Disentangling detrimental sand fly-mite interactions in a closed laboratory sand fly colony: implications for vector-borne disease studies and guidelines for overcoming severe mite infestations.

BACKGROUND: Vector sand fly colonies are a critical component of studies aimed at improving the understanding of the neglected tropical disease leishmaniasis and alleviating its global impact. However, among laboratory-colonized arthropod vectors of infectious diseases, the labor-intensive nature of sand fly rearing coupled with the low number of colonies worldwide has generally discouraged the widespread use of sand flies in laboratory settings. Among the different factors associated with the low productivity of sand fly colonies, mite infestations are a significant factor. Sand fly colonies are prone to infestation by mites, and the physical interactions between sand flies and mites and metabolites have a negative impact on sand fly larval development. METHODS: Mites were collected from sand fly larval rearing pots and morphologically identified using taxonomic keys. Upon identification, they were photographed with a scanning electron microscope. Several mite control measures were adopted in two different laboratories, one at the Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases-National Institutes of Health (Rockville, MD, USA), and the other at the University of Calgary (Calgary, AB, Canada). RESULTS: The mite species associated with sand fly colonies in the two laboratories were morphologically identified as Tyrophagus sp. and Stratiolaelaps scimitus. While complete eradication of mites in sand fly colonies is considered unrealistic, drastically reducing their population has been associated with higher sand fly productivity. CONCLUSIONS: We report a case of detrimental interaction between sand flies and Tyrophagus sp. and S. scimitus in a closed laboratory sand fly colony, discuss their impact on sand fly production and provide guidelines for limiting the mite population size in a closed laboratory colony leading to improved sand fly yields.

Fiebre y exantema tras picadura de garrapata / Fever and rash after tick bite

Las garrapatas duras se han convertido en los principales vectores de enfermedades infecciosas en el mundo industrializado, pudiendo transmitir a través de su picadura bacterias, virus y protozoos, además de causar procesos alérgicos y tóxicos. Dentro de las enfermedades transmitidas por garrapatas, las más frecuentes en nuestro medio son: la fiebre botonosa mediterránea, la enfermedad de Lyme y la enfermedad de Debonel/Tibola. La fiebre botonosa mediterránea es la rickettsiosis más frecuente en Europa. Se ha observado un aumento de los casos en los últimos años, en probable relación con el aumento de temperatura global. (AU)

Hard ticks have become the main vectors of infectious diseases in the industrialized world, being able to transmit bacteria, viruses and protozoa through their bite, as well as causing allergic and toxic processes. Among the tick-borne disease the most frequent in our setting are boutonneuse fever, Lyme disease and Debonel/Tibola disease. Boutonneuse fever is the most common rickettsiosis in Europe. An increase in cases has been observed in recent years, probably related to the increase in global temperature. (AU)

VectorNet: collaborative mapping of arthropod disease vectors in Europe and surrounding areas since 2010.

BackgroundArthropod vectors such as ticks, mosquitoes, sandflies and biting midges are of public and veterinary health significance because of the pathogens they can transmit. Understanding their distributions is a key means of assessing risk. VectorNet maps their distribution in the EU and surrounding areas.AimWe aim to describe the methodology underlying VectorNet maps, encourage standardisation and evaluate output.Methods: Vector distribution and surveillance activity data have been collected since 2010 from a combination of literature searches, field-survey data by entomologist volunteers via a network facilitated for each participating country and expert validation. Data were collated by VectorNet members and extensively validated during data entry and mapping processes.ResultsAs of 2021, the VectorNet archive consisted of ca 475,000 records relating to > 330 species. Maps for 42 species are routinely produced online at subnational administrative unit resolution. On VectorNet maps, there are relatively few areas where surveillance has been recorded but there are no distribution data. Comparison with other continental databases, namely the Global Biodiversity Information Facility and VectorBase show that VectorNet has 5-10 times as many records overall, although three species are better represented in the other databases. In addition, VectorNet maps show where species are absent. VectorNet's impact as assessed by citations (ca 60 per year) and web statistics (58,000 views) is substantial and its maps are widely used as reference material by professionals and the public.ConclusionVectorNet maps are the pre-eminent source of rigorously validated arthropod vector maps for Europe and its surrounding areas.

Diversity and distribution of mosquitoes (Diptera: Culicidae) in Peru and their relationship with metaxenic diseases / Diversidad y distribución de mosquitos (Diptera: Culicidae) en el Perú y su relación con las enfermedades metaxénicas

Abstract Introduction: Tropical Andean and Amazonian ecosystems shape the great biodiversity found in Peru. However, studies on mosquitoes (Diptera: Culicidae) in the country are scarce, resulting in an information gap regarding their diversity, ecology, distribution, and abundance. Objective: To search for documented evidence on the diversity and distribution of mosquitoes (Diptera: Culicidae) in Peru, and to establish -in relation to species of medical relevance- their potential link with the epidemiological cycle of some metaxenic diseases. Materials and methods: A literature review was conducted in Google Scholar, ScienceDirect, PubMed, and SciELO, as well as in 7 databases specialized in insect taxonomy, using specific terms and their combinations by means of Boolean operators ("AND" and "OR"). Search strategy: study types: original research articles, review articles, books, and book chapters; publication period: no initial date - June 2020; languages: English, Spanish, and Portuguese. Results: A total of 99 studies were retrieved after the initial search, 84 in the 4 electronic databases and 15 in the specialized databases, of which 41 met the inclusion criteria. One additional record was included due to its relevance to the objective of the review. The studies included were original articles (n=28), review articles (n=6), books (n=5), book chapters (n=2), and undergraduate theses (n=1). Regarding the language of publication, 25 (59.52%) were published in English and 17 (40.48%) in Spanish. Conclusions: In Peru, the greatest diversity of mosquito genera is found in the departments of Loreto, Huánuco, and Madre de Dios. Knowledge of the diversity of mosquitoes in Peru that have the potential to cause health issues is relatively scarce and is focused on Aedes aegypti and some species of the Anopheles and Culex genera, thus overlooking a rich diversity distributed in the Amazonian forests, high jungle, and inter-Andean valleys.

Resumen Introducción. Los ecosistemas tropicales andinos y amazónicos determinan la gran biodiversidad presente en Perú. Sin embargo, los estudios sobre mosquitos (Diptera: Culicidae) en el país son escasos, lo que resulta en un vacío de información en cuanto a su diversidad, ecología, distribución y abundancia. Objetivos. Buscar evidencia documentada sobre la diversidad y la distribución de mosquitos en el Perú, y establecer -con relación a las especies de importancia médica- su potencial vínculo con el ciclo epidemiológico de algunas enfermedades metaxénicas. Materiales y métodos. Se realizó una revisión de la literatura en Google Académico, ScienceDirect, PubMed y Scielo, así como en siete bases de datos especializadas en taxonomía de insectos, usando términos específicos y diferentes combinaciones con operadores booleanos ("AND" y "OR"). Estrategia de búsqueda: tipos de estudios: artículos originales de investigación, artículos de revisión, libros y capítulos de libros; periodo de publicación: sin límite inicial a junio del 2020; idiomas: inglés, español y portugués. Resultados. La búsqueda inicial arrojó 99 estudios, 84 en las 4 bases de datos electrónicas y 15 en las bases de datos especializadas, de los cuales 41 cumplieron los criterios de inclusión. También se incluyó un registro adicional debido a su relevancia con el objetivo de la revisión. Los estudios incluidos fueron artículos originales (n=28), artículos de revisión (n=6), libros (n=5), capítulos de libro (n=2) y tesis de pregrado (n=1). Respecto al idioma de publicación, 25 (59.52%) estaban en inglés y 17 (40.48%), en español. Conclusiones. En Perú, la mayor diversidad de géneros de mosquitos se encuentra presente en los departamentos de Loreto, Huánuco y Madre de Dios. El conocimiento sobre la diversidad de mosquitos en Perú que tienen el potencial de generar problemas de salud es relativamente escaso y se centra en el Aedes aegypti y algunas especies de los géneros Anopheles y Culex, dejando de lado una rica diversidad distribuida en los bosques amazónicos, la selva alta y los valles interandinos.

Yellow Fever: Roles of Animal Models and Arthropod Vector Studies in Understanding Epidemic Emergence.

Yellow fever virus (YFV) is a mosquito-borne flavivirus circulating throughout the tropical and sub-tropical regions of Africa and South America. It is responsible for an estimated 30,000 deaths annually, and while there is a highly successful vaccine, coverage is incomplete, and there is no approved treatment for YFV infection. Despite advancements in the field, animal models for YFV infection remain scarce, and care must be taken to select an appropriate model for a given hypothesis. Small animal models require either adapted YFV strains or immunocompromised hosts. Non-human primates (NHPs) recapitulate human disease, but they require specialized facilities and training, are often in short supply and cost-prohibitive, and can present ethical concerns. The limitations in studying the mosquito vectors for YFV infection include inconsistency in the laboratory environment, the requirement for a high containment insectary, and difficulty in maintaining sylvatic mosquitoes. In this review, we discuss the roles of animal models and arthropod vector studies in understanding epidemic emergence.

Vector-Borne Viral Diseases as a Current Threat for Human and Animal Health-One Health Perspective.

Over the last decades, an increase in the emergence or re-emergence of arthropod-borne viruses has been observed in many regions. Viruses such as dengue, yellow fever, or zika are a threat for millions of people on different continents. On the other hand, some arboviruses are still described as endemic, however, they could become more important in the near future. Additionally, there is a group of arboviruses that, although important for animal breeding, are not a direct threat for human health. Those include, e.g., Schmallenberg, bluetongue, or African swine fever viruses. This review focuses on arboviruses and their major vectors: mosquitoes, ticks, biting midges, and sandflies. We discuss the current knowledge on arbovirus transmission, ecology, and methods of prevention. As arboviruses are a challenge to both human and animal health, successful prevention and control are therefore only possible through a One Health perspective.

The ecological significance of arthropod vectors of plant, animal, and human pathogens.

Vector management is a cornerstone in the fight against vector-borne pathogens. However, the impact on ecosystem functioning of reducing or eliminating arthropod vector populations remains poorly understood. Vectors are members of complex ecological communities, and recent studies suggest that their population suppression alters food web dynamics (bottom-up and top-down trophic cascades), inter- and intraspecific competition, and plant pollination. Other possible overlooked roles are also proposed. In this review, with examples from vectors of plant, animal, and human pathogens, we highlight that, although the ecological roles of most vector species might be redundant with other non-vector species, changes in vector abundance alter biotic interactions and, thus, are unlikely to be neutral in terms of ecosystem functioning.

A Comparative Study of Body Lice and Bed Bugs Reveals Factors Potentially Involved in Differential Vector Competence for the Relapsing Fever Spirochete Borrelia recurrentis.

Borrelia recurrentis is the causative agent of louse-borne relapsing fever and the only Borrelia species transmitted by an insect rather than a tick vector. While bed bugs (Cimex lectularius L.) are not established vectors of any human pathogens, a recent study reported that they may be competent vectors of B. recurrentis. However, many aspects of infection and transmission remain unclear in this possible secondary vector. Here, we carried out several quantitative laboratory studies to gain a better understanding of the host suitability of bed bugs relative to the established body louse vector as well as the factors that may affect the ability of bed bugs to transmit the pathogen. We fed bed bugs B. recurrentis and estimated the level and duration of infection in the hemolymph using live imaging. We performed quantitative PCR (qPCR) to examine whole-body spirochete levels and the occurrence of vertical transmission to progeny. We also developed an assay to compare the amounts of force required to release infectious hemolymph from recently engorged bed bugs and body lice. Finally, we analyzed humoral antibacterial activity in the hemolymph, hemolymph pH, and hemocyte activity in both insect species. Our results confirm that within 24 h of ingestion, B. recurrentis can penetrate the midgut epithelium of bed bugs and enter the hemolymph, overcoming a major host barrier, as in body lice. Once in the hemolymph, spirochetes remain visible for at least 4 days. Moreover, we show that bed bugs are more physically susceptible to crushing than body lice, suggesting that crushing is a feasible route for the natural dissemination of B. recurrentis from the hemolymph of bed bugs, as for body lice. Nonetheless, our data also indicate that bed bugs are suboptimal hosts for B. recurrentis, as the bacterium does not appear to proliferate to high levels or stably colonize the hemolymph and exhibits pleomorphism in this environment. In particular, our data suggest that hemolymph pH and unique cellular immune responses, rather than humoral effectors, may be involved in limiting spirochete survival in bed bugs. Notably, we document the formation of extracellular DNA traps by bed bug hemocytes for the first time. For these reasons, while bed bugs may be capable of limited transmission given their ecology, vector competence is probably minimal relative to body lice. Additional mechanistic studies of human pathogen infection of bed bugs may provide much-needed insight into the biological factors that restrict their ability to act as vectors and may reveal novel mechanisms of immunity.

Competitive Exclusion of Phytopathogenic Serratia marcescens from Squash Bug Vectors by the Gut Endosymbiont Caballeronia.

Many insects harbor microbial symbiotic partners that offer protection against pathogens, parasitoids, and other natural enemies. Mounting evidence suggests that these symbiotic microbes can play key roles in determining infection outcomes in insect vectors, making them important players in the quest to develop novel vector control strategies. Using the squash bug Anasa tristis, we investigated how the presence of Caballeronia symbionts affected the persistence and intensity of phytopathogenic Serratia marcescens within the insect vector. We reared insects aposymbiotically and with different Caballeronia isolates, infected them with S. marcescens, and then sampled the insects periodically to assess the intensity and persistence of pathogen infection. Squash bugs harboring Caballeronia consistently had much lower-intensity infections and cleared S. marcescens significantly faster than their aposymbiotic counterparts. These patterns held even when we reversed the timing of exposure to symbiont and pathogen. Taken together, these results indicate that Caballeronia symbionts play an essential role in S. marcescens infection outcomes in squash bugs and could be used to alter vector competence to enhance agricultural productivity in the future. IMPORTANCE Insect-microbe symbioses have repeatedly been shown to profoundly impact an insect's ability to vector pathogens to other hosts. The use of symbiotic microbes to control insect vector populations is of growing interest in agricultural settings. Our study examines how symbiotic microbes affect the dynamics of a plant pathogen infection within the squash bug vector Anasa tristis, a well-documented pest of squash and other cucurbit plants and a vector of Serratia marcescens, the causative agent of cucurbit yellow vine disease. We provide evidence that the symbiont Caballeronia prevents successful, long-term establishment of S. marcescens in the squash bug. These findings give us insight into symbiont-pathogen dynamics within the squash bug that could ultimately determine its ability to transmit pathogens and be leveraged to interrupt disease transmission in this system.

Evolutionary Insights into the Microneme-Secreted, Chitinase-Containing High-Molecular-Weight Protein Complexes Involved in Plasmodium Invasion of the Mosquito Midgut.

While general mechanisms by which Plasmodium ookinetes invade the mosquito midgut have been studied, details regarding the interface of the ookinete, specifically its barriers to invasion, such as the proteolytic milieu, the chitin-containing, protein cross-linked peritrophic matrix, and the midgut epithelium, remain to be understood. Here, we review our knowledge of Plasmodium chitinases and the mechanisms by which they mediate ookinetes crossing the peritrophic matrix. The integration of new genomic insights into previous findings advances our understanding of Plasmodium evolution. Recently obtained Plasmodium species genomic data enable identification of the conserved residues in the experimentally demonstrated hetero-multimeric, high-molecular-weight complex comprised of a short chitinase covalently linked to binding partners, von Willebrand factor A domain-related protein (WARP) and secreted ookinete adhesive protein (SOAP). Artificial intelligence-based high-resolution structural modeling using the DeepMind AlphaFold algorithm yielded highly informative three-dimensional structures and insights into how short chitinases, WARP, and SOAP may interact at the atomic level to form the ookinete-secreted peritrophic matrix invasion complex. Elucidating the significance of the divergence of ookinete-secreted micronemal proteins among Plasmodium species may lead to a better understanding of the ookinete invasion machinery and the coevolution of Plasmodium-mosquito interactions.

Lineage Divergence and Vector-Specific Adaptation Have Driven Chikungunya Virus onto Multiple Adaptive Landscapes.

Previous studies have shown that the adaptation of Indian Ocean lineage (IOL) chikungunya virus (CHIKV) strains for Aedes albopictus transmission was mediated by an E1-A226V substitution, followed by either a single substitution in E2 or synergistic substitutions in the E2 and E3 envelope glycoproteins. Here, we examined whether Asian lineage strains, including those that descended from the 2014 Caribbean introduction, are likely to acquire these A. albopictus-adaptive E2 substitutions. Because Asian lineage strains cannot adapt through the E1-A226V substitution due to an epistatic constraint, we first determined that the beneficial effect of these E2 mutations in IOL strains is independent of E1-A226V. We then introduced each of these E2 adaptive mutations into the Asian lineage backbone to determine if they improve infectivity for A. albopictus. Surprisingly, our results indicated that in the Asian lineage backbone, these E2 mutations significantly decreased CHIKV fitness in A. albopictus. Furthermore, we tested the effects of these mutations in Aedes aegypti and observed different results from those in A. albopictus, suggesting that mosquito species-specific factors that interact with the envelope proteins are involved in vector infection efficiency. Overall, our results indicate that the divergence between Asian lineage and IOL CHIKVs has led them onto different adaptive landscapes with differing potentials to expand their vector host range. IMPORTANCE Since its introduction into the Caribbean in October 2013, CHIKV has rapidly spread to almost the entire neotropical region. However, its potential to further spread globally, including into more temperate climates, depends in part on its ability to be transmitted efficiently by Aedes albopictus, which can survive colder winters than A. aegypti. We examined in an Asian lineage backbone A. albopictus-adaptive mutations that arose from 2005 to 2009 in Indian Ocean lineage (IOL) strains. Our results predict that the Asian CHIKV lineage now in the Americas will not readily adapt for enhanced A. albopictus transmission via the same mechanisms or adaptive mutations used previously by IOL strains. The vector species- and CHIKV lineage-specific effects caused by adaptive CHIKV envelope glycoprotein substitutions may elucidate our understanding of the mechanisms of mosquito infection and spread.

Tick gut barriers impacting tick-microbe interactions and pathogen persistence.

Ticks are regarded as one of the most ancient, unique, and highly evolved ectoparasites. They can parasitize diverse vertebrates and transmit a number of widespread infections. Once acquired from infected hosts, many tick-borne pathogens, like Borrelia burgdorferi, are confined within the tick gut lumen and are surrounded by discrete gut barriers. Such barriers include the peritrophic membrane (PM) and the dityrosine network (DTN), which are in close contact with resident microbiota and invading pathogens, influencing their survival within the vector. Herein, we review our current state of knowledge about tick-microbe interactions involving the PM and DTN structures. As a model, we will focus on Ixodes ticks, their microbiome, and the pathogen of Lyme disease. We will address the most salient findings on the structural and physiological roles of these Ixodes gut barriers on microbial interactions, with a comparison to analogous functions in other model vectors, such as mosquitoes. We will distill how this information could be leveraged towards a better understanding of the basic mechanisms of gut biology and tick-microbial interactions, which could contribute to potential therapeutic strategies in response to ticks and tick-borne infections.

Presence of two species of the Amblyomma cajennense complex (Acari: Ixodidae) and probable zones of sympatry in northwestern Colombia.

The A. cajennense tick complex has been thought to be the main vector of Rickettsia rickettsii in Central and South America. Studies in Colombia have determined the presence of species A. patinoi and A. mixtum of the A. cajennense complex, but it is unknown which species of this complex exist in northwestern Colombia. Our aim was to identify the species of the A. cajennense complex that are present in northwestern Colombia. We sampled ticks of A. cajennense sensu lato infesting equids. Females identified according to the morphology of their genital pore were selected for genetic confirmation. Specimens from each locality were selected to perform molecular and genetic analysis. Specimens were analyzed from five departments (Antioquia, Bolívar, Córdoba, Magdalena, and Sucre). Morphologically 65 specimens were identified as A. patinoi and 5 as A. mixtum. Molecular analysis allowed to confirm the morphological identification of 27 specimens. In this study A. patinoi was widely distributed in the departments of Antioquia, Bolívar, and Córdoba with allopatric and sympatric distribution in some places. These two species in the region could have unexpected effects on the epidemiology of rickettsiosis.

The genus Anaplasma: drawing back the curtain on tick-pathogen interactions.

Tick-borne illnesses pose a serious concern to human and veterinary health and their prevalence is on the rise. The interactions between ticks and the pathogens they carry are largely undefined. However, the genus Anaplasma, a group of tick-borne bacteria, has been instrumental in uncovering novel paradigms in tick biology. The emergence of sophisticated technologies and the convergence of entomology with microbiology, immunology, metabolism and systems biology has brought tick-Anaplasma interactions to the forefront of vector biology with broader implications for the infectious disease community. Here, we discuss the use of Anaplasma as an instrument for the elucidation of novel principles in arthropod-microbe interactions. We offer an outlook of the primary areas of study, outstanding questions and future research directions.

A Case for Systematic Quality Management in Mosquito Control Programmes in Europe.

The recent spread of invasive mosquito species, such as Aedes albopictus and the seasonal sporadic transmission of autochthonous cases of arboviral diseases (e.g., dengue, chikungunya, Zika) in temperate areas, such as Europe and North America, highlight the importance of effective mosquito-control interventions to reduce not only nuisance, but also major threats for public health. Local, regional, and even national mosquito control programs have been established in many countries and are executed on a seasonal basis by either public or private bodies. In order for these interventions to be worthwhile, funding authorities should ensure that mosquito control is (a) planned by competent scientific institutions addressing the local demands, (b) executed following the plan that is based on recommended and effective methods and strategies, (c) monitored regularly by checking the efficacy of the implemented actions, (d) evaluated against the set of targets, and (e) regularly improved according to the results of the monitoring. Adherence to these conditions can only be assured if a formal quality management system is adopted and enforced that ensures the transparency of effectiveness of the control operation. The current paper aims at defining the two components of this quality management system, quality assurance and quality control for mosquito control programs with special emphasis on Europe, but applicable over temperate areas.

Crop protection practices and viral zoonotic risks within a One Health framework.

Recent viral zoonotic epidemics have been attributed partially to the negative impact of human activities on ecosystem biodiversity. Agricultural activities, particularly conventional crop protection (CP) practices, are a major threat to global biodiversity, ecosystem health and human health. Here we review interactions between CP practices and viral zoonoses (VZs), the first time this has been done. It should be noted that a) VZs stand at the interface between human, animal and ecosystem health; b) some VZs involve arthropod vectors that are affected by CP practices; and c) some crop pests, or their natural enemies are vertebrate reservoirs/carriers of certain VZs, and their contact with humans or domestic animals is affected by CP practices. Our review encompasses examples highlighting interactions between VZs and CP practices, both efficiency improvement-based (i.e. conventional with agrochemical insecticides and rodenticides), substitution-based (i.e. mainly with physical/mechanical or biopesticidal pest control), and redesign-based (i.e. mainly with conservation biological pest control, including some forms of crop-livestock integration). These CP practices mainly target arthropod and vertebrate pests. They also target, to a lesser extent, weeds and plant pathogens. Conventional and some physical/mechanical control methods and some forms of biopesticidal and crop-livestock integration practices were found to have mixed outcomes in terms of VZ risk management. Conversely, practices based on biological control by habitat conservation of arthropod or vertebrate natural enemies, falling within the Agroecological Crop Protection (ACP) framework, result in VZ prevention at various scales (local to global, and short-term to long-term). ACP addresses major global challenges including climate resilience, biodiversity conservation and animal welfare, and helps integrate plant health within the extended "One Health" concept.

No evidence for African swine fever virus DNA in haematophagous arthropods collected at wild boar baiting sites in Estonia.

African swine fever (ASF) is a highly pathogenic viral disease affecting all Suidae, with Ornithodoros moubata complex soft ticks acting as the biological arthropod vectors of the causative agent, African swine fever virus (ASFV). While ASFV is also transmissible via direct contact, pig products and fomites, other arthropods may be involved in virus transmission and persistence. Therefore, we checked various groups of blood-feeding arthropods collected during summer 2017 in wild boar habitats on the Estonian Island of Saaremaa for the presence of ASFV. Saaremaa had the highest ASF infection prevalences in Estonia in 2017, with an incidence of 9% among hunted wild boar. In addition to ASFV, we tested for other selected pathogens. In total, 784 ticks, 6,274 culicoid biting midges, 77 tabanids and 757 mosquitoes were tested as individuals or pools. No ASFV-DNA was found in any of them although about 20% of the tick samples tested positive for swine DNA. By contrast, tick-borne encephalitis virus-RNA was detected in one out of 37 tick pools (2.7%) and Borrelia burgdorferi s.l.-DNA in 20 individual ticks and 17 tick pools (25.2% of all samples). No Schmallenberg virus was detected in the Culicoides specimens. In conclusion, we found no evidence for Ixodes ricinus ticks, Culicoides punctatus and Obsoletus complex biting midges, Aedes spp., Anopheles spp. and Culiseta annulata mosquitoes, and Haematopota pluvialis tabanids playing a role in ASFV transmission in the wild boar population in Estonia.

Modernizing the Toolkit for Arthropod Bloodmeal Identification.

Understanding vertebrate-vector interactions is vitally important for understanding the transmission dynamics of arthropod-vectored pathogens and depends on the ability to accurately identify the vertebrate source of blood-engorged arthropods in field collections using molecular methods. A decade ago, molecular techniques being applied to arthropod blood meal identification were thoroughly reviewed, but there have been significant advancements in the techniques and technologies available since that time. This review highlights the available diagnostic markers in mitochondrial and nuclear DNA and discusses their benefits and shortcomings for use in molecular identification assays. Advances in real-time PCR, high resolution melting analysis, digital PCR, next generation sequencing, microsphere assays, mass spectrometry, and stable isotope analysis each offer novel approaches and advantages to bloodmeal analysis that have gained traction in the field. New, field-forward technologies and platforms have also come into use that offer promising solutions for point-of-care and remote field deployment for rapid bloodmeal source identification. Some of the lessons learned over the last decade, particularly in the fields of DNA barcoding and sequence analysis, are discussed. Though many advancements have been made, technical challenges remain concerning the prevention of sample degradation both by the arthropod before the sample has been obtained and during storage. This review provides a roadmap and guide for those considering modern techniques for arthropod bloodmeal identification and reviews how advances in molecular technology over the past decade have been applied in this unique biomedical context.

Chikungunya Virus Replication Rate Determines the Capacity of Crossing Tissue Barriers in Mosquitoes.

Chikungunya virus (CHIKV) is a reemerging and rapidly spreading pathogen transmitted by mosquitoes. The emergence of new epidemic variants of the virus is associated with genetic evolutionary traits, including duplication of repeated RNA elements in the 3' untranslated region (UTR) that seemingly favor transmission by mosquitoes. The transmission potential of a given variant results from a complex interplay between virus populations and anatomical tissue barriers in the mosquito. Here, we used the wild-type CHIKV Caribbean strain and an engineered mutant harboring a deletion in the 3' UTR to dissect the interactions of virus variants with the anatomical barriers that impede transmission during the replication cycle of the virus in Aedes mosquitoes. Compared to the 3'-UTR mutant, we observed that the wild-type virus had a short extrinsic incubation period (EIP) after an infectious blood meal and was expectorated into mosquito saliva much more efficiently. We found that high viral titers in the midgut are not sufficient to escape the midgut escape barrier. Rather, viral replication kinetics play a crucial role in determining midgut escape and the transmission ability of CHIKV. Finally, competition tests in mosquitoes coinfected with wild-type and mutant viruses revealed that both viruses successfully colonized the midgut, but wild-type viruses effectively displaced mutant viruses during systemic infection due to their greater efficiency of escaping from the midgut into secondary tissues. Overall, our results uncover a link between CHIKV replication kinetics and the effect of bottlenecks on population diversity, as slowly replicating variants are less able to overcome the midgut escape barrier.IMPORTANCE It is well established that selective pressures in mosquito vectors impose population bottlenecks for arboviruses. Here, we used a CHIKV Caribbean lineage mutant carrying a deletion in the 3' UTR to study host-virus interactions in vivo in the epidemic mosquito vector Aedes aegypti We found that the mutant virus had a delayed replication rate in mosquitoes, which lengthened the extrinsic incubation period (EIP) and reduced fitness relative to the wild-type virus. As a result, the mutant virus displayed a reduced capacity to cross anatomical barriers during the infection cycle in mosquitoes, thus reducing the virus transmission rate. Our findings show how selective pressures act on CHIKV noncoding regions to select variants with shorter EIPs that are preferentially transmitted by the mosquito vector.