22. Bedoya, RJU; Mitzey, AM; Obraztsova, M; Lowenberger, C. (2005) Molecular cloning and transcriptional activation of lysozyme-encoding cDNAs in the mosquito Aedes aegypti.Insect Molecular Biology 14: 89-94 Molecular cloning and transcriptional activation of lysozyme-encoding cDNAs in the mosquito Aedes aegypti
lysozyme; insect immunity
Lysozymes are enzymes characterized by their ability to break down bacterial cell walls. In insects certain lysozymes are only found in the midgut, whereas others are only found in the haemolymph and fat body after immune challenge. We identified two lysozyme-encoding cDNAs from Aedes aegypti. Both deduced protein sequences are basic in nature, contain 148 amino acids including eight highly conserved cysteine residues, and their genomic sequences contain a single intron. Transcriptional profiles indicated that the predominant form is constitutively expressed and up-regulated upon immune challenge and blood feeding in adult mosquitoes. The second form is expressed during early developmental stages, larvae and pupae, and at low levels in adults after immune challenge. Lysozymes in Aedes aegypti play both roles, defined by the spatial and temporal regulation of their expression.
21. Bartholomay, LC; Fuchs, JF; Cheng, LL; Beck, ET; Vizioli, J; Lowenberger, C; Christensen, BM. (2004) Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti.Insect Molecular Biology 13: 125-132 Reassessing the role of defensin in the innate immune response of the mosquito, Aedes aegypti
Defensin is the predominant inducible immune peptide in Aedes aegypti. In spite of its activity against Gram-positive bacteria in vitro, defensin expression is detected in mosquitoes inoculated with Gram-positive or negative bacteria, or with filarial worms. Defensin transcription and expression are dependent upon bacterial dose; however, translation is inconsistent with transcription because peptide is detectable only in mosquitoes inoculated with large doses. In vitro translation assays provide further evidence for post-transcriptional regulation of defensin. Clearance assays show that a majority of bacteria are cleared before defensin is detected. In gene silencing experiments, no significant difference in mortality was observed between defensin-deficient and control mosquitoes after bacteria inoculation. These studies suggest that defensin may have an alternative function in mosquito immunity.
20. Boulanger, N; Lowenberger, C; Volf, P; Ursic, R; Sigutova, L; Sabatier, L; Svobodova, M; Beverley, SM; Spath, G; Brun, R; Pesson, B; Bulet, P. (2004) Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major.Infection and Immunity 72: 7140-7146 Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major
Antimicrobial peptides are major components of the innate immune response of epithelial cells. In insect vectors, these peptides may play a role in the control of gut pathogens. We have analyzed antimicrobial peptides produced by the sand fly Phlebotomus duboscqi, after challenge by injected bacteria or feeding with bacteria or the protozoan parasite Leishmania major. A new hemolymph peptide with antimicrobial activity was identified and shown to be a member of the insect defensin family. Interestingly, this defensin exhibits an antiparasitic activity against the promastigote forms of L. major, which reside normally within the sand fly midgut. P. duboscqi defensin could be induced by both hemolymph or gut infections. Defensin mRNA was induced following infection by wild-type L. major, and this induction was much less following infections with L. major knockout mutants that survive poorly in sand flies, due to specific deficiencies in abundant cell surface glycoconjugates containing phosphoglycans (including lipophosphoglycan). The ability of gut pathogens to induce gut as well as fat body expression of defensin raises the possibility that this antimicrobial peptide might play a key role in the development of parasitic infections.
19. Lopez, L; Morales, G; Ursic, R; Wolff, M; Lowenberger, C. (2003) Isolation and characterization of a novel insect defensin from Rhodnius prolixus, a vector of Chagas disease.Insect Biochemistry and Molecular Biology 33: 439-447 Isolation and characterization of a novel insect defensin from Rhodnius prolixus, a vector of Chagas disease
Rhodnius prolixus; defensin; innate immunity; antimicrobial peptides; Chagas disease
An antimicrobial peptide belonging to the defensin family of small cationic peptides associated with innate immunity in insects was isolated from the hemolymph of Rhodnius prolixus, a vector of Chagas disease. This peptide, designated R. prolixus defensin A, was purified and sequenced. The active peptide contains 43 residues and aligns well with other insect defensins. However the pre-pro region of the sequence has little shared identity with other insect defensins. We have identified 3 isoforms of R. prolixus defensin from cDNA clones obtained from RNA isolated from the whole bodies of immune activated insects. Northern analysis and Real-Time Quantitative PCR indicate that there is a very low baseline transcription of this peptide in naive insects, and that transcription increases significantly in the fat body of immune activated insects. In addition there is a delayed induction of transcription of this peptide in the intestine 24 h post activation suggesting that the midgut/intestine of this species is active in the immune response against pathogens. (C) 2003 Elsevier Science Ltd. All rights reserved.
18.Lowenberger, C. (2001) Innate immune response of Aedes aegypti.Insect Biochemistry and Molecular Biology 31 (Sp. Iss. SI): 219-229 Innate immune response of Aedes aegypti
Aedes aegypti; innate immunity; defensin; cecropin
Insects are able to protect themselves from invasion by pathogens by a rapid and potent arsenal of inducible immune peptides. This fast, extremely effective response is part of the innate immunity exhibited by all insects and many invertebrates, and shows striking similarities with the innate immune response of vertebrates. In Aedes aegypti invasion of the hemocoel by bacteria elicits the production of defensins, cecropins, a peptide active only against Gram-negative bacteria, and several other peptides that we are now characterizing. However, not all insects utilize the same peptides in the same concentrations, which may reflect the pathogens to which they may have been exposed through evolutionary time. These protective measures we see in mosquitoes are the current state of the evolution of a rapid immune response that has contributed to the success of insects in inhabiting essentially every niche on earth. The molecules involved in the response of Aedes aegypti to pathogens, and the potential role of these peptides against eukaryotic parasites ingested and transmitted by mosquitoes are discussed. (C) 2001 Elsevier Science Ltd. All rights reserved.
16. Ferdig, MT; Taft, AS; Smartt, CT; Lowenberger, CA; Li, J; Zhang, J; Christensen, BM. (2000) Aedes aegypti dopa decarboxylase: gene structure and regulation.Insect Molecular Biology 9: 231-239 Aedes aegypti dopa decarboxylase: gene structure and regulation
Aedes aegypti; dopa decarboxylase; gene structure; gene expression; ovary development
Dopa decarboxylase converts L-dopa to dopamine, a precursor molecule for diverse biological activities in insects including neurotransmission and a variety of tanning reactions required for development, reproduction and defence against parasites. Herein, we report the cloning and sequencing of the Aedes aegypti Ddc gene, including 2.1 kb of the upstream promoter region. The transcribed region of the gene spans more than 16 kb and contains five exons. In site hybridization localizes the blood-meal-induced ovarian transcription of this gene to the follicular epithelial cells surrounding individual oocytes. Ovary tissue transcription of Ddc is increased in response to injection of 20-hydroxyecdysone to levels equal to those observed for blood-fed controls, however coinjection with the translational inhibitor cycloheximide negates the effect, indicating an indirect regulatory role for this hormone. Clusters of putative ecdysone-responsive elements and zinc-finger binding domains for the products of Broad-Complex gene family are identified in the 5'-promoter region. These elements are discussed in the context of common insect Ddc regulatory mechanisms.
15. Vizioli, J; Bulet, P; Charlet, M; Lowenberger, C; Blass, C; Muller, HM; Dimopoulos, G; Hoffmann, J; Kafatos, FC; Richman, A. (2000) Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae.Insect Molecular Biology 9: 75-84 Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae
Anopheles gambiae; cecropin; malaria; antimicrobial peptide; innate immunity
Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.
14.Lowenberger, C; Charlet, M; Vizioli, J; Kamal, S; Richman, A; Christensen, BM; Bulet, P. (1999) Antimicrobial activity spectrum, cDNA cloning, and mRNA expression of a newly isolated member of the cecropin family from the mosquito vector Aedes aegypti.Journal of Biological Chemistry 274: 20092-20097 Antimicrobial activity spectrum, cDNA cloning, and mRNA expression of a newly isolated member of the cecropin family from the mosquito vector Aedes aegypti
An antimicrobial peptide belonging to the cecropin family was isolated from the hemolymph of bacteria-challenged adult Aedes aegypti. This new peptide, named cecropin A, was purified to homogeneity and fully characterized after cDNA cloning. The 34-residue A. aegypti cecropin A is different from the majority of reported insect cecropins in that it is devoid of a tryptophan residue and C-terminal amidation. The importance of these two structural features on the activity spectrum was investigated using a chemically synthesized peptide. A comparison of the antimicrobial activity spectrum of A. aegypti and Drosophila cecropin A showed a lower activity for the mosquito molecule. A. aegypti cecropin mRNA expression was not detected by Northern blot or reverse transcription-polymerase chain reaction analysis in any immature stage of the mosquito, nor in naive adults, but it was observed in challenged adults 6 h after bacteria inoculation, and it continued over 7-10 days.
13.Lowenberger, CA; Kamal, S; Chiles, J; Paskewitz, S; Bulet, P; Hoffmann, JA; Christensen, BM. (1999) Mosquito - Plasmodium interactions in response to immune activation of the vector.Experimental Parasitology 91: 59-69 Mosquito - Plasmodium interactions in response to immune activation of the vector
Aedes aegypti; Anopheles gambiae; Plasmodium gallinaceum; Plasmodium berghei; Diptera; Culicidae; mosquito; Liverpool; vector competence; refractoriness; insect immunity; defensin; malaria
During the development of Plasmodium sp, within the mosquito midgut, the parasite undergoes a series of developmental changes. The elongated ookinete migrates through the layers of the midgut where it forms the oocyst under the basal lamina. We demonstrate here that if Aedes aegypti or Anopheles gambiae, normally susceptible to Plasmodium gallinaceum and P. berghei, respectively, are immune activated by the injection of bacteria into the hemocoel, and subsequently are fed on an infectious bloodmeal, there is a significant reduction in the prevalence and mean intensity of infection of oocysts on the midgut. Only those mosquitoes immune activated prior to, or immediately after, parasite ingestion exhibit this reduction in parasite development. Mosquitoes immune activated 2-5 days after bloodfeeding show no differences in parasite burdens compared with naive controls. Northern analyses reveal that transcriptional activity for mosquito defensins is not detected in the whole bodies of Ae. aegypti from 4 h to 10 days after ingesting I? gallinaceum, suggesting that parasite ingestion; passage from the food bolus through the midgut, oocyst formation, and subsequent release of sporozoites into the hemolymph do not induce the production of defensin. However, reverse transcriptase-PCR of RNA isolated solely from the midguts of Ae. aegypti indicates that transcription of mosquito defensins occurs in the midguts of naive mosquitoes and those ingesting an infectious or noninfectious bloodmeal. Bacteria-challenged Ae. aegypti showed high levels of mature defensin in the hemolymph that correlate with a lower prevalence and mean intensity of infection with oocysts. Because few oocysts were found on the midgut of immune-activated mosquitoes, the data suggest,that some factor, induced by bacterial challenge, kills the parasite at a preoocyst stage. (C) 1999 Academic Press.
12.Lowenberger, CA; Smartt, CT; Bulet, P; Ferdig, MT; Severson, DW; Hoffmann, JA; Christensen, BM. (1999) Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti.Insect Molecular Biology 8: 107-118 Insect immunity: molecular cloning, expression, and characterization of cDNAs and genomic DNA encoding three isoforms of insect defensin in Aedes aegypti
Aedes aegypti; defensin; insect immunity
Aedes aegypti were immune activated by injection with bacteria, and the expression of insect defensins was measured over time. Northern analyses indicated that defensin transcriptional activity continued for at least 21 days after bacterial injection, and up to 10 days after saline inoculation. Mature defensin levels in the haemolymph reached approximately 45 mu M at 24 h post inoculation, cDNAs encoding the preprodefensins of three previously described mature Ae, aegypti defensins were amplified by PCR, cloned and sequenced. Genomic crones were amplified using primers designed against the cDNA sequence. Sequence comparison indicates that there is significant inter- and intra-isoform variability in the signal peptide and prodefensin sequences of defensin genes. Preprodefensin sequences of isoforms A and B are very similar, consisting of a signal peptide region of twenty amino acids, a prodefensin region of thirty-eight amino acids and a forty amino acid mature peptide domain. The sequence encoding isoform C is significantly different, comprising a signal peptide region of twenty-three amino acids, a prodefensin region of thirty-six amino acids, and the mature protein domain of forty amino acids. Analysis of the genomic clones of each isoform revealed one intron spatially conserved in the prodefensin region of all sequences. The intron in isoforms A and B is 64 nt long, and except for a 4 nt substitution in one clone, these intron sequences are identical. The intron in isoform C is 76 nt long and does not share significant identity with the intron sequences of isoforms A or B, The defensin gene mapped to chromosome 3, between two known loci, bit and LF168.
11. Smartt, CT; Chiles, J; Lowenberger, C; Christensen, BM. (1998) Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene.Insect Biochemistry and Molecular Biology 28: 935-945 Biochemical analysis of a blood meal-induced Aedes aegypti glutamine synthetase gene
Aedes aegypti; midgut glutamine synthetase; peritrophic matrix
Glutamine synthetase (GS) in the mosquito, Aedes aegypti, is induced in the midgut following a blood meal. Mosquito GS message is detected as soon as 1 h post-blood feeding and remains stable for 18 h. Using a PCR product encoding mosquito GS, a lambda gt10 adult female mosquito cDNA library was screened. A cDNA clone, pCl5A2, encoding the full translation product of mosquito GS was isolated and sequence analyses performed. Mosquito GS cDNA is 2.5 kb in length and its putative translation product shares all the conserved regions characteristic of the GS gene family, including the presumed ATP biding site. Glutamine synthetase activity in the mosquito midgut is highest at 18 h post-blood feeding. Activity can be detected over a broad pH range, from 6.0 to 7.5. Unlike other cellular GS enzymes, mosquito GS is not active in the presence of ATP. Very low dosages (0.05 mM) of L-methionine S-sulfoximine are sufficient to partially inhibit mosquito GS activity. Inhibition of GS disrupts the normal formation of the midgut peritrophic matrix, suggesting that GS enzyme might be involved in the initial pathway of chitin synthesis. The unique expression pattern and inducible nature of the mosquito GS gene make it an interesting candidate for studying promoter function. Additionally, the blood meal activation of the GS gene makes this a potentially valuable tool in mosquito transformation studies. (C) 1998 Elsevier Science Ltd. All rights reserved.
10. Beerntsen, BT; Lowenberger, CA; Klinkhammer, JA; Christensen, LA; Christensen, BM. (1996) Influence of anesthetics on the peripheral blood microfilaremia of Brugia malayi in the Mongolian Jird, Meriones unguiculatus.Journal of Parasitology 82: 327-330 Influence of anesthetics on the peripheral blood microfilaremia of Brugia malayi in the Mongolian Jird, Meriones unguiculatus
The effects of anesthetics on the peripheral blood microfilaremia of Brugia malayi in Meriones unguiculatus were investigated. Microfilaremias were assessed by orbital puncture prior to and following the use, either individually or in tandem, of ether, Rompun(R), Ketaset(R), and sodium pentobarbital. Results indicate that the peripheral microfilaremia varied dramatically, depending on the anesthetic administered. Although microfilaremias were not affected by an initial ether exposure, counts of microfilariae increased significantly when jirds received Rompun and Ketaset, or Ketaset alone. Administration of sodium pentobarbital did not increase the number of microfilariae observed in the peripheral blood. The mode of action differs between these drugs and is likely responsible for the different effects observed. Consequently, studies involving vector-parasite interactions should take precautions to prevent parasite-induced vector mortality due to the ingestion of large numbers of microfilariae induced in the peripheral bloodstream by certain anesthetics.
9.Lowenberger, CA; Ferdig, MT; Bulet, P; Khalili, S; Hoffmann, JA; Christensen, BM. (1996) Aedes aegypti: Induced antibacterial proteins reduce the establishment and development of Brugia malayi.Experimental Parasitology 83: 191-201 Aedes aegypti: Induced antibacterial proteins reduce the establishment and development of Brugia malayi
The effect of host immune activation on the development of Brugia malayi in one susceptible and four refractory strains of Aedes aegypti and in Armigeres subalbatus was assessed. A. aegypti that were immune activated by the injection of saline or bacteria 24 hr before feeding on a B. malayi-infected gerbil had significantly reduced prevalences and mean intensities of infection from those of naive controls when exposed to bloodmeals with low (105 mf/20 mu l) and medium (160 mf/20 mu l) microfilaremias. At a higher microfilaremia (237 mf/20 mu l) there were no significant differences in mean intensities, suggesting that the number of parasites ingested may affect the host's ability to mount an effective defense response. Because the major immune proteins in A. aegypti are defensins, we did Northern analyses of fat body RNA 8 hr after immune activation or bloodfeeding. All mosquitoes demonstrated rapid transcriptional activity for defensins following immune activation by intrathoracic inoculation with either saline or bacteria. However, no strain of A. aegypti, susceptible or refractory to B. malayi, nor Ar. subalbatus produced defensin transcripts after bloodfeeding on an uninfected or a B. malayi-infected gerbil. These data suggest that inducible immune proteins of mosquitoes can reduce the prevalence and mean intensity of infections with ingested parasites, but these proteins are not expressed routinely after parasite ingestion and midgut penetration and probably do not contribute to existing refractory mechanisms. Immune proteins such as defensins, however, represent potential candidates to genetically engineer mosquitoes for resistance to filarial worms. (C) 1996 Academic Press, Inc.
6. Willott, E; Lowenberger, C; Christensen, BM; Kanost, MR. (1995) Monoclonal antibodies against Manduca sexta hemocytes bind Aedes aegypti hemocytes: Characterization of six monoclonal antibodies that bind hemocytes from both species.Developmental and Comparative Immunology 19: 451-461 Monoclonal antibodies against Manduca sexta hemocytes bind Aedes aegypti hemocytes: Characterization of six monoclonal antibodies that bind hemocytes from both species
Aedes aegypti; hemocytes; Manduca sexta; insect; mosquito; monoclonal antibody; immune response
The mechanisms by which hemocytes mediate a mosquito's defense response to parasites or pathogens are not well understood. This is due in part to difficulty in collecting intact mosquito hemocytes for experiments and to a lack of reagents, such as antibodies. Our objectives were to collect adult Aedes aegypti hemocytes under conditions suitable for immunofluorescence microscopy, and to test whether monoclonal antibodies, generated against larval Manduca sexta hemocytes, bind adult Ae. aegypti hemocytes, We present immunofluorescence micrographs of M. sexta and Ae, aegypti hemocytes stained by sir monoclonal antibodies, Two antibodies, MS11 and MS32, immunolocalized to hemocyte nuclei in both species. On Western blots, these antibodies generate one signal at similar to 40 kDa and four others between 10 and 25 kDa, Immunofluorescence staining patterns of the other four antibodies were more complex, That these antibodies bind hemocytes from both species suggests significant molecular similarities exist between hemocytes from evolutionarily divergent species.
