Pathogens Of Vertebrates Research Articles

Characterization of the Blood Bacterial Microbiota in Lowland Tapirs (Tapirus terrestris), a Vulnerable Species in Brazil

Microbiome studies targeting hypervariable regions of the 16S rRNA gene are suitable for understanding interactions between animals and their associated bacteria. While many studies focus on the gut microbiome, assessments of blood microbiota remain scarce despite the prevalence of blood-borne pathogens in vertebrates. This study aimed to investigate the bacterial community in blood samples from 79 living and 7 road-killed lowland tapirs (Tapirus terrestris), a vulnerable species, sampled in two biomes in midwestern Brazil: Pantanal and Cerrado. Animals were categorized by condition (living or road-killed), sex, age, and biome. V3–V4 16S rRNA fragments were obtained from 86 blood samples and 4 negative controls. After filtering contaminants, 13,742,198 sequences representing 2146 ASVs were analyzed. Alpha diversity significantly differed by condition, while beta diversity differed by condition, site, and age (adults vs. sub-adults). For living animals (79/86 samples), alpha diversity showed no significant differences, but beta diversity differed by age. Different vector-borne bacterial pathogens, including Anaplasmataceae, Bartonella, and Borrelia spp., were detected. Additionally, evidence of transient translocation of microbial communities from other body regions to the bloodstream was observed. Amplification of bacterial 16S rRNA from blood samples of wild T. terrestris provided novel information about the diversity of blood-borne microbiota of lowland tapirs, members of a poorly studied mammalian family. Next-generation sequencing proved to be a valuable tool for screening potential vector-borne pathogens in this host.

One Health Farming: Noninvasive monitoring reveals links between farm vertebrate richness and pathogen markers in outdoor hoofstock

Outdoor farming contributes to biodiversity conservation and enhances animal welfare, but also raises biosafety concerns due to livestock contact with potentially infected wildlife. Thus, there is a need to assess the balance between vertebrate species richness on farms, visits by wildlife species posing a biosafety risk, and pathogen circulation in open-air farming systems. We explored these links in a pilot study involving 15 open-air hoofstock farms (6 cattle, 5 small ruminant, and 4 pig farms), where we conducted interviews and risk point inspections and used two noninvasive tools: short-term camera trap (CT) deployment and environmental nucleic acid detection (ENAD). CTs were deployed to assess the richness of birds and mammals, as well as to determine the percentage of CTs detecting defined risk species. We also collected livestock feces and used sponges to sample surfaces for environmental DNA (eDNA), testing for nine pathogen markers. Total vertebrate richness ranged from 18 to 42 species, with waterholes significantly contributing to farm vertebrate richness, since 48.2 % of all wild vertebrates were detected at waterbodies, and 28.6 % were exclusively detected at waterholes. Pathogen markers detected at risk points correlated with those detected in livestock samples. Notably, the frequency of uidA marker detection correlated with the total number of pathogen markers detected per farm. Overall marker richness, an indicator of pathogen diversity, varied between farms, being higher in small ruminant farms compared to cattle or pig farms. At the farm level, wild vertebrate richness was negatively correlated with the richness of pathogen markers detected at risk points. Additionally, risk points with a higher probability of detecting more pathogen markers had lower vertebrate richness. Although CT-based assessments of vertebrate richness and ENAD-based pathogen marker detection are only indicators of actual biodiversity and farm health, respectively, our findings suggest that farmland vertebrate communities provide important ecosystem services and may help limit the circulation of multi-host pathogens.

Mosquito bloodmeals can be used to determine vertebrate diversity, host preference, and pathogen exposure in humans and wildlife

The surveillance and detection of zoonotic pathogens in animals is essential for predicting disease transmission pathways and the risks of spillover, but challenges include the costs, ethics and technical expertise required for vertebrate trapping, serum sampling and antibody or virus screening. Surveillance using haematophagous arthropods as a sampling tool offers a unique opportunity to obtain blood samples from a wide range of vertebrate species, allowing the study of host-mosquito associations, and host exposure to pathogens. We explored vertebrate diversity and potential Ross River virus (RRV) transmission pathways by analysing blood-fed mosquitoes collected in Brisbane, Australia. Host origins were identified using barcode sequencing, and host exposure to RRV was assessed using a modified plaque reduction neutralisation test. In total, 480 blood-fed mosquitoes were collected between February 2021 and May 2022. The host origins of 346 (72%) bloodmeals were identified, with humans (73%) and cattle (9%) comprising the dominant hosts. RRV seroprevalence was high in both vertebrate species with evidence of RRV exposure in 70% (21/30) of cattle and 52% (132/253) of humans. This is a novel, non-invasive method of estimating seroprevalence in vertebrate host populations. Our results highlight the potential of blood-fed mosquitoes to provide species-specific insights into pathogen transmission dynamics.

Microbiota diversity and anti-Pseudogymnoascus destructans bacteria isolated from Myotis pilosus skin during late hibernation.

Symbiotic microorganisms that reside on the host skin serve as the primary defense against pathogens in vertebrates. Specifically, the skin microbiome of bats may play a crucial role in providing resistance against Pseudogymnoascus destructans (Pd), the pathogen causing white-nose syndrome. However, the epidermis symbiotic microbiome and its specific role in resisting Pd in highly resistant bats in Asia are still not well understood. In this study, we collected and characterized skin microbiota samples of 19 Myotis pilosus in China and explored the differences between Pd-positive and negative individuals. We identified inhibitory effects of these bacteria through cultivation methods. Our results revealed that the Simpson diversity index of the skin microbiota for positive individuals was significantly lower than that of negative individuals, and the relative abundance of Pseudomonas was significantly higher in positive bats. Regardless of whether individuals were positive or negative for Pd, the relative abundance of potentially antifungal genera in skin microbiota was high. Moreover, we successfully isolated 165 microbes from bat skin and 41 isolates from positive individuals able to inhibit Pd growth compared to only 12 isolates from negative individuals. A total of 10 genera of Pd-inhibiting bacteria were screened, among which the genera Algoriella, Glutamicibacter, and Psychrobacter were newly discovered as Pd-inhibiting genera. These Pd-inhibiting bacteria metabolized a variety of volatile compounds, including dimethyl trisulfide, dimethyl disulfide, propylene sulfide, 2-undecanone, and 2-nonanone, which were able to completely inhibit Pd growth at low concentrations.IMPORTANCERecently, white-nose syndrome has caused the deaths of millions of hibernating bats, even threatening some with regional extinction. Bats in China with high resistance to Pseudogymnoascus destructans can provide a powerful reference for studying the management of white-nose syndrome and understanding the bats against the pathogen's intrinsic mechanisms. This study sheds light on the crucial role of host symbiotic skin microorganisms in resistance to pathogenic fungi and highlights the potential for harnessing natural defense mechanisms for the prevention and treatment of white-nose syndrome. In addition, this may also provide promising candidates for the development of bioinsecticides and fungicides that offer new avenues for addressing fungal diseases in wildlife and agricultural environments.

The hidden RNA viruses in Blattodea (cockroaches and termites).

The insect order Blattodea (cockroaches and termites) has drawn substantial research attention for their dietary habits and lifestyle of living with or around humans. In the present study, we focused on the discovery of RNA viruses hidden in Blattodea insects using the publicly available RNA sequencing datasets. Overall, 136 distinctive RNA viruses were identified from 36 Blattodea species, of which more than 70 % were most closely related to the invertebrate-associated viral groups within Picornavirales, Sobelivirales, Bunyaviricetes, Jingchuvirales, Durnavirales, Lispiviridae, Orthomyxoviridae, Permutotetraviridae, Flaviviridae and Muvirales. Several viruses were associated with pathogens of vertebrates (Paramyxoviridae), plants (Tymovirales), protozoa (Totiviridae), fungi (Narnaviridae) and bacteria (Norzivirales). Collectively, 93 complete or near-complete viral genomes were retrieved from the datasets, and several viruses appeared to have remarkable temporal and spatial distributions. Interestingly, the newly identified Periplaneta americana dicistrovirus displayed a remarkable distinct bicistronic genome arrangement from the well-recognized dicistroviruses with the translocated structural and non-structural polyprotein encoding open reading frames over the genome. These results significantly enhance our knowledge of RNA virosphere in Blattodea insects, and the novel genome architectures in dicistroviruses and other RNA viruses may break our stereotypes in the understanding of the genomic evolution and the emergence of potential novel viral species.

No Ranaviral DNA Found in Australian Freshwater Turtles, 2014-19, Despite Previous Serologic Evidence.

Ranaviruses are pathogens of ectothermic vertebrates (fish, amphibians, and reptiles). Turtles are the most common group of reptiles reported with ranaviral infections. However, there have been no surveys for wild ranaviral infection in any turtles from the suborder Pleurodira, despite ranaviral distributions and experimentally susceptible pleurodiran turtle populations overlapping in several areas, including Australia. We assayed 397 pooled blood samples from six Australian freshwater turtle species collected from five different sites in northern Australia between 2014 and 2019. Historical serologic surveys in the area had found antiranaviral antibodies; however, we did not detect any ranaviral DNA in our samples. Discrepancies between historical serologic and our molecular results may be explained by low viral prevalence during the years that these samples were collected, survivorship bias, or possibly an age class bias in sampling.

Rickettsia induces strong cytoplasmic incompatibility in a predatory insect.

Rickettsia, a group of intracellular bacteria found in eukaryotes, exhibits diverse lifestyles, with some acting as vertebrate pathogens transmitted by arthropod vectors and others serving as maternally transmitted arthropod endosymbionts, some of which manipulate host reproduction for their own benefit. Two phenotypes, namely male-killing and parthenogenesis induction are known as Rickettsia-induced host reproductive manipulations, but it remains unknown whether Rickettsia can induce other types of host manipulation. In this study, we discovered that Rickettsia induced strong cytoplasmic incompatibility (CI), in which uninfected females produce no offspring when mated with infected males, in the predatory insect Nesidiocoris tenuis (Hemiptera: Miridae). Molecular phylogenetic analysis revealed that the Rickettsia strain was related to Rickettsia bellii, a common insect endosymbiont. Notably, this strain carried plasmid-encoded homologues of the CI-inducing factors (namely cifA-like and cifB-like genes), typically found in Wolbachia, which are well-known CI-inducing endosymbionts. Protein domain prediction revealed that the cifB-like gene encodes PD-(D/E)XK nuclease and deubiquitinase domains, which are responsible for Wolbachia-induced CI, as well as ovarian tumour-like (OTU-like) cysteine protease and ankyrin repeat domains. These findings suggest that Rickettsia and Wolbachia endosymbionts share underlying mechanisms of CI and that CI-inducing ability was acquired by microbes through horizontal plasmid transfer.

Persist or Perish: Can Bats Threatened with Extinction Persist and Recover from White-nose Syndrome?

Emerging mycoses are an increasing concern in wildlife and human health. Given the historical rarity of fungal pathogens in warm-bodied vertebrates, there is a need to better understand how to manage mycoses and facilitate recovery in affected host populations. We explore challenges to host survival and mechanisms of host recovery in three bat species (Myotis lucifugus, Perimyotis subflavus, and M. septentrionalis) threatened with extinction by the mycosis, white-nose syndrome (WNS) as it continues to spread across North America. We present evidence from the literature that bats surviving WNS are exhibiting mechanisms of avoidance (by selecting microclimates within roosts) and tolerance (by increasing winter fat reserves), which may help avoid costs of immunopathology incurred by a maladaptive host resistance response. We discuss management actions for facilitating species recovery that take into consideration disease pressures (e.g., environmental reservoirs) and mechanisms underlying persistence, and suggest strategies that alleviate costs of immunopathology and target mechanisms of avoidance (protect or create refugia) and tolerance (increase body condition). We also propose strategies that target population and species-level recovery, including increasing reproductive success and reducing other stressors (e.g., wind turbine mortality). The rarity of fungal pathogens paired with the increasing frequency of emerging mycoses in warm-bodied vertebrate systems, including humans, requires a need to challenge common conventions about how diseases operate, how hosts respond, and how these systems could be managed to increase probability of recovery in host populations.

An obligate microsporidian parasite modulates defense against opportunistic bacterial infection in the yellow fever mosquito, Aedes aegypti.

The microsporidium Edhazardia aedis is a parasite of the yellow fever mosquito, Aedes aegypti. This mosquito transmits multiple viruses to humans in the United States and around the world, including dengue, yellow fever, and Zika viruses. Hundreds of millions of people worldwide will become infected with one of these viruses each year. E. aedis infection significantly reduces the lifespan of Ae. aegypti and is therefore a promising novel biocontrol agent. Here, we show that when the mosquito is infected with this parasite, it is also significantly more susceptible to infection by an opportunistic bacterial pathogen, Serratia marcescens. This novel discovery suggests the mosquito's ability to control infection by other microbes is impacted by the presence of the parasite.

Limited associations between MHC diversity and reproductive success in a bird species with biparental care.

The selective pressure from pathogens on individuals can have direct consequences on reproduction. Genes from the major histocompatibility complex (MHC) are central to the vertebrate adaptive immune system and pathogen resistance. In species with biparental care, each sex has distinct reproductive roles and levels of investment, and due to a trade-off with immunity, one can expect different selective regimes acting upon the MHC of each parent. Here, we addressed whether couples combine each other's variation at MHC loci to increase their breeding success. Specifically, we used a 23-year dataset from a barn owl population (Tyto alba) to understand how MHC class Iα and IIβ functional divergence and supertypes of each parent were associated with clutch size and fledging success. We did not detect associations between MHC diversity and supertypes with the clutch size or with the fledging success. In addition, to understand the relative contribution from the MHC of the genetic parents and the social parents, we analyzed the fledging success using only a cross-fostered dataset. We found several associations of weak-to-moderate effect sizes between the father's MHC and fledging success: (i) lower MHC-Iα divergence in the genetic father increases fledging success, which might improve paternal care during incubation, and (ii) one and two MHC-IIβ DAB2 supertypes in the social father decrease and increase, respectively, fledging success, which may affect the paternal care after hatching. Furthermore, fledging success increased when both parents did not carry MHC-IIβ DAB1 supertype 2, which could suggest conditional effects of this supertype. Although our study relied on a substantial dataset, we showed that the associations between MHC diversity and reproductive success remain scarce and of complex interpretation in the barn owl. Moreover, our results highlighted the need to incorporate more than one proxy of reproductive success and several MHC classes to capture more complex associations.

Antimicrobial Peptides (AMP) in the Cell-Free Culture Media of Xenorhabdus budapestensis and X. szentirmaii Exert Anti-Protist Activity against Eukaryotic Vertebrate Pathogens including Histomonas meleagridis and Leishmania donovani Species.

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.

Species- and strain-specific differences in the phagocytosis of Prototheca: insights from live-cell imaging.

The genus Prototheca is an extremely unusual group of achlorophyllic, obligately heterotrophic algae. Six species have been identified as pathogens of vertebrates, including cattle and humans. In cattle, P. bovis is the main infectious pathogen and is associated with bovine mastitis. In contrast, human infections typically involve P. wickerhamii and are associated with a spectrum of varying clinical presentations. Prototheca spp. enter the host from the environment and are therefore likely to be initially recognized by cells of the innate immune system. However, little is known about the nature of the interaction between Prototheca spp. and host phagocytes. In the present study, we adopt a live-cell imaging approach to investigate these interactions over time. Using environmental and clinical strains, we show that P. bovis cells are readily internalized and processed by macrophages, whereas these immune cells struggle to internalize P. wickerhamii. Serum opsonization of P. wickerhamii only marginally improves phagocytosis, suggesting that this species (but not P. bovis) may have evolved mechanisms to evade phagocytosis. Furthermore, we show that inhibition of the kinases Syk or PI3K, which are both critical for innate immune signaling, drastically reduces the uptake of P. bovis. Finally, we show that genetic ablation of MyD88, a signaling adaptor critical for Toll-like receptor signaling, has little impact on uptake but significantly prolongs phagosome maturation once P. bovis is internalized. Together, our data suggest that these two pathogenic Prototheca spp. have very different host-pathogen interactions which have potential therapeutic implications for the treatment of human and animal disease.

Rapid Evolution of Glycan Recognition Receptors Reveals an Axis of Host-Microbe Arms Races beyond Canonical Protein-Protein Interfaces.

Detection of microbial pathogens is a primary function of many mammalian immune proteins. This is accomplished through the recognition of diverse microbial-produced macromolecules including proteins, nucleic acids, and carbohydrates. Pathogens subvert host defenses by rapidly changing these structures to avoid detection, placing strong selective pressures on host immune proteins that repeatedly adapt to remain effective. Signatures of rapid evolution have been identified in numerous immunity proteins involved in the detection of pathogenic protein substrates, but whether similar signals can be observed in host proteins engaged in interactions with other types of pathogen-derived molecules has received less attention. This focus on protein-protein interfaces has largely obscured the study of fungi as contributors to host-pathogen conflicts, despite their importance as a formidable class of vertebrate pathogens. Here, we provide evidence that mammalian immune receptors involved in the detection of microbial glycans have been subject to recurrent positive selection. We find that rapidly evolving sites in these genes cluster in key functional domains involved in carbohydrate recognition. Further, we identify convergent patterns of substitution and evidence for balancing selection in one particular gene, MelLec, which plays a critical role in controlling invasive fungal disease. Our results also highlight the power of evolutionary analyses to reveal uncharacterized interfaces of host-pathogen conflict by identifying genes, like CLEC12A, with strong signals of positive selection across mammalian lineages. These results suggest that the realm of interfaces shaped by host-microbe conflicts extends beyond the world of host-viral protein-protein interactions and into the world of microbial glycans and fungi.

Sargasso Sea Vibrio bacteria: Underexplored potential pathovars in a perturbed habitat

We fully sequenced the genomes of 16 Vibrio cultivars isolated from eel larvae, plastic marine debris (PMD), the pelagic brown macroalga Sargassum, and seawater samples collected from the Caribbean and Sargasso Seas of the North Atlantic Ocean. Annotation and mapping of these 16 bacterial genome sequences to a PMD-derived Vibrio metagenome-assembled genome created for this study showcased vertebrate pathogen genes closely-related to cholera and non-cholera pathovars. Phenotype testing of cultivars confirmed rapid biofilm formation, hemolytic, and lipophospholytic activities, consistent with pathogenic potential. Our study illustrates that open ocean vibrios represent a heretofore undescribed group of microbes, some representing potential new species, possessing an amalgam of pathogenic and low nutrient acquisition genes, reflecting their pelagic habitat and the substrates and hosts they colonize.

Regulatory roles of cytokines in T and B lymphocytes-mediated immunity in teleost fish

T and B lymphocytes (T and B cells) are immune effector cells that play critical roles in adaptive immunity and defend against external pathogens in most vertebrates, including teleost fish. In mammals, the development and immune response of T and B cells is associated with cytokines including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors during pathogenic invasion or immunization. Given that teleost fish have evolved a similar adaptive immune system to mammals with T and B cells bearing unique receptors (B-cell receptors (BCRs) and T-cell receptors (TCRs)) and that cytokines in general have been identified, whether the regulatory roles of cytokines in T and B cell-mediated immunity are evolutionarily conserved between mammalians and teleost fish is a fascinating question. Thus, the purpose of this review is to summarize the current knowledge of teleost cytokines and T and B cells as well as the regulatory roles of cytokines on these two types of lymphocytes. This may provide important information on the parallelisms and dissimilarities of the functions of cytokines in bony fish versus higher vertebrates, which may aid in the evaluation and development of adaptive immunity-based vaccines or immunostimulants.

Two-speed genome evolution drives pathogenicity in fungal pathogens of animals

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd, with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

Evolution of viruses in immunized populations of vertebrates

Scientists have described thousands of species of viruses, many of which are pathogens of vertebrates. Given that vertebrates have their highly sophisticated adaptive immune systems capable of memorizing pathogens, interaction with such systems should theoretically be one of the most important factors influencing the evolution of viruses. The review focuses on how acquired immunity (infection-induced and vaccine-induced) affects the most important medical characteristics of viral pathogens – transmissibility, infectivity, and virulence. Both known real examples of the evolution of viruses in immunized populations, as well as theoretical articles and the results of mathematical modeling, are considered. Special attention is paid to the SARS-CoV-2 pandemic. Methodological recommendations are given for creating vaccines and conducting vaccination campaigns in the light of the raised evolutionary issues.

Analysis of the Type 4 Effectome across the Genus Rickettsia

Rickettsia are obligate intracellular bacteria primarily carried by arthropod hosts. The genus Rickettsia contains several vertebrate pathogens vectored by hematophagous arthropods. Despite the potential for disease, our understanding of Rickettsias are limited by the difficulties associated with growing and manipulating obligate intracellular bacteria. To aid with this, our lab conducted an analysis of eight genomes and three plasmids from across the genus Rickettsia. Using OPT4e, a learning algorithm-based program designed to identify effector proteins secreted by the type 4 secretion system, we generated a putative effectome for the genus. We then consolidated effectors into homolog sets to identify effectors unique to Rickettsia with different life strategies or evolutionary histories. We also compared predicted effectors to non-effectors for differences in G+C content and gene splitting. Based on this analysis, we predicted 1571 effectors across the genus, resulting in 604 homolog sets. Each species had unique homolog sets, while 42 were present in all eight species analyzed. Effectors were flagged in association with pathogenic, tick and flea-borne Rickettsia. Predicted effectors also varied in G+C content and frequency of gene splitting as compared to non-effectors. Species effector repertoires show signs of expansion, degradation, and horizontal acquisition associated with lifestyle and lineage.

Diversity, composition, altitude, and seasonality of high-altitude windborne migrating mosquitoes in the Sahel: Implications for disease transmission.

Recent studies have reported Anopheles mosquitoes captured at high-altitude (40-290 m above ground) in the Sahel. Here, we describe this migration modality across genera and species of African Culicidae and examine its implications for disease transmission and control. As well as Anopheles, six other genera-Culex, Aedes, Mansonia, Mimomyia, Lutzia, and Eretmapodites comprised 90% of the 2,340 mosquitoes captured at altitude. Of the 50 molecularly confirmed species (N = 2,107), 33 species represented by multiple specimens were conservatively considered high-altitude windborne migrants, suggesting it is a common migration modality in mosquitoes (31-47% of the known species in Mali), and especially in Culex (45-59%). Overall species abundance varied between 2 and 710 specimens/species (in Ae. vittatus and Cx. perexiguus, respectively). At altitude, females outnumbered males 6:1, and 93% of the females have taken at least one blood meal on a vertebrate host prior to their departure. Most taxa were more common at higher sampling altitudes, indicating that total abundance and diversity are underestimated. High-altitude flight activity was concentrated between June and November coinciding with availability of surface waters and peak disease transmission by mosquitoes. These hallmarks of windborne mosquito migration bolster their role as carriers of mosquito-borne pathogens (MBPs). Screening 921 mosquitoes using pan-Plasmodium assays revealed that thoracic infection rate in these high-altitude migrants was 2.4%, providing a proof of concept that vertebrate pathogens are transported by windborne mosquitoes at altitude. Fourteen of the 33 windborne mosquito species had been reported as vectors to 25 MBPs in West Africa, which represent 32% of the MBPs known in that region and include those that inflict the heaviest burden on human and animal health, such as malaria, yellow fever, dengue, and Rift Valley fever. We highlight five arboviruses that are most likely affected by windborne mosquitoes in West Africa: Rift Valley fever, O'nyong'nyong, Ngari, Pangola, and Ndumu. We conclude that the study of windborne spread of diseases by migrating insects and the development of surveillance to map the sources, routes, and destinations of vectors and pathogens is key to understand, predict, and mitigate existing and new threats of public health.

Variation in within-host replication kinetics among virus genotypes provides evidence of specialist and generalist infection strategies across three salmonid host species.

Theory of the evolution of pathogen specialization suggests that a specialist pathogen gains high fitness in one host, but this comes with fitness loss in other hosts. By contrast, a generalist pathogen does not achieve high fitness in any host, but gains ecological fitness by exploiting different hosts, and has higher fitness than specialists in nonspecialized hosts. As a result, specialist pathogens are predicted to have greater variation in fitness across hosts, and generalists would have lower fitness variation across hosts. We test these hypotheses by measuring pathogen replicative fitness as within-host viral loads from the onset of infection to the beginning of virus clearance, using the rhabdovirus infectious hematopoietic necrosis virus (IHNV) in salmonid fish. Based on field prevalence and virulence studies, the IHNV subgroups UP, MD, and L are specialists, causing infection and mortality in sockeye salmon, steelhead, and Chinook salmon juveniles, respectively. The UC subgroup evolved naturally from a UP ancestor and is a generalist infecting all three host species but without causing severe disease. We show that the specialist subgroups had the highest peak and mean viral loads in the hosts in which they are specialized, and they had low viral loads in nonspecialized hosts, resulting in large variation in viral load across hosts. Viral kinetics show that the mechanisms of specialization involve the ability to both maximize early virus replication and avoid clearance at later times, with different mechanisms of specialization evident in different host-virus combinations. Additional nuances in the data included different fitness levels for nonspecialist interactions, reflecting different trade-offs for specialist viruses in other hosts. The generalist UC subgroup reached intermediate viral loads in all hosts and showed the smallest variation in fitness across hosts. The evolution of the UC generalist from an ancestral UP sockeye specialist was associated with fitness increases in steelhead and Chinook salmon, but only slight decreases in fitness in sockeye salmon, consistent with low- or no-cost generalism. Our results support major elements of the specialist-generalist theory, providing evidence of a specialist-generalist continuum in a vertebrate pathogen. These results also quantify within-host replicative fitness trade-offs resulting from the natural evolution of specialist and generalist virus lineages in multi-host ecosystems.