Obligate Blood Feeders Research Articles

Revisiting the paradigm of anhematophagy in male mosquitoes.

Female mosquitoes are reproductively obligate bloodfeeders which feed on vertebrate blood to obtain nutrients required for egg production (driving transmission of vector-borne pathogens in the process), and which rely on plant sugars for their non-reproductive energy requirements. Male mosquitoes, on the other hand, are thought to rely exclusively on plant sugars for their energetic needs; indeed, this dichotomy is one of the central tenets of medical entomology. Here, we show that male Culex tarsalis and Aedes aegypti mosquitoes will readily take blood from a membrane feeder when reared under dehydration conditions with no toxic effects. Mosquitoes with impaired humidity detection do not increase their bloodfeeding rates when dehydrated compared to wild-type controls. While conventionally reared males ignore a human host, dehydrated males are attracted to and attempt to probe, with some success, although they cannot access host capillaries. However, they will take blood from a vertebrate host wound. When fed a blood meal containing West Nile virus, male mosquitoes can become infected with and orally transmit the pathogen at rates and titers equivalent to females. These data suggest that under some circumstances male mosquitoes may be able to probe and/or ingest blood and transmit pathogens to vertebrate hosts, and that their role in maintaining pathogen transmission cycles should be re-examined.

Laboratory maintenance and care of Rhodnius prolixus (Hemiptera: Reduviidae) and other Triatominae.

Triatomines (Hemiptera: Reduviidae) are hematophagous insects that transmit Trypanosoma cruzi, the etiological agent for Chagas disease, to humans and other mammals. As medically important vectors, species such as Rhodnius prolixus (Hemiptera: Reduviidae) have long been used as a model organism for physiological studies. Laboratory rearing of triatomines is needed to support vector and parasite research. Many environmental conditions, such as suitable housing containers, light source and duration, temperature, humidity, and density, must be addressed when adapting triatomines from a natural habitat for artificial rearing to create conditions for optimal growth and survival. Food source is also an important factor, as triatomines are considered the obligate blood feeders. Parasites and pathogens present risks not only for triatomines but also for the laboratorians handling them. Equipping an insectary space should apply best practices to ensure community, personnel, and insect health. Various triatomine colonies have been maintained in the Centers for Disease Control and Prevention (CDC) Entomology Branch insectary for over 25 years and have more recently been made available to the research community through the Biodefense and Emerging Infections Research Resources Repository (BEI Resources). The CDC Rhodnius prolixus genome has been characterized and thus represents an opportunity for continued model organism research. In addition to fulfilling requests for live triatomines, inquiries are received for support in establishing new and troubleshooting existing laboratory colonies. To accompany the extensive MR4 manual, Methods in Anopheles Research, procedures for triatomine husbandry have been developed and are shared here to address the aforementioned topics.

Updating the northern tsetse distribution limit in Chad in the context of global change

Tsetse flies (Diptera: Glossinidae) are obligate bloodfeeders that occur exclusively in Sub-Saharan Africa, where they are the vectors of trypanosomes causing HAT (human African trypanosomiasis) and AAT (African animal trypanosomiasis). In Chad, tsetse flies occur only in the most southern part of the country because of its favorable bioclimatic conditions. However, despite the importance of HAT and AAT in this country, very little is known about the current tsetse distribution, in particular its northern limit, which is of key importance for the surveillance of these diseases. A total of 217 biconical traps were deployed in 2021 and 2022 from the West to the East around the formerly known northern limit, resulting in 1,024 tsetse caught belonging to three different taxa: Glossina morsitans submorsitans (57%), G. tachinoides (39%) and G. fuscipes fuscipes (4%). In addition to the information gathered on the presence/absence of each tsetse taxon, we show a strong North-South shift of the northen tsetse distribution limit as compared to the previous works from 1966 to 1996, and a growing spatial fragmentation in more and more discrete pockets of tsetse presence. This North-South shift of the northern tsetse distribution limit in Chad is the likely consequence of the combined effect of severe draughts that affected the country, and increasing human pressure on land. This update of the tsetse northern limit will be of help to the national programmes in charge of HAT and AAT.

Key molecules regulating the blood meals of Rhipicephalus sanguineus (Acari: Ixodidae) revealed by transcriptomics.

Rhipicephalus sanguineus, a repulsive obligate blood feeder, is a three-host tick inflicting tremendous damage. Blood-sucking initiates tick-pathogen-host interactions along with alterations in the expression levels of numerous bioactive ingredients. Key molecules regulating blood meals were identified using the transcriptomic approach. A total number of 744 transcripts showed statistically significantly differential expression including 309 significantly upregulated transcripts and 435 significantly downregulated transcripts in semiengorged female ticks compared to unfed ticks, all collected in 2021. The top 10 differentially upregulated transcripts with explicit functional annotations included turripeptide OL55-like protein, valine tRNA ligase-like protein and ice-structuring glycoprotein-like protein. The top 10 differentially down-regulated transcripts were uncharacterized proteins. Gene Ontology (GO) enrichment analysis revealed four associated terms in the cellular component category and 16 in the molecular function category among the top 20 terms. Differentially expressed genes (DEGs) were enriched in GO terms ID 0000323 (lytic vacuole) and ID 0005773 (vacuole). The top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included metabolism, cellular processes, organismal systems and human diseases. The DEGs were enriched in the KEGG term ID: ko-04142 (lysosome pathway) associated with intracellular digestion in the tick midgut epithelium. Molecular markers annotated via comparative transcriptomic profiling were expected to be candidate markers for the purpose of tick control.

No evidence for direct thermal carryover effects on starvation tolerance in the obligate blood-feeder, Glossina morsitans morsitans.

Thermal stress during development can prime animals to cope better with similar conditions in later life. Alternatively, negative effects of thermal stress can persist across life stages and result in poorer quality adults (negative carryover effects). As mean temperatures increase due to climate change, evidence for such effects across diverse taxa is required. Using Glossina morsitans morsitans, a species of tsetse fly and vector of trypanosomiasis, we asked whether (i) adaptive developmental plasticity allows flies to survive for longer under food deprivation when pupal and adult temperatures are matched; or (ii) temperature stress during development persists into adulthood, resulting in a greater risk of death. We did not find any advantage of matched pupal and adult temperature in terms of improved starvation tolerance, and no direct negative carryover effects were observed. There was some evidence for indirect carryover effects-high pupal temperature produced flies of lower body mass, which, in turn, resulted in greater starvation risk. However, adult temperature had the largest impact on starvation tolerance by far: flies died 60% faster at 31°C than those experiencing 25°C, consequently reducing survival time from a median of 8 (interquartile range (IQR) 7-9) to 5 (IQR 5-5.25) days. This highlights differences in temperature sensitivity between life stages, as there was no direct effect of pupal temperature on starvation tolerance. Therefore, for some regions of sub-Saharan Africa, climate change may result in a higher mortality rate in emerging tsetse while they search for their first blood meal. This study reinforces existing evidence that responses to temperature are life stage specific and that plasticity may have limited capacity to buffer the effects of climate change.

Arsenophonus symbiosis with louse flies: multiple origins, coevolutionary dynamics, and metabolic significance.

Arsenophonus is a widespread insect symbiont with life strategies that vary from parasitism to obligate mutualism. In insects living exclusively on vertebrate blood, mutualistic Arsenophonus strains are presumed to provide B vitamins missing in the insect host diet. Hippoboscidae, obligate blood feeders related to tsetse flies, have been previously suggested to have acquired Arsenophonus symbionts in several independent events. Based on comparative genomic analyses of 11 Hippoboscidae-associated strains, 9 of them newly assembled, we reveal a wide range of their genomic characteristics and phylogenetic affiliations. Phylogenetic patterns and genomic traits split the strains into two different types. Seven strains display characteristics of obligate mutualists with significantly reduced genomes and long phylogenetic branches. The remaining four strains cluster on short branches, and their genomes resemble those of free-living bacteria or facultative symbionts. Both phylogenetic positions and genomic traits indicate that evolutionary history of the Hippoboscidae-Arsenophonus associations is a mixture of short-term coevolutions with at least four independent origins. The comparative approach to a reconstruction of B vitamin pathways across the available Arsenophonus genomes has produced two kinds of patterns. On one hand, it indicates the different importance of individual B vitamins in the host-symbiont interaction. While some (riboflavin, pantothenate, and folate) seem to be synthesized by all Hippoboscidae-associated obligate symbionts, pathways for others (thiamine, nicotinamide, and cobalamin) are mostly missing. On the other hand, the broad comparison has produced patterns that can serve as bases for further assessments of the pathways' completeness and functionality. IMPORTANCE Insects that live exclusively on vertebrate blood utilize symbiotic bacteria as a source of essential compounds, e.g., B vitamins. In louse flies, the most frequent symbiont originated in genus Arsenophonus, known from a wide range of insects. Here, we analyze genomic traits, phylogenetic origins, and metabolic capacities of 11 Arsenophonus strains associated with louse flies. We show that in louse flies, Arsenophonus established symbiosis in at least four independent events, reaching different stages of symbiogenesis. This allowed for comparative genomic analysis, including convergence of metabolic capacities. The significance of the results is twofold. First, based on a comparison of independently originated Arsenophonus symbioses, it determines the importance of individual B vitamins for the insect host. This expands our theoretical insight into insect-bacteria symbiosis. The second outcome is of methodological significance. We show that the comparative approach reveals artifacts that would be difficult to identify based on a single-genome analysis.

Bat blunders

Bat blunders

Malpighian tubules of Rhodnius prolixus: More than post-prandial diuresis.

Rhodnius prolixus, a major vector of Chagas disease, may be considered the model upon which the foundations of insect physiology and biochemistry were built. It is an obligate blood feeder in which the blood meal triggers growth, development and reproduction. The blood meal also triggers a post-prandial diuresis to maintain osmotic homeostasis. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in this diuresis, and much has been learned about diuresis in R. prolixus, and in other model insects. But the post-genomic era has brought new insights, identifying functions quite apart from diuresis for Malpighian tubules. Indeed, microarrays, transcriptomes, and proteomics have revealed the major roles that Malpighian tubules play in immunity, detoxification, pesticide resistance, and in tolerance to overall stress. This is particularly relevant to R. prolixus since gorging on blood creates several challenges in addition to osmotic balance. Xenobiotics may be present in the blood or toxins may be produced by metabolism of blood; and these must be neutralized and excreted. These processes have not been well described at the molecular level for Malpighian tubules of R. prolixus. This paper will review the involvement of Malpighian tubules in immunity and detoxification, identifying new aspects for Malpighian tubule physiology of R. prolixus by virtue of a transcriptome analysis. The transcriptome analysis indicates the potential of Malpighian tubules of R. prolixus to mount a robust innate immune response, and to contribute to antioxidant production and heme detoxification.

Marine vampires: Persistent, internal associations between bacteria and blood-feeding marine annelids and crustaceans.

Persistent bacterial presence is believed to play an important role in host adaptation to specific niches that would otherwise be unavailable, including the exclusive consumption of blood by invertebrate parasites. Nearly all blood-feeding animals examined so far host internal bacterial symbionts that aid in some essential aspect of their nutrition. Obligate blood-feeding (OBF) invertebrates exist in the oceans, yet symbiotic associations between them and beneficial bacteria have not yet been explored. This study describes the microbiome of 6 phylogenetically-diverse species of marine obligate blood-feeders, including leeches (both fish and elasmobranch specialists; e.g., Pterobdella, Ostreobdella, and Branchellion), isopods (e.g., Elthusa and Nerocila), and a copepod (e.g., Lernanthropus). Amplicon sequencing analysis revealed the blood-feeding invertebrate microbiomes to be low in diversity, compared to host fish skin surfaces, seawater, and non-blood-feeding relatives, and dominated by only a few bacterial genera, including Vibrio (100% prevalence and comprising 39%-81% of the average total recovered 16S rRNA gene sequences per OBF taxa). Vibrio cells were localized to the digestive lumen in and among the blood meal for all taxa examined via fluorescence microscopy. For Elthusa and Branchellion, Vibrio cells also appeared intracellularly within possible hemocytes, suggesting an interaction with the immune system. Additionally, Vibrio cultivated from four of the obligate blood-feeding marine taxa matched the dominant amplicons recovered, and all but one was able to effectively lyse vertebrate blood cells. Bacteria from 2 additional phyla and 3 families were also regularly recovered, albeit in much lower abundances, including members of the Oceanospirillaceae, Flavobacteriacea, Porticoccaceae, and unidentified members of the gamma-and betaproteobacteria, depending on the invertebrate host. For the leech Pterobdella, the Oceanospirillaceae were also detected in the esophageal diverticula. For two crustacean taxa, Elthusa and Lernanthropus, the microbial communities associated with brooded eggs were very similar to the adults, indicating possible direct transmission. Virtually nothing is known about the influence of internal bacteria on the success of marine blood-feeders, but this evidence suggests their regular presence in marine parasites from several prominent groups.

Perspective on Biology and Management of Bed Bugs: Introduction.

Bed bugs are an important group of medical and urban insect pests. They are obligate blood-feeders. Their bites may cause skin irritation and allergic reactions and, under some circumstances, may lead to mental and other health issues. Despite numerous discoveries on the biology of these obnoxious pests and progress in control strategies over the last two decades, bed bugs continue to preferentially plague those from low socioeconomic communities because the poor generally could not afford effective control options. As a result, such infestations in poorer communities serve as a reservoir for wider society. This Special Collection of the Perspective on Biology and Management of Bed Bugs presents nine original research papers on bed bug detection, insecticide performance and resistance, nonchemical treatment, fungal biopesticides, and pest management procurement and contracts. We hope that these investigative findings will spur research on safer, more affordable, and effective control options in the future.

Effects of Wolbachia elimination and B-vitamin supplementation on bed bug development and reproduction

Obligate blood feeders, such as Cimex lectularius (common bed bug), have symbiotic associations with nutritional endosymbionts that produce B-vitamins. To quantify the symbiont’s contribution to host fitness in these obligate mutualisms, the symbiont must be eliminated and its absence rigorously confirmed. We developed and validated procedures for complete elimination of Wolbachia (Wb) in bed bugs and quantified development and reproduction in bed bugs with and without Wb and with and without B-vitamins supplementation. Aposymbiotic bed bugs had slower nymphal development, reduced adult survivorship, smaller adult size, fewer eggs per female, and lower hatch rate than bed bugs that harbored Wb. In aposymbiotic bed bugs that were fed B-vitamins-supplemented blood, nymph development time, adult survivorship and hatch rate recovered to control levels, but adult size and egg number only partially recovered. These results underscore the nutritional dependence of bed bugs on their Wb symbiont and suggest that Wb may provide additional nutritional benefits beyond the B-vitamin mix that we investigated.

Fasciola hepatica Gastrodermal Cells Selectively Release Extracellular Vesicles via a Novel Atypical Secretory Mechanism.

The liver fluke, Fasciola hepatica, is an obligate blood-feeder, and the gastrodermal cells of the parasite form the interface with the host’s blood. Despite their importance in the host–parasite interaction, in-depth proteomic analysis of the gastrodermal cells is lacking. Here, we used laser microdissection of F. hepatica tissue sections to generate unique and biologically exclusive tissue fractions of the gastrodermal cells and tegument for analysis by mass spectrometry. A total of 226 gastrodermal cell proteins were identified, with proteases that degrade haemoglobin being the most abundant. Other detected proteins included those such as proton pumps and anticoagulants which maintain a microenvironment that facilitates digestion. By comparing the gastrodermal cell proteome and the 102 proteins identified in the laser microdissected tegument with previously published tegument proteomic datasets, we showed that one-quarter of proteins (removed by freeze–thaw extraction) or one-third of proteins (removed by detergent extraction) previously identified as tegumental were instead derived from the gastrodermal cells. Comparative analysis of the laser microdissected gastrodermal cells, tegument, and F. hepatica secretome revealed that the gastrodermal cells are the principal source of secreted proteins, as well as showed that both the gastrodermal cells and the tegument are likely to release subpopulations of extracellular vesicles (EVs). Microscopical examination of the gut caeca from flukes fixed immediately after their removal from the host bile ducts showed that selected gastrodermal cells underwent a progressive thinning of the apical plasma membrane which ruptured to release secretory vesicles en masse into the gut lumen. Our findings suggest that gut-derived EVs are released via a novel atypical secretory route and highlight the importance of the gastrodermal cells in nutrient acquisition and possible immunomodulation by the parasite.

Assessment of different Acaricides resistance and uses of ethanobotanical agents for controlling ticks activity: a Review

Ticks are obligate blood feeding ectoparasites of vertebrates incurring huge production loss in livestock industry & creating serious public health problems in the world. This review explores the acaricides resistance & ethnoveterinary practices to control ticks infestation. Long-term use of acaricides has generated resistance in many tick species. The use of phyto extract might play an important role in addressing the ticksmanifestation as ethnoveterinary plant has demonstrated repellent & toxic effects on ticks. They are of course less expensive, but they are often regarded as being more effective& therefore can be developed further for tick control & management. In regard to our literature survey the biological & parasitic activities of the plant species & status of acaricides resistance are given herein. It is hoped that our review would help to facilitate selection for further investigation of Plants & Acaracides with relatively high levels of potency & a wide range of biological activities

Fluid Secretion by Malpighian Tubules of Rhodnius prolixus: Neuroendocrine Control With New Insights From a Transcriptome Analysis.

Rhodnius prolixus (the kissing bug and a major vector of Chagas disease) is an obligate blood feeder that in the case of the fifth instar consumes up to 10 times its unfed body weight in a single 20-minute feed. A post-prandial diuresis is initiated, within minutes of the start of gorging, in order to lower the mass and concentrate the nutrients of the meal. Thus, R. prolixus rapidly excretes a fluid that is high in NaCl content and hypo-osmotic to the hemolymph, thereby eliminating 50% of the volume of the blood meal within 3 hours of gorging. In R. prolixus, as with other insects, the Malpighian tubules play a critical role in diuresis. Malpighian tubules are not innervated, and their fine control comes under the influence of the neuroendocrine system that releases amines and neuropeptides as diuretic or antidiuretic hormones. These hormones act upon the Malpighian tubules via a variety of G protein-coupled receptors linked to second messenger systems that influence ion transporters and aquaporins; thereby regulating fluid secretion. Much has been discovered about the control of diuresis in R. prolixus, and other model insects, using classical endocrinological studies. The post-genomic era, however, has brought new insights, identifying novel diuretic and antidiuretic hormone-signaling pathways whilst also validating many of the classical discoveries. This paper will focus on recent discoveries into the neuroendocrine control of the rapid post-prandial diuresis in R. prolixus, in order to emphasize new insights from a transcriptome analysis of Malpighian tubules taken from unfed and fed bugs.

Argasid Ticks of Palearctic Bats: Distribution, Host Selection, and Zoonotic Importance.

The soft ticks (Ixodida: Argasidae) are ectoparasites of terrestrial vertebrates with a wide geographic distribution, occurring on all continents. These ticks are obligate blood-feeders, most of them show high degrees of host-specialization and several species in arid and tropical regions are important parasites of livestock and men. Species commonly occurring on domestic animals and man are generally well-known, with many studies focusing on their ecology, distribution or vectorial role. However, wildlife-specialist soft ticks are less studied. Nearly half of all soft tick species are bat specialists, with five species (Carios vespertilionis, Chiropterargas boueti, Chiropterargas confusus, Reticulinasus salahi, and Secretargas transgariepinus) occurring in the Western Palearctic. There is no comprehensive study on the distribution, hosts or pathogens in these soft ticks, although most species were shown to carry several viral, bacterial, or protozoan pathogens and also to occasionally infest humans. Based on a literature survey and 1,120 distinct georeferenced records, we present here the geographical range, host selection and vectorial potential for bat-specialist soft ticks occurring in the Western Palearctic (chiefly Europe, North Africa, and the Middle East). Carios vespertilionis shows the largest distribution range and was found on most host species, being ubiquitous wherever crevice-roosting bats occur. All the other species were located only in areas with Mediterranean climate, with Ch. boueti, Chiropteraragas confusus, and R. salahi are missing entirely from Europe. These three species have a host spectrum of bats roosting primarily in caves, while S. transgariepinus and Ca. vespertilionis is feeding primarily on crevice-roosting bat species. All but one of these soft tick species are known to feed on humans and may be vectors of important disease agents (Rickettsia spp., Borrelia spp., Bartonella spp., Ehrlichia spp., Babesia spp., several nairo-, and flaviviruses). As several crevice-roosting bat species show a continuous adaptation to human-altered areas, with certain species becoming common city-dwellers in the Western Palearctic, the study of bat specialist soft ticks is also important from an epidemiologic point of view.

Louse flies in Azorean and mainland populations of four Passeriformes species: A new perspective to parasite Island syndromes

Hippoboscid flies, also known as louse flies, are obligate blood-feeders ectoparasites of birds and mammals. By studying louse fly parasites of four Passeriformes species, Eurasian blackbird (Turdus merula), Eurasian blackcap (Sylvia atricapilla), common chaffinch (Fringilla coelebs) and European robin (Erithacus rubecula), with dissimilar time of colonization of Azores islands, we tested whether: (i) island host populations have lower parasite richness than the mainland one; (ii) island host populations undergo higher parasite prevalence, mean intensities and mean abundance than the mainland one; (iii) island parasite diversity are composed exclusively by specific parasites and (iv) parasite richness is positively correlated with the island area and proximity to the continent. For these purposes, 775 birds were sampled for presence of louse flies, by modified fumigation chamber method, from Azores Islands (São Miguel, Terceira and Flores) and Portugal mainland. Insular and mainland parasite assemblages were statistically compared. We record for the first time to Azores, Ornithomya fringillina and Icosta minor from Portugal mainland. Louse flies had highest prevalence and abundance from Azores Islands compared to those observed in mainland birds, especially blackbirds. The insular parasite diversity of Azores blackbirds, blackcaps and chaffinches was richer than the one observed in mainland population. None of the hippoboscid flies observed on the islands and mainland were host-specific. Thus, our findings provide an upgrade of parasite island syndromes knowledge, in the context of the ectoparasites, namely to the hippoboscid flies case.

Collecting and Monitoring for Northern Fowl Mite (Acari: Macronyssidae) and Poultry Red Mite (Acari: Dermanyssidae) in Poultry Systems.

The two most economically important poultry ectoparasites are the northern fowl mite, Ornithonyssus sylviarum (Canestrini and Fanzago), and the poultry red mite, Dermanyssus gallinae (De Geer). Both mites are obligate blood feeders but differ in where they reside. Sampling methods thus focus on-host, especially the vent feathers, for northern fowl mite and off-host, especially cracks and crevices near the nighttime roosting areas, for poultry red mite. Much remains unknown, however, about the basic biology and ecology of both mites. Here we discuss mite detection, quantification, and decision making and provide thoughts on future directions for research.

Parasitic mites alter chicken behaviour and negatively impact animal welfare

The northern fowl mite, Ornithonyssus sylviarum, is one of the most common and damaging ectoparasites of poultry. As an obligate blood feeding mite, the northern fowl mite can cause anaemia, slower growth, and decreased egg production of parasitized birds. However, the impact of mites or other ectoparasites on hen behaviour or welfare is not well studied. Here, we use activity sensors (three-axis accelerometers) affixed to individual birds to continuously record hen movement before, during, and after infestation with mites. Movements recorded by sensors were identified to specific bird behaviours through a previously trained algorithm, with frequency of these behaviours recorded for individual birds. Hen welfare was also determined before, during, and after mite infestation of hens using animal-based welfare metrics. Northern fowl mites significantly increased hen preening behaviour and resulted in increased skin lesions of infested birds.

An insight into the sialome, mialome and virome of the horn fly, Haematobia irritans

BackgroundThe horn fly (Haematobia irritans) is an obligate blood feeder that causes considerable economic losses in livestock industries worldwide. The control of this cattle pest is mainly based on insecticides; unfortunately, in many regions, horn flies have developed resistance. Vaccines or biological control have been proposed as alternative control methods, but the available information about the biology or physiology of this parasite is rather scarce.ResultsWe present a comprehensive description of the salivary and midgut transcriptomes of the horn fly (Haematobia irritans), using deep sequencing achieved by the Illumina protocol, as well as exploring the virome of this fly. Comparison of the two transcriptomes allow for identification of uniquely salivary or uniquely midgut transcripts, as identified by statistically differential transcript expression at a level of 16 x or more. In addition, we provide genomic highlights and phylogenetic insights of Haematobia irritans Nora virus and present evidence of a novel densovirus, both associated to midgut libraries of H. irritans.ConclusionsWe provide a catalog of protein sequences associated with the salivary glands and midgut of the horn fly that will be useful for vaccine design. Additionally, we discover two midgut-associated viruses that infect these flies in nature. Future studies should address the prevalence, biological effects and life cycles of these viruses, which could eventually lead to translational work oriented to the control of this economically important cattle pest.

Surface sterilization methods impact measures of internal microbial diversity in ticks

BackgroundTicks are obligate blood feeders transmitting major pathogens worldwide. Over the past few years, considerable research efforts have focused on the diversity, distribution and impact of gut and intracellular bacterial symbionts on tick development and tick-borne pathogen transmission. The study of this internal microbiome requires the use of a sterilization method to remove external (i.e. cuticular) microbes present on the tick’s surface and to avoid any further contamination. Several sterilization methods exist, including ethanol- or bleach-based treatments that are both effective in killing microbes but with different potential effects on DNA denaturation.MethodsWe examined how these different sterilization methods impact the measure of internal microbial diversity hosted by the Cayenne tick Amblyomma cajennense (sensu stricto). Bacterial barcoding investigations based on 16S rRNA gene sequences were conducted on two batches of 50 individuals each: Ticks of the first batch were sterilized with bleach diluted at 1% and the second batch with 70% ethanol. Tick external microbiome was also determined from cuticle smearing and water samples used for tick washing.ResultsBacterial barcoding investigations showed major differences between ethanol- and bleach-treated specimens. Both methods led to the detection of major intracellular bacteria associated with A. cajennense (s.s.) but ethanol-treated ticks always harbored a higher bacterial diversity than bleach-treated ticks. Further examinations of tick gut and tick external microbiome revealed that ethanol-based surface sterilization method is inefficient to eliminate the DNA of external bacteria.ConclusionsWe herein provide evidence that studies investigating the internal microbiome of ticks should consider bleach as the gold standard to efficiently remove cuticular bacterial DNA. Indeed, this method does not impact the internal bacterial diversity hosted by ticks and is thus a better method than the ethanol-based one for studying the internal microbiome.