ABSTRACT
Parasitic nematodes have an intimate, chronic and lifelong exposure to vertebrate tissues. Here we mined 41 published parasitic nematode transcriptomes from vertebrate hosts and identified 91 RNA viruses across 13 virus orders from 24 families in ~70% (28 out of 41) of parasitic nematode species, which include only 5 previously reported viruses. We observe widespread distribution of virus-nematode associations across multiple continents, suggesting an ancestral acquisition event and host-virus co-evolution. Characterization of viruses of Brugia malayi (BMRV1) and Onchocerca volvulus (OVRV1) shows that these viruses are abundant in reproductive tissues of adult parasites. Importantly, the presence of BMRV1 RNA in B. malayi parasites mounts an RNA interference response against BMRV1 suggesting active viral replication. Finally, BMRV1 and OVRV1 were found to elicit antibody responses in serum samples from infected jirds and infected or exposed humans, indicating direct exposure to the immune system.
ABSTRACT
The human malaria parasite Plasmodium falciparum is responsible for the majority of mortality and morbidity caused by malaria infection and differs from other human malaria species in the degree of accumulation of parasite-infected red blood cells in the microvasculature, known as cytoadherence or sequestration. In P. falciparum, cytoadherence is mediated by a protein called PfEMP1 which, due to its exposure to the host immune system, undergoes antigenic variation resulting in the expression of different PfEMP1 variants on the infected erythrocyte membrane. These PfEMP1s contain various combinations of adhesive domains, which allow for the differential engagement of a repertoire of endothelial receptors on the host microvasculature, with specific receptor usage associated with severe disease. We used a co-culture model of cytoadherence incubating human brain microvascular endothelial cells with erythrocytes infected with two parasite lines expressing different PfEMP1s that demonstrate different binding profiles to vascular endothelium. We determined the transcriptional profile of human brain microvascular endothelial cells (HBMEC) following different incubation periods with infected erythrocytes, identifying different transcriptional profiles of pathways previously found to be involved in the pathology of severe malaria, such as inflammation, apoptosis and barrier integrity, induced by the two PfEMP1 variants.
Subject(s)
Malaria, Falciparum , Plasmodium falciparum , Humans , Endothelial Cells/metabolism , Coculture Techniques , Protozoan Proteins/metabolism , Malaria, Falciparum/parasitology , Erythrocytes/parasitology , Endothelium, Vascular/metabolism , Cell AdhesionABSTRACT
Lymphatic filariasis is a vector-borne neglected tropical disease prioritized for global elimination. The filarial nematodes that cause the disease host a symbiotic bacterium, Wolbachia, which has been targeted using antibiotics, leading to cessation of parasite embryogenesis, waning of circulating larvae (microfilariae [mf]), and gradual cure of adult infection. One of the benefits of the anti-Wolbachia mode of action is that it avoids the rapid killing of mf, which can drive inflammatory adverse events. However, mf depleted of Wolbachia persist for several months in circulation, and thus patients treated with antibiotics are assumed to remain at risk for transmitting infections. Here, we show that Wolbachia-depleted mf rapidly lose the capacity to develop in the mosquito vector through a defect in exsheathment and inability to migrate through the gut wall. Transcriptomic and Western blotting analyses demonstrate that chitinase, an enzyme essential for mf exsheathment, is down-regulated in Wolbachia-depleted mf and correlates with their inability to exsheath and escape the mosquito midgut. Supplementation of in vitro cultures of Wolbachia-depleted mf with chitinase enzymes restores their ability to exsheath to a similar level to that observed in untreated mf. Our findings elucidate a mechanism of rapid transmission-blocking activity of filariasis after depletion of Wolbachia and adds to the broad range of biological processes of filarial nematodes that are dependent on Wolbachia symbiosis.
Subject(s)
Anti-Bacterial Agents , Chitinases , Elephantiasis, Filarial , Microfilariae , Wolbachia , Animals , Anti-Bacterial Agents/pharmacology , Chitinases/genetics , Elephantiasis, Filarial/transmission , Humans , Microfilariae/enzymology , Microfilariae/growth & development , Microfilariae/microbiology , Mosquito Vectors/parasitology , Wolbachia/drug effects , Wolbachia/geneticsABSTRACT
Wolbachia, a widespread bacterium that can reduce pathogen transmission in mosquitoes, has recently been reported to be present in Anopheles (An.) species. In wild populations of the An. gambiae complex, the primary vectors of Plasmodium malaria in Sub-Saharan Africa, Wolbachia DNA sequences at low density and infection frequencies have been detected. As the majority of studies have used highly sensitive nested PCR as the only method of detection, more robust evidence is required to determine whether Wolbachia strains are established as endosymbionts in Anopheles species. Here, we describe high-density Wolbachia infections in geographically diverse populations of An. moucheti and An. demeilloni. Fluorescent in situ hybridization localized a heavy infection in the ovaries of An. moucheti, and maternal transmission was observed. Genome sequencing of both Wolbachia strains obtained genome depths and coverages comparable to those of other known infections. Notably, homologs of cytoplasmic incompatibility factor (cif) genes were present, indicating that these strains possess the capacity to induce the cytoplasmic incompatibility phenotype, which allows Wolbachia to spread through host populations. These strains should be further investigated as candidates for use in Wolbachia biocontrol strategies in Anopheles aiming to reduce the transmission of malaria.
Subject(s)
Anopheles , Malaria , Wolbachia , Animals , Anopheles/genetics , In Situ Hybridization, Fluorescence , Maternal Inheritance , Mosquito Vectors , Wolbachia/geneticsABSTRACT
Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.
Subject(s)
Cyclohexanones/therapeutic use , Drug Repositioning , Infection Control/methods , Nitrobenzoates/therapeutic use , Trypanosomiasis, African/prevention & control , 4-Hydroxyphenylpyruvate Dioxygenase/antagonists & inhibitors , 4-Hydroxyphenylpyruvate Dioxygenase/metabolism , Animals , Bees/drug effects , Female , Humans , Insecticides/therapeutic use , Male , Metabolome/drug effects , Mice , Models, Theoretical , Neglected Diseases/prevention & control , Orphan Drug Production , Rats , Rats, Wistar , Toxicity Tests , Trypanosomiasis, African/transmission , Tsetse Flies/drug effects , Tsetse Flies/metabolism , Tyrosine/metabolismABSTRACT
Skin snip evaluation for onchocerciasis has insufficient sensitivity when skin microfilarial (mf) densities are low, such as following ivermectin treatment. Mf density is suitable for assessing microfilaricidal efficacy but only serves as an indirect indicator of macrofilaricidal activity. We assessed circulating nucleic acids from Onchocerca volvulus as an alternative to skin snips. We screened a plasma sample set of infected individuals followed up at four, 12 and 21 months after microfilaricidal (ivermectin, n = four), macrofilaricidal (doxycycline, n = nine), or combination treatment (n = five). Two parasite-derived miRNAs, cel-miR-71-5p and bma-lin-4, and O-150 repeat DNA were assessed. Highly abundant DNA repeat families identified in the O. volvulus genome were also evaluated. miRNAs were detected in two of 72 plasma samples (2.8%) and two of 47 samples (4.3%) with microfilaridermia using RT-qPCR. O-150 DNA was detected in eight (44.4%) baseline samples by qPCR and the number of positives declined post-treatment. One doxycycline-treated individual remained O-150 positive. However, only 11 (23.4%) samples with microfilaridermia were qPCR-positive. Analysis by qPCR showed novel DNA repeat families were comparatively less abundant than the O-150 repeat. Circulating parasite-derived nucleic acids are therefore insufficient as diagnostic tools or as biomarkers of treatment efficacy for O. volvulus.
Subject(s)
Biomarkers/blood , Circulating MicroRNA/blood , DNA/blood , Onchocerca volvulus/physiology , Onchocerciasis/drug therapy , Onchocerciasis/genetics , Adult , Animals , Humans , Male , Onchocerciasis/blood , Parasites/genetics , Treatment OutcomeABSTRACT
IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts whose expression is associated with insecticide resistance phenotypes in Anopheles gambiae mosquitoes. The application can be used online or downloaded and used locally by anyone. The local application can be modified to add new insecticide resistance datasets generated from multiple -omics platforms. This guide demonstrates how to add new datasets and handle missing data. Furthermore, IR-TEx can be completely and easily recoded to use-omics datasets from any experimental data, making it a valuable resource to many researchers. The protocol illustrates the utility of IR-TEx in identifying new insecticide resistance candidates using the the microsomal glutathione transferase, GSTMS1, as an example. This transcript is upregulated in multiple pyrethroid resistant populations from Côte D'Ivoire and Burkina Faso. The identification of co-correlated transcripts provides further insight into the putative roles of this gene.
Subject(s)
Anopheles/pathogenicity , Malaria/transmission , Transcriptome/physiology , Animals , Big Data , Mosquito Control/methodsABSTRACT
The asp viper Vipera aspis aspis is a venomous snake found in France, and despite its medical importance, the complete toxin repertoire produced is unknown. Here, we used a venomics approach to decipher the composition of its venom. Transcriptomic analysis revealed 80 venom-annotated sequences grouped into 16 gene families. Among the most represented toxins were snake venom metalloproteases (23%), phospholipases A2 (15%), serine proteases (13%), snake venom metalloprotease inhibitors (13%) and C-type lectins (12%). LC-MS of venoms revealed similar profiles regardless of the method of extraction (milking vs defensive bite). Proteomic analysis validated 57 venom-annotated transcriptomic sequences (>70%), including one for each of the 16 families, but also identified 7 sequences not initially annotated as venom proteins, including a serine protease, a disintegrin, a glutaminyl-peptide cyclotransferase, a proactivator polypeptide-like and 3 aminopeptidases. Interestingly, phospholipases A2 were the dominant proteins in the venom, among which included an ammodytoxin B-like sequence, which may explain the reported neurotoxicity following some asp viper envenomations. In total, 87 sequences were retrieved from the Vipera aspis aspis transcriptome and proteome, constituting a valuable resource that will help in understanding the toxinological basis of clinical signs of envenoming and for the mining of useful pharmacological compounds. BIOLOGICAL SIGNIFICANCE: The asp viper (Vipera aspis aspis) causes several hundred envenomations annually in France, including unusual cases with neurological signs, resulting in one death per year on average. Here, we performed a proteotranscriptomic analysis of V. a. aspis venom in order to provide a better understanding of its venom composition. We found that, as in other Vipera species, phospholipase A2 dominates in the venom, and the presence of a sequence related to ammodytoxin B may explain the reported neurotoxicity following some asp viper envenomations. Thus, this study will help in informing the toxinological basis of clinical signs of envenoming.
Subject(s)
Proteomics , Viperidae , Animals , France , Humans , Metalloproteases/genetics , Phospholipases A2 , Viper VenomsABSTRACT
Snakebite is a neglected tropical disease that causes 138,000 deaths each year. Neurotoxic snake venoms contain small neurotoxins, including three-finger toxins (3FTxs), which can cause rapid paralysis in snakebite victims by blocking postsynaptic transmission via nicotinic acetylcholine receptors (nAChRs). These toxins are typically weakly immunogenic and thus are often not effectively targeted by current polyclonal antivenom therapies. We investigated whether nAChR mimics, also known as acetylcholine binding proteins (AChBPs), could effectively capture 3FTxs and therefore be developed as a novel class of snake-generic therapeutics for combatting neurotoxic envenoming. First, we identified the binding specificities of 3FTx from various medically important elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from Lymnaea stagnalis and a humanized neuronal α7 version (α7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while α7-AChBP alone did not protect against Naja haje (Egyptian cobra) venom lethality in vivo, it significantly prolonged survival times when coadministered with a nonprotective dose of antivenom. Thus, nAChR mimics are capable of neutralizing specific venom toxins and may be useful adjunct therapeutics for improving the safety and affordability of existing snakebite treatments by reducing therapeutic doses. Our findings justify exploring the future development of AChBPs as potential snakebite treatments.
ABSTRACT
We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8â¯kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. BIOLOGICAL SIGNIFICANCE: Population level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.
Subject(s)
Mass Spectrometry/methods , Viper Venoms/chemistry , Age Factors , Animals , Antivenins/chemistry , Biodiversity , Metalloproteases/analysis , Phospholipases A2/analysis , Proteomics/methods , Species Specificity , Transcriptome , Turkey , Viper Venoms/enzymology , ViperidaeABSTRACT
While envenoming by the southern African shield-nosed or coral snakes (genus Aspidelaps) has caused fatalities, bites are uncommon. Consequently, this venom is not used in the mixture of snake venoms used to immunise horses for the manufacture of regional SAIMR (South African Institute for Medical Research) polyvalent antivenom. Aspidelaps species are even excluded from the manufacturer's list of venomous snakes that can be treated by this highly effective product. This leaves clinicians, albeit rarely, in a therapeutic vacuum when treating envenoming by these snakes. This is a significantly understudied small group of nocturnal snakes and little is known about their venom compositions and toxicities. Using a murine preclinical model, this study determined that the paralysing toxicity of venoms from Aspidelaps scutatus intermedius, A. lubricus cowlesi and A. l. lubricus approached that of venoms from highly neurotoxic African cobras and mambas. This finding was consistent with the cross-genus dominance of venom three-finger toxins, including numerous isoforms which showed extensive interspecific variation. Our comprehensive analysis of venom proteomes showed that the three Aspidelaps species possess highly similar venom proteomic compositions. We also revealed that the SAIMR polyvalent antivenom cross-reacted extensively in vitro with venom proteins of the three Aspidelaps. Importantly, this cross-genus venom-IgG binding translated to preclinical (in a murine model) neutralisation of A. s. intermedius venom-induced lethality by the SAIMR polyvalent antivenom, at doses comparable with those that neutralise venom from the cape cobra (Naja nivea), which the antivenom is directed against. Our results suggest a wider than anticipated clinical utility of the SAIMR polyvalent antivenom, and here we seek to inform southern African clinicians that this readily available antivenom is likely to prove effective for victims of Aspidelaps envenoming. BIOLOGICAL SIGNIFICANCE: Coral and shield-nosed snakes (genus Aspidelaps) comprise two species and several subspecies of potentially medically important venomous snakes distributed in Namibia, Botswana, Zimbabwe, Mozambique and South Africa. Documented human fatalities, although rare, have occurred from both A. lubricus and A. scutatus. However, their venom proteomes and the pathological effects of envenomings by this understudied group of nocturnal snakes remain uncharacterised. Furthermore, no commercial antivenom is made using venom from species of the genus Aspidelaps. To fill this gap, we have conducted a transcriptomics-guided comparative proteomics analysis of the venoms of the intermediate shield-nose snake (A. s. intermedius), southern coral snake (A. l. lubricus), and Cowle's shield snake (A. l. cowlesi); investigated the mechanism of action underpinning lethality by A. s. intermedius in the murine model; and assessed the in vitro immunoreactivity of the SAIMR polyvalent antivenom towards the venom toxins of A. l. lubricus and A. l. cowlesi, and the in vivo capability of this antivenom at neutralising the lethal effect of A. s. intermedius venom. Our data revealed a high degree of conservation of the global composition of the three Aspidelaps venom proteomes, all characterised by the overwhelming predominance of neurotoxic 3FTxs, which induced classical signs of systemic neurotoxicity in mice. The SAIMR polyvalent antivenom extensively binds to Aspidelaps venom toxins and neutralised, with a potency of 0.235 mg venom/mL antivenom, the lethal effect of A. s. intermedius venom. Our data suggest that the SAIMR antivenom could be a useful therapeutic tool for treating human envenomings by Aspidelaps species.
Subject(s)
Antivenins , Coral Snakes/metabolism , Elapid Venoms , Reptilian Proteins , Animals , Antivenins/immunology , Antivenins/pharmacology , Elapid Venoms/chemistry , Elapid Venoms/immunology , Elapid Venoms/metabolism , Elapid Venoms/toxicity , Horses , Humans , Immunization , Male , Mice , Reptilian Proteins/chemistry , Reptilian Proteins/immunology , Reptilian Proteins/metabolism , Reptilian Proteins/toxicity , Snake Bites/drug therapy , Snake Bites/immunology , Snake Bites/metabolism , South AfricaABSTRACT
BACKGROUND: To understand more about changes to the molecular components that occur when host endothelium interacts with Plasmodium falciparum-infected erythrocytes, a combined technique of protein separation (1D Blue-Native electrophoresis) and mass spectrometry of infected erythrocytes with endothelial cells (EC) in a co-culture system has been used. METHODS: Native proteins were extracted from co-cultures and identified by mass spectrometry. Proteomic data from different parasite strains, either adhesion proficient (to endothelial cells) or non-adherent, were analysed in parallel to reveal protein associations linked to cytoadherence. Informatic approaches were developed to facilitate this comparison. RESULTS: Blue-Native gel separation and LC/MS/MS identification revealed major differences in samples produced from endothelial cell co-culture with adherent and non-adherent parasite strains. This approach enabled us to identify protein associations seen only with the adhesion proficient parasite strain. CONCLUSIONS: The combination of proteomic and analytical approaches has identified differences between adherent and non-adherent parasite lines in co-culture with EC, providing potential candidates for complexes or associations formed during cytoadherence involved in cell structure, signalling and apoptosis.
Subject(s)
Cell Adhesion , Electrophoresis , Endothelial Cells/parasitology , Erythrocytes/parasitology , Plasmodium falciparum/physiology , Protozoan Proteins/physiology , Chromatography, Liquid , Coculture Techniques , Humans , Proteomics , Tandem Mass SpectrometryABSTRACT
Mambas (genus Dendroaspis) are among the most feared of the medically important elapid snakes found in sub-Saharan Africa, but many facets of their biology, including the diversity of venom composition, remain relatively understudied. Here, we present a reconstruction of mamba phylogeny, alongside genus-wide venom gland transcriptomic and high-resolution top-down venomic analyses. Whereas the green mambas, D. viridis, D. angusticeps, D. j. jamesoni and D. j. kaimosae, express 3FTx-predominant venoms, black mamba (D. polylepis) venom is dominated by dendrotoxins I and K. The divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas makes it plausible that this major difference in venom composition is due to dietary variation. The pattern of intrageneric venom variability across Dendroaspis represented a valuable opportunity to investigate, in a genus-wide context, the variant toxicity of the venom, and the degree of paraspecific cross-reactivity between antivenoms and mamba venoms. To this end, the immunological profiles of the five mamba venoms were assessed against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview of which available antivenoms may be more efficacious in neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the notably different potency and pharmacological profiles of Dendroaspis venoms at locus resolution. This understanding will allow selection and design of toxin immunogens with a view to generating a safer and more efficacious pan-specific antivenom against any mamba envenomation. BIOLOGICAL SIGNIFICANCE: The mambas (genus Dendroaspis) comprise five especially notorious medically important venomous snakes endemic to sub-Saharan Africa. Their highly potent venoms comprise a high diversity of pharmacologically active peptides, including extremely rapid-acting neurotoxins. Previous studies on mamba venoms have focused on the biochemical and pharmacological characterisation of their most relevant toxins to rationalize the common neurological and neuromuscular symptoms of envenomings caused by these species, but there has been little work on overall venom composition or comparisons between them. Only very recently an overview of the composition of the venom of two Dendroaspis species, D. angusticeps and D. polylepis, has been unveiled through venomics approaches. Here we present the first genus-wide transcriptomic-proteomic analysis of mamba venom composition. The transcriptomic analyses described in this paper have contributed 29 (D. polylepis), 23 (D. angusticeps), 40 (D. viridis), 25 (D. j. jamesoni) and 21 (D. j. kaimosae), novel full-length toxin sequences to the non-redundant Dendroaspis sequence database. The mamba genus-wide venomic analysis demonstrated that major D. polylepis venom components are Kunitz-fold family toxins. This feature is unique in relation to the relatively conserved three-finger toxin (3FTx)-dominated venom compositions of the green mambas. Venom variation was interpreted in the context of dietary variation due to the divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas. Additionally, the degree of cross-reactivity conservation of mamba venoms was assessed by antivenomics against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview to infer which available antivenoms may be capable of neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the pharmacological profiles of mamba venoms at locus resolution. This understanding will contribute to the generation of a safer and more efficacious pan-Dendroaspis therapeutic antivenom against any mamba envenomation.
Subject(s)
Antivenins/immunology , Dendroaspis , Elapid Venoms/chemistry , Africa South of the Sahara , Animals , Diet , Elapid Venoms/immunology , Elapid Venoms/toxicity , Elapidae , Humans , Phylogeny , Species Specificity , TranscriptomeABSTRACT
BACKGROUND: Malaria control in Africa is dependent upon the use insecticides but intensive use of a limited number of chemicals has led to resistance in mosquito populations. Increased production of enzymes that detoxify insecticides is one of the most potent resistance mechanisms. Several metabolic enzymes have been implicated in insecticide resistance but the processes controlling their expression have remained largely elusive. RESULTS: Here, we show that the transcription factor Maf-S regulates expression of multiple detoxification genes, including the key insecticide metabolisers CYP6M2 and GSTD1 in the African malaria vector Anopheles gambiae. Attenuation of this transcription factor through RNAi induced knockdown reduced transcript levels of these effectors and significantly increased mortality after exposure to the pyrethroid insecticides and DDT (permethrin: 9.2% to 19.2% (p = 0.015), deltamethrin: 3.9% to 21.6% (p = 0.036) and DDT: 1% to 11.7% (p = <0.01), whilst dramatically decreasing mortality induced by the organophosphate malathion (79.6% to 8.0% (p = <0.01)). Additional genes regulated by Maf-S were also identified providing new insight into the role of this transcription factor in insects. CONCLUSION: Maf-S is a key regulator of detoxification genes in Anopheles mosquitoes. Disrupting this transcription factor has opposing effects on the mosquito's response to different insecticide classes providing a mechanistic explanation to the negative cross resistance that has been reported between pyrethroids and organophosphates.
Subject(s)
Anopheles/genetics , Anopheles/metabolism , Insect Proteins/metabolism , Insect Vectors/genetics , Insect Vectors/metabolism , Insecticide Resistance/genetics , Maf Transcription Factors/metabolism , Animals , Anopheles/drug effects , Data Mining , Gene Expression Regulation/drug effects , Gene Knockdown Techniques , Insect Proteins/deficiency , Insect Proteins/genetics , Insect Vectors/drug effects , Maf Transcription Factors/deficiency , Maf Transcription Factors/genetics , Malaria/transmissionABSTRACT
Venom systems have evolved on multiple occasions across the animal kingdom, and they can act as key adaptations to protect animals from predators [1]. Consequently, venomous animals serve as models for a rich source of mimicry types, as non-venomous species benefit from reductions in predation risk by mimicking the coloration, body shape, and/or movement of toxic counterparts [2-5]. The frequent evolution of such deceitful imitations provides notable examples of phenotypic convergence and are often invoked as classic exemplars of evolution by natural selection. Here, we investigate the evolution of fangs, venom, and mimetic relationships in reef fishes from the tribe Nemophini (fangblennies). Comparative morphological analyses reveal that enlarged canine teeth (fangs) originated at the base of the Nemophini radiation and have enabled a micropredatory feeding strategy in non-venomous Plagiotremus spp. Subsequently, the evolution of deep anterior grooves and their coupling to venom secretory tissue provide Meiacanthus spp. with toxic venom that they effectively employ for defense. We find that fangblenny venom contains a number of toxic components that have been independently recruited into other animal venoms, some of which cause toxicity via interactions with opioid receptors, and result in a multifunctional biochemical phenotype that exerts potent hypotensive effects. The evolution of fangblenny venom has seemingly led to phenotypic convergence via the formation of a diverse array of mimetic relationships that provide protective (Batesian mimicry) and predatory (aggressive mimicry) benefits to other fishes [2, 6]. Our results further our understanding of how novel morphological and biochemical adaptations stimulate ecological interactions in the natural world.
Subject(s)
Biological Evolution , Fishes/physiology , Predatory Behavior , Venoms , Adaptation, Physiological , Animals , Phenotype , Pigmentation , Proteome/analysis , Selection, GeneticABSTRACT
BACKGROUND: Non-front-fanged colubroid snakes comprise about two-thirds of extant ophidian species. The medical significance of the majority of these snakes is unknown, but at least five species have caused life-threatening or fatal human envenomings. However, the venoms of only a small number of species have been explored. METHODS: A combined venomic and venom gland transcriptomic approach was employed to characterise of venom of Dispholidus typus (boomslang), the snake that caused the tragic death of Professor Karl Patterson Schmidt. The ability of CroFab™ antivenom to immunocapture boomslang venom proteins was investigated using antivenomics. RESULTS: Transcriptomic-assisted proteomic analysis identified venom proteins belonging to seven protein families: three-finger toxin (3FTx); phospholipase A2 (PLA2); cysteine-rich secretory proteins (CRISP); snake venom (SV) serine proteinase (SP); C-type lectin-like (CTL); SV metalloproteinases (SVMPs); and disintegrin-like/cysteine-rich (DC) proteolytic fragments. CroFab™ antivenom efficiently immunodepleted some boomslang SVMPs. CONCLUSIONS: The present work is the first to address the overall proteomic profile of D. typus venom. This study allowed us to correlate the toxin composition with the toxic activities of the venom. The antivenomic analysis suggested that the antivenom available at the time of the unfortunate accident could have exhibited at least some immunoreactivity against the boomslang SVMPs responsible for the disseminated intravascular coagulation syndrome that caused K.P. Schmidt's fatal outcome. GENERAL SIGNIFICANCE: This study may stimulate further research on other non-front-fanged colubroid snake venoms capable of causing life-threatening envenomings to humans, which in turn should contribute to prevent fatal human accidents, such as that unfortunately suffered by K.P. Schmidt.
Subject(s)
Antivenins/immunology , Salivary Glands/metabolism , Snake Venoms/genetics , Snakes/genetics , Transcriptome/genetics , Animals , Humans , Lectins, C-Type/genetics , Metalloproteases/genetics , Phospholipases A2/genetics , Proteome/genetics , Proteomics/methods , Snake Venoms/immunology , Snakes/immunology , TreesABSTRACT
BACKGROUND: Snake venoms contain many proteinaceous toxins that can cause severe pathology and mortality in snakebite victims. Interestingly, mRNA encoding such toxins can be recovered directly from venom, although yields are low and quality is unknown. It also remains unclear whether such RNA contains information about toxin isoforms and whether it is representative of mRNA recovered from conventional sources, such as the venom gland. Answering these questions will address the feasibility of using venom-derived RNA for future research relevant to biomedical and antivenom applications. METHODOLOGY/PRINCIPAL FINDINGS: Venom was extracted from several species of snake, including both members of the Viperidae and Elapidae, and either lyophilized or immediately added to TRIzol reagent. TRIzol-treated venom was incubated at a range of temperatures (4-37°C) for a range of durations (0-48 hours), followed by subsequent RNA isolation and assessments of RNA quantity and quality. Subsequently, full-length toxin transcripts were targeted for PCR amplification and Sanger sequencing. TRIzol-treated venom yielded total RNA of greater quantity and quality than lyophilized venom, and with quality comparable to venom gland-derived RNA. Full-length sequences from multiple Viperidae and Elapidae toxin families were successfully PCR amplified from TRIzol-treated venom RNA. We demonstrated that venom can be stored in TRIzol for 48 hours at 4-19°C, and 8 hours at 37°C, at minimal cost to RNA quality, and found that venom RNA encoded multiple toxin isoforms that seemed homologous (98-99% identity) to those found in the venom gland. CONCLUSIONS/SIGNIFICANCE: The non-invasive experimental modifications we propose will facilitate the future investigation of venom composition by using venom as an alternative source to venom gland tissue for RNA-based studies, thus obviating the undesirable need to sacrifice snakes for such research purposes. In addition, they expand research horizons to rare, endangered or protected snake species and provide more flexibility to performing fieldwork on venomous snakes in tropical conditions.
Subject(s)
Elapidae/physiology , RNA, Messenger/chemistry , Snake Venoms/chemistry , Viperidae/physiology , Amino Acid Sequence , Animals , Guanidines/chemistry , Phenols/chemistry , RNA, Messenger/genetics , Specimen Handling , Temperature , Tropical ClimateABSTRACT
Cnidarian venom research has lagged behind other toxinological fields due to technical difficulties in recovery of the complex venom from the microscopic nematocysts. Here we report a newly developed rapid, repeatable and cost effective technique of venom preparation, using ethanol to induce nematocyst discharge and to recover venom contents in one step. Our model species was the Australian box jellyfish (Chironex fleckeri), which has a notable impact on public health. By utilizing scanning electron microscopy and light microscopy, we examined nematocyst external morphology before and after ethanol treatment and verified nematocyst discharge. Further, to investigate nematocyst content or "venom" recovery, we utilized both top-down and bottom-up transcriptomics-proteomics approaches and compared the proteome profile of this new ethanol recovery based method to a previously reported high activity and recovery protocol, based upon density purified intact cnidae and pressure induced disruption. In addition to recovering previously characterized box jellyfish toxins, including CfTX-A/B and CfTX-1, we recovered putative metalloproteases and novel expression of a small serine protease inhibitor. This study not only reveals a much more complex toxin profile of Australian box jellyfish venom but also suggests that ethanol extraction method could augment future cnidarian venom proteomics research efforts.
Subject(s)
Cnidarian Venoms/genetics , Cubozoa/chemistry , Serine Proteinase Inhibitors/genetics , Animals , Australia , Cnidarian Venoms/chemistry , Microscopy, Electron, Scanning , Nematocyst/metabolism , Proteome/chemistry , Proteomics , Serine Proteinase Inhibitors/chemistry , TranscriptomeABSTRACT
BACKGROUND: Within many research areas, such as transcriptomics, the millions of short DNA fragments (reads) produced by current sequencing platforms need to be assembled into transcript sequences before they can be utilized. Despite recent advances in assembly software, creating such transcripts from read data harboring isoform variation remains challenging. This is because current approaches fail to identify all variants present or they create chimeric transcripts within which relationships between co-evolving sites and other evolutionary factors are disrupted. We present VTBuilder, a tool for constructing non-chimeric transcripts from read data that has been sequenced from sources containing isoform complexity. RESULTS: We validated VTBuilder using reads simulated from 54 Sanger sequenced transcripts (SSTs) expressed in the venom gland of the saw scaled viper, Echis ocellatus. The SSTs were selected to represent genes from major co-expressed toxin groups known to harbor isoform variants. From the simulated reads, VTBuilder constructed 55 transcripts, 50 of which had a greater than 99% sequence similarity to 48 of the SSTs. In contrast, using the popular assembler tool Trinity (r2013-02-25), only 14 transcripts were constructed with a similar level of sequence identity to just 11 SSTs. Furthermore VTBuilder produced transcripts with a similar length distribution to the SSTs while those produced by Trinity were considerably shorter. To demonstrate that our approach can be scaled to real world data we assembled the venom gland transcriptome of the African puff adder Bitis arietans using paired-end reads sequenced on Illumina's MiSeq platform. VTBuilder constructed 1481 transcripts from 5 million reads and, following annotation, all major toxin genes were recovered demonstrating reconstruction of complex underlying sequence and isoform diversity. CONCLUSION: Unlike other approaches, VTBuilder strives to maintain the relationships between co-evolving sites within the constructed transcripts, and thus increases transcript utility for a wide range of research areas ranging from transcriptomics to phylogenetics and including the monitoring of drug resistant parasite populations. Additionally, improving the quality of transcripts assembled from read data will have an impact on future studies that query these data. VTBuilder has been implemented in java and is available, under the GPL GPU V0.3 license, from http:// http://www.lstmed.ac.uk/vtbuilder .