Knowledge, Attitudes and Practices About Zika Virus Infection Among Women of Reproductive Age in Central Brooklyn, New York City, USA.

Zika virus (ZIKV) is spread by mosquitos, sexual intercourse and vertically during pregnancy. The 2015-2016 ZIKV epidemic infected millions in the Americas and resulted in thousands of infants born with malformations. Though the clusters of severe birth defects have subsided since 2017, ZIKV transmission remains a concern throughout Latin America and the Caribbean. Travel-associated and sexually-transmitted Zika, therefore, remain potential routes of transmission for women of reproductive age and their partners. This is particularly true for communities with high immigrant and foreign-born populations in Central Brooklyn, New York. Limited information has been collected on the perception by this population of ZIKV and how high-risk women engage in preventive practices. Using a survey adapted from the WHO, we assessed engagement in mosquito-related preventive practices while traveling. Data from 483 respondents on knowledge and perceived ZIKV concern, along with demographics as correlates of engagement in preventive practices were collected using a convenience sample between September 2020 and January 2021. Data were collected via a multipronged approach using social media in REDCap. Our findings show that being white/not Hispanic, pregnant, knowledgeable and concerned about ZIKV, and having enough information about ZIKV were all significantly associated with an increased likelihood of engaging in preventive practices while traveling. Multivariable logistic modeling revealed that knowledge was significantly associated with an increased likelihood of engaging in preventive practices while traveling (AOR = 1.90, 95% CI [1.28-2.83]). These findings underscore the importance of directing tailored health education efforts to vulnerable populations.

Excess mortality in northern Haiti during the 2010 cholera epidemic.

In the course of infectious disease outbreaks, barriers to accessing health care can contribute to preventable mortality. According to the Ministry of Health of Haiti (Ministère de la Santé Publique et de la Population [MSPP]), the 2010 cholera epidemic caused 7,936 deaths from October 2010 to December 2012 in Haiti alone. We seek to quantify the excess mortality attributable to patients not seeking care during the cholera outbreak in the Nord Department in 2010-2012. Using data from a community-based retrospective survey conducted by Doctors Without Borders (Médecins Sans Frontières [MSF]) in Northern Haiti, we used logistic regression to examine the association between healthcare utilization and fatality among household members with watery diarrhea in the Communes of Borgne, Pilate, Plaisance, and Port-Margot in the Nord Department. We found that failing to seek care resulted in a 5-fold increase in the case fatality ratio among infected individuals (26%) versus those who sought care (5%). Common concerns noted for why care was not sought included travel distance to treatment centers, not attributing watery diarrhea episodes to cholera, and being unsure where to seek health care for their watery diarrhea episodes within their Communes. In conclusion, addressing transportation and information needs could increase healthcare utilization and reduce lives lost during an outbreak.

The genome of the Wollemi pine, a critically endangered "living fossil" unchanged since the Cretaceous, reveals extensive ancient transposon activity.

We present the genome of the living fossil, Wollemia nobilis, a southern hemisphere conifer morphologically unchanged since the Cretaceous. Presumed extinct until rediscovery in 1994, the Wollemi pine is critically endangered with less than 60 wild adults threatened by intensifying bushfires in the Blue Mountains of Australia. The 12 Gb genome is among the most contiguous large plant genomes assembled, with extremely low heterozygosity and unusual abundance of DNA transposons. Reduced representation and genome re-sequencing of individuals confirms a relictual population since the last major glacial/drying period in Australia, 120 ky BP. Small RNA and methylome sequencing reveal conservation of ancient silencing mechanisms despite the presence of thousands of active and abundant transposons, including some transferred horizontally to conifers from arthropods in the Jurassic. A retrotransposon burst 8-6 my BP coincided with population decline, possibly as an adaptation enhancing epigenetic diversity. Wollemia, like other conifers, is susceptible to Phytophthora, and a suite of defense genes, similar to those in loblolly pine, are targeted for silencing by sRNAs in leaves. The genome provides insight into the earliest seed plants, while enabling conservation efforts.

Host adaptation drives genetic diversity in a vector-borne disease system.

The range of hosts a pathogen can infect is a key trait, influencing human disease risk and reservoir host infection dynamics. Borrelia burgdorferi sensu stricto (Bb), an emerging zoonotic pathogen, causes Lyme disease and is widely considered a host generalist, commonly infecting mammals and birds. Yet the extent of intraspecific variation in Bb host breadth, its role in determining host competence, and potential implications for human infection remain unclear. We conducted a long-term study of Bb diversity, defined by the polymorphic ospC locus, across white-footed mice, passerine birds, and tick vectors, leveraging long-read amplicon sequencing. Our results reveal strong variation in host breadth across Bb genotypes, exposing a spectrum of genotype-specific host-adapted phenotypes. We found support for multiple niche polymorphism, maintaining Bb diversity in nature and little evidence of temporal shifts in genotype dominance, as would be expected under negative frequency-dependent selection. Passerine birds support the circulation of several human-invasive strains (HISs) in the local tick population and harbor greater Bb genotypic diversity compared with white-footed mice. Mouse-adapted Bb genotypes exhibited longer persistence in individual mice compared with nonadapted genotypes. Genotype communities infecting individual mice preferentially became dominated by mouse-adapted genotypes over time. We posit that intraspecific variation in Bb host breadth and adaptation helps maintain overall species fitness in response to transmission by a generalist vector.

Structural evolution of an immune evasion determinant shapes pathogen host tropism.

Modern infectious disease outbreaks often involve changes in host tropism, the preferential adaptation of pathogens to specific hosts. The Lyme disease-causing bacterium Borrelia burgdorferi (Bb) is an ideal model to investigate the molecular mechanisms of host tropism, because different variants of these tick-transmitted bacteria are distinctly maintained in rodents or bird reservoir hosts. To survive in hosts and escape complement-mediated immune clearance, Bb produces the outer surface protein CspZ that binds the complement inhibitor factor H (FH) to facilitate bacterial dissemination in vertebrates. Despite high sequence conservation, CspZ variants differ in human FH-binding ability. Together with the FH polymorphisms between vertebrate hosts, these findings suggest that minor sequence variation in this bacterial outer surface protein may confer dramatic differences in host-specific, FH-binding-mediated infectivity. We tested this hypothesis by determining the crystal structure of the CspZ-human FH complex, and identifying minor variation localized in the FH-binding interface yielding bird and rodent FH-specific binding activity that impacts infectivity. Swapping the divergent region in the FH-binding interface between rodent- and bird-associated CspZ variants alters the ability to promote rodent- and bird-specific early-onset dissemination. We further linked these loops and respective host-specific, complement-dependent phenotypes with distinct CspZ phylogenetic lineages, elucidating evolutionary mechanisms driving host tropism emergence. Our multidisciplinary work provides a novel molecular basis for how a single, short protein motif could greatly modulate pathogen host tropism.

Phylogenomic Diversity Elucidates Mechanistic Insights into Lyme Borreliae-Host Association.

Host association-the selective adaptation of pathogens to specific host species-evolves through constant interactions between host and pathogens, leaving a lot yet to be discovered on immunological mechanisms and genomic determinants. The causative agents of Lyme disease (LD) are spirochete bacteria composed of multiple species of the Borrelia burgdorferi sensu lato complex, including B. burgdorferi (Bb), the main LD pathogen in North America-a useful model for the study of mechanisms underlying host-pathogen association. Host adaptation requires pathogens' ability to evade host immune responses, such as complement, the first-line innate immune defense mechanism. We tested the hypothesis that different host-adapted phenotypes among Bb strains are linked to polymorphic loci that confer complement evasion traits in a host-specific manner. We first examined the survivability of 20 Bb strains in sera in vitro and/or bloodstream and tissues in vivo from rodent and avian LD models. Three groups of complement-dependent host-association phenotypes emerged. We analyzed complement-evasion genes, identified a priori among all strains and sequenced and compared genomes for individual strains representing each phenotype. The evolutionary history of ospC loci is correlated with host-specific complement-evasion phenotypes, while comparative genomics suggests that several gene families and loci are potentially involved in host association. This multidisciplinary work provides novel insights into the functional evolution of host-adapted phenotypes, building a foundation for further investigation of the immunological and genomic determinants of host association. IMPORTANCE Host association is the phenotype that is commonly found in many pathogens that preferential survive in particular hosts. The Lyme disease (LD)-causing agent, B. burgdorferi (Bb), is an ideal model to study host association, as Bb is mainly maintained in nature through rodent and avian hosts. A widespread yet untested concept posits that host association in Bb strains is linked to Bb functional genetic variation conferring evasion to complement, an innate defense mechanism in vertebrate sera. Here, we tested this concept by grouping 20 Bb strains into three complement-dependent host-association phenotypes based on their survivability in sera and/or bloodstream and distal tissues in rodent and avian LD models. Phylogenomic analysis of these strains further correlated several gene families and loci, including ospC, with host-specific complement-evasion phenotypes. Such multifaceted studies thus pave the road to further identify the determinants of host association, providing mechanistic insights into host-pathogen interaction.

Cellular and immunological mechanisms influence host-adapted phenotypes in a vector-borne microparasite.

Predicting pathogen emergence and spillover risk requires understanding the determinants of a pathogens' host range and the traits involved in host competence. While host competence is often considered a fixed species-specific trait, it may be variable if pathogens diversify across hosts. Balancing selection can lead to maintenance of pathogen polymorphisms (multiple-niche-polymorphism; MNP). The causative agent of Lyme disease, Borrelia burgdorferi (Bb), provides a model to study the evolution of host adaptation, as some Bb strains defined by their outer surface protein C (ospC) genotype, are widespread in white-footed mice and others are associated with non-rodent vertebrates (e.g. birds). To identify the mechanisms underlying potential strain × host adaptation, we infected American robins and white-footed mice, with three Bb strains of different ospC genotypes. Bb burdens varied by strain in a host-dependent fashion, and strain persistence in hosts largely corresponded to Bb survival at early infection stages and with transmission to larvae (i.e. fitness). Early survival phenotypes are associated with cell adhesion, complement evasion and/or inflammatory and antibody-mediated removal of Bb, suggesting directional selective pressure for host adaptation and the potential role of MNP in maintaining OspC diversity. Our findings will guide future investigations to inform eco-evolutionary models of host adaptation for microparasites.

Host tropism determination by convergent evolution of immunological evasion in the Lyme disease system.

Pathogens possess the ability to adapt and survive in some host species but not in others-an ecological trait known as host tropism. Transmitted through ticks and carried mainly by mammals and birds, the Lyme disease (LD) bacterium is a well-suited model to study such tropism. Three main causative agents of LD, Borrelia burgdorferi, B. afzelii, and B. garinii, vary in host ranges through mechanisms eluding characterization. By feeding ticks infected with different Borrelia species, utilizing feeding chambers and live mice and quail, we found species-level differences in bacterial transmission. These differences localize on the tick blood meal, and specifically complement, a defense in vertebrate blood, and a polymorphic bacterial protein, CspA, which inactivates complement by binding to a host complement inhibitor, Factor H (FH). CspA selectively confers bacterial transmission to vertebrates that produce FH capable of allele-specific recognition. CspA is the only member of the Pfam54 gene family to exhibit host-specific FH-binding. Phylogenetic analyses revealed convergent evolution as the driver of such uniqueness, and that FH-binding likely emerged during the last glacial maximum. Our results identify a determinant of host tropism in Lyme disease infection, thus defining an evolutionary mechanism that shapes host-pathogen associations.

Using fisher-contributed secondary fins to fill critical shark-fisheries data gaps.

Developing-world shark fisheries are typically not assessed or actively managed for sustainability; one fundamental obstacle is the lack of species and size-composition catch data. We tested and implemented a new and potentially widely applicable approach for collecting these data: mandatory submission of low-value secondary fins (anal fins) from landed sharks by fishers and use of the fins to reconstruct catch species and size. Visual and low-cost genetic identification were used to determine species composition, and linear regression was applied to total length and anal fin base length for catch-size reconstruction. We tested the feasibility of this approach in Belize, first in a local proof-of-concept study and then scaling it up to the national level for the 2017-2018 shark-fishing season (1,786 fins analyzed). Sixteen species occurred in this fishery. The most common were the Caribbean reef (Carcharhinus perezi), blacktip (C. limbatus), sharpnose (Atlantic [Rhizoprionodon terraenovae] and Caribbean [R. porosus] considered as a group), and bonnethead (Sphyrna cf. tiburo). Sharpnose and bonnethead sharks were landed primarily above size at maturity, whereas Caribbean reef and blacktip sharks were primarily landed below size at maturity. Our approach proved effective in obtaining critical data for managing the shark fishery, and we suggest the tools developed as part of this program could be exported to other nations in this region and applied almost immediately if there were means to communicate with fishers and incentivize them to provide anal fins. Outside the tropical Western Atlantic, we recommend further investigation of the feasibility of sampling of secondary fins, including considerations of time, effort, and cost of species identification from these fins, what secondary fin type to use, and the means with which to communicate with fishers and incentivize participation. This program could be a model for collecting urgently needed data for developing-world shark fisheries globally. Article impact statement: Shark fins collected from fishers yield data critical to shark fisheries management in developing nations.

Uso de Aletas Secundarias Proporcionadas por Pescadores para Llenar Vacíos Importantes de Información sobre las Pesquerías de Tiburones Resumen Con frecuencia no se evalúan las pesquerías de tiburones del mundo en desarrollo ni cuentan con un manejo activo de sustentabilidad. Uno de los principales obstáculos para esto es la falta de información sobre las especies y la composición de los tamaños en las capturas. Probamos e implementamos una estrategia nueva y potencialmente aplicable en todas partes para la recolección de estos datos: la entrega obligatoria de las aletas secundarias de bajo valor económico (aletas anales) obtenidas de los tiburones desembarcados por parte de los pescadores y el uso de estas aletas para reconstruir las especies y tamaños en la captura. Usamos identificaciones genéticas de bajo costo e identificaciones visuales para determinar la composición de las especies y aplicamos una regresión lineal a la longitud total y a la de la base de la aleta anal para la reconstrucción del tamaño en captura. Probamos la viabilidad de esta estrategia en Belice, primero en un estudio de prueba de concepto y después subiendo al nivel nacional para la temporada de pesca de tiburón 2017-2018 (1,786 aletas analizadas). Se registraron 16 especies en esta pesquería. Las más comunes fueron Carcharhinus perezi, C. limbatus, Rhizoprionodon terraenovae y R. porosus (consideradas como un grupo) y Sphyrna cf. tiburo. Las últimas tres especies fueron desembarcadas principalmente por encima del tamaño maduro, mientras que con las dos primeras especies lo hacían por debajo del tamaño maduro. Nuestra estrategia demostró ser efectiva en la obtención de información crítica para el manejo de la pesquería de tiburones y sugerimos que las herramientas desarrolladas como parte de este programa puedan ser exportadas a otras naciones en esta región y aplicadas casi de manera inmediata si existen los medios para comunicarse con los pescadores e incentivarlos a proporcionar las aletas anales. Fuera del Atlántico Occidental tropical, recomendamos una mayor investigación de la viabilidad del muestreo de aletas secundarias, incluyendo la consideración del tiempo, esfuerzo y costo de la identificación de especies a partir de estas aletas; cuál tipo de aleta secundaria utilizar; y los medios mediante los cuales comunicarse con los pescadores e incentivarlos a participar. Este programa podría ser un modelo para la recolección de información de necesidad urgente para las pesquerías del mundo en desarrollo.

Dynamically evolving novel overlapping gene as a factor in the SARS-CoV-2 pandemic.

Understanding the emergence of novel viruses requires an accurate and comprehensive annotation of their genomes. Overlapping genes (OLGs) are common in viruses and have been associated with pandemics but are still widely overlooked. We identify and characterize ORF3d, a novel OLG in SARS-CoV-2 that is also present in Guangxi pangolin-CoVs but not other closely related pangolin-CoVs or bat-CoVs. We then document evidence of ORF3d translation, characterize its protein sequence, and conduct an evolutionary analysis at three levels: between taxa (21 members of Severe acute respiratory syndrome-related coronavirus), between human hosts (3978 SARS-CoV-2 consensus sequences), and within human hosts (401 deeply sequenced SARS-CoV-2 samples). ORF3d has been independently identified and shown to elicit a strong antibody response in COVID-19 patients. However, it has been misclassified as the unrelated gene ORF3b, leading to confusion. Our results liken ORF3d to other accessory genes in emerging viruses and highlight the importance of OLGs.

Genomic Epidemiology of Vancomycin-Resistant Enterococcus faecium (VREfm) in Latin America: Revisiting The Global VRE Population Structure.

Little is known about the population structure of vancomycin-resistant Enterococcus faecium (VREfm) in Latin America (LATAM). Here, we provide a complete genomic characterization of 55 representative Latin American VREfm recovered from 1998-2015 in 5 countries. The LATAM VREfm population is structured into two main clinical clades without geographical clustering. Using the LATAM genomes, we reconstructed the global population of VREfm by including 285 genomes from 36 countries spanning from 1946 to 2017. In contrast to previous studies, our results show an early branching of animal related isolates and a further split of clinical isolates into two sub-clades within clade A. The overall phylogenomic structure of clade A was highly dependent on recombination (54% of the genome) and the split between clades A and B was estimated to have occurred more than 2,765 years ago. Furthermore, our molecular clock calculations suggest the branching of animal isolates and clinical clades occurred ~502 years ago whereas the split within the clinical clade occurred ~302 years ago (previous studies showed a more recent split between clinical an animal branches around ~74 years ago). By including isolates from Latin America, we present novel insights into the population structure of VREfm and revisit the evolution of these pathogens.

Pre-detection history of extensively drug-resistant tuberculosis in KwaZulu-Natal, South Africa.

Antimicrobial-resistant (AMR) infections pose a major threat to global public health. Similar to other AMR pathogens, both historical and ongoing drug-resistant tuberculosis (TB) epidemics are characterized by transmission of a limited number of predominant Mycobacterium tuberculosis (Mtb) strains. Understanding how these predominant strains achieve sustained transmission, particularly during the critical period before they are detected via clinical or public health surveillance, can inform strategies for prevention and containment. In this study, we employ whole-genome sequence (WGS) data from TB clinical isolates collected in KwaZulu-Natal, South Africa to examine the pre-detection history of a successful strain of extensively drug-resistant (XDR) TB known as LAM4/KZN, first identified in a widely reported cluster of cases in 2005. We identify marked expansion of this strain concurrent with the onset of the generalized HIV epidemic 12 y prior to 2005, localize its geographic origin to a location in northeastern KwaZulu-Natal ∼400 km away from the site of the 2005 outbreak, and use protein structural modeling to propose a mechanism for how strain-specific rpoB mutations offset fitness costs associated with rifampin resistance in LAM4/KZN. Our findings highlight the importance of HIV coinfection, high preexisting rates of drug-resistant TB, human migration, and pathoadaptive evolution in the emergence and dispersal of this critical public health threat. We propose that integrating whole-genome sequencing into routine public health surveillance can enable the early detection and local containment of AMR pathogens before they achieve widespread dispersal.

Whole genome sequencing identifies bacterial factors affecting transmission of multidrug-resistant tuberculosis in a high-prevalence setting.

Whole genome sequencing (WGS) can elucidate Mycobacterium tuberculosis (Mtb) transmission patterns but more data is needed to guide its use in high-burden settings. In a household-based TB transmissibility study in Peru, we identified a large MIRU-VNTR Mtb cluster (148 isolates) with a range of resistance phenotypes, and studied host and bacterial factors contributing to its spread. WGS was performed on 61 of the 148 isolates. We compared transmission link inference using epidemiological or genomic data and estimated the dates of emergence of the cluster and antimicrobial drug resistance (DR) acquisition events by generating a time-calibrated phylogeny. Using a set of 12,032 public Mtb genomes, we determined bacterial factors characterizing this cluster and under positive selection in other Mtb lineages. Four of the 61 isolates were distantly related and the remaining 57 isolates diverged ca. 1968 (95%HPD: 1945-1985). Isoniazid resistance arose once and rifampin resistance emerged subsequently at least three times. Emergence of other DR types occurred as recently as within the last year of sampling. We identified five cluster-defining SNPs potentially contributing to transmissibility. In conclusion, clusters (as defined by MIRU-VNTR typing) may be circulating for decades in a high-burden setting. WGS allows for an enhanced understanding of transmission, drug resistance, and bacterial fitness factors.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Outer surface protein polymorphisms linked to host-spirochete association in Lyme borreliae.

Lyme borreliosis is caused by multiple species of the spirochete bacteria Borrelia burgdorferi sensu lato. The spirochetes are transmitted by ticks to vertebrate hosts, including small- and medium-sized mammals, birds, reptiles, and humans. Strain-to-strain variation in host-specific infectivity has been documented, but the molecular basis that drives this differentiation is still unclear. Spirochetes possess the ability to evade host immune responses and colonize host tissues to establish infection in vertebrate hosts. In turn, hosts have developed distinct levels of immune responses when invaded by different species/strains of Lyme borreliae. Similarly, the ability of Lyme borreliae to colonize host tissues varies among different spirochete species/strains. One potential mechanism that drives this strain-to-strain variation of immune evasion and colonization is the polymorphic outer surface proteins produced by Lyme borreliae. In this review, we summarize research on strain-to-strain variation in host competence and discuss the evidence that supports the role of spirochete-produced protein polymorphisms in driving this variation in host specialization. Such information will provide greater insights into the adaptive mechanisms driving host and Lyme borreliae association, which will lead to the development of interventions to block pathogen spread and eventually reduce Lyme borreliosis health burden.

Selective constraint and adaptive potential of West Nile virus within and among naturally infected avian hosts and mosquito vectors.

Arthropod-borne viruses are among the most genetically constrained RNA viruses, yet they have a remarkable propensity to adapt and emerge. We studied wild birds and mosquitoes naturally infected with West Nile virus (WNV) in a 'hot spot' of virus transmission in Chicago, IL, USA. We generated full coding WNV genome sequences from spatiotemporally matched bird and mosquito samples using high-throughput sequencing, allowing a molecular evolutionary assessment with deep coverage. Mean FST among samples was 0.66 (±0.02 SE) and was bimodal, with mean nucleotide diversity being higher between samples (interhost πN = 0.001; πS = 0.024) than within them (intrahost πN < 0.0001; πS < 0.001). Eight genomic sites with FST > 1.01 (in the PrM, NS2a, NS3, NS4b, and 5'-noncoding genomic regions) showed bird versus mosquito variant frequency differences of >30 per cent and/or polymorphisms fixed in ≥5 host or vector individuals, suggesting host tropism for these variants. However, phylogenetic analyses demonstrated a lack of grouping by bird or mosquito, most inter-sample differences were synonymous (mean interhost πN/πS = 0.04), and there was no significant difference between hosts and vectors in either their nucleotide diversities or levels of purifying selection (mean intrahost πN/πS = 0.28 in birds and πN/πS = 0.21 in mosquitoes). This finding contrasts with the 'trade-off' and 'selective sieve' hypotheses that have been proposed and tested in the laboratory, which predict strong host versus vector effects on WNV genetic variation, with heightened selective constraint in birds alternating with heightened viral diversity in mosquitoes. Overall, our data show WNV to be highly selectively constrained within and between both hosts and vectors but still able to vary at a limited number of sites across the genome. Such site-specific plasticity in the face of overall selective constraint may offer a mechanism whereby highly constrained viruses such as WNV and its relatives can still adapt and emerge.

Transient influence of blood meal and natural environment on blacklegged tick bacterial communities.

Blacklegged ticks (Ixodes scapularis) spend the majority of their life cycle off host, typically in woodland habitat, but require a blood meal at each of three life stages (larva, nymph, adult) to reach maturity and reproduce. Blood feeding usually lasts for several days each time and as blood is imbibed, a range of known pathogens from the host may also be acquired. Using next generation sequencing of 16S rRNA gene amplicons, we examined the influence of host blood meal on the internal bacterial community within nymphal blacklegged ticks across host-seeking, feeding, blood meal digestion, and after molting into the adult stage. Results demonstrate bacterial community structuring across host and ticks with 287 taxa found exclusively in ticks, suggesting the field environment plays a significant role in shaping the internal tick microbiome. A decrease in bacterial diversity was noted from unfed nymphs through feeding/digestion and after molting into adults, suggesting that bacterial species are lost during the corresponding physiological changes. The similarity in biochemical pathways across the different tick categories suggests that the loss of bacterial taxa does not mirror a large change in microbial function. Ticks likely lose bacterial taxa after feeding, but continual exposure to bacteria from the field environment counters this loss.

Historical mitochondrial diversity in African leopards (Panthera pardus) revealed by archival museum specimens.

Once found throughout Africa and Eurasia, the leopard (Panthera pardus) was recently uplisted from Near Threatened to Vulnerable by the International Union for the Conservation of Nature (IUCN). Historically, more than 50% of the leopard's global range occurred in continental Africa, yet sampling from this part of the species' distribution is only sparsely represented in prior studies examining patterns of genetic variation at the continental or global level. Broad sampling to determine baseline patterns of genetic variation throughout the leopard's historical distribution is important, as these measures are currently used by the IUCN to direct conservation priorities and management plans. By including data from 182 historical museum specimens, faecal samples from ongoing field surveys, and published sequences representing sub-Saharan Africa, we identify previously unrecognized genetic diversity in African leopards. Our mtDNA data indicates high levels of divergence among regional populations and strongly differentiated lineages in West Africa on par with recent studies of other large vertebrates. We provide a reference benchmark of genetic diversity in African leopards against which future monitoring can be compared. These findings emphasize the utility of historical museum collections in understanding the processes that shape present biodiversity. Additionally, we suggest future research to clarify African leopard taxonomy and to differentiate between delineated units requiring monitoring or conservation action.

Genetic identification of mammalian meal source in dung beetle gut contents.

Coprophagous dung beetles are a numerically and functionally important group. Their obligatory use of mammalian dung has broad ecological implications, including providing economically and epidemiologically relevant ecosystem services. Beetle-mammal ecological networks are critically important in determining the resilience of dung beetle communities and the supply of beetle-mediated ecosystem functions. However, our understanding of dung beetle trophic networks remains incomplete. Here we report on a pilot study to evaluate the effectiveness of DNA-based analyses in identifying the source of dung beetle meals. Using beetles collected from dung piles of known provenance, we hypothesized that molecular analysis of gut content would correctly identify the mammal host, and that beetle body size would increase the odds of successful detection of mammalian DNA. We analyzed 90 specimens belonging to six beetle species. Most samples yielded mtDNA sequences from the expected mammalian species, suggesting that these methods can be an efficient tool for the investigation of dung beetle diet.