Positive associations matter: Microbial relationships drive tick microbiome composition.

Untangling how factors such as environment, host, associations among bacterial species and dispersal predict microbial composition is a fundamental challenge. In this study, we use complementary machine-learning approaches to quantify the relative role of these factors in shaping microbiome variation of the blacklegged tick Ixodes scapularis. I. scapularis is the most important vector for Borrelia burgdorferi (the causative agent for Lyme disease) in the U.S. as well as a range of other important zoonotic pathogens. Yet the relative role of the interactions between pathogens and symbionts compared to other ecological forces is unknown. We found that positive associations between microbes where the occurrence of one microbe increases the probability of observing another, including between both pathogens and symbionts, was by far the most important factor shaping the tick microbiome. Microclimate and host factors played an important role for a subset of the tick microbiome including Borrelia (Borreliella) and Ralstonia, but for the majority of microbes, environmental and host variables were poor predictors at a regional scale. This study provides new hypotheses on how pathogens and symbionts might interact within tick species, as well as valuable predictions for how some taxa may respond to changing climate.

Growth Dynamics and Antibiotic Elimination of Symbiotic Rickettsia buchneri in the Tick Ixodes scapularis (Acari: Ixodidae).

Rickettsia buchneri is the principal symbiotic bacterium of the medically significant tick Ixodes scapularis This species has been detected primarily in the ovaries of adult female ticks and is vertically transmitted, but its tissue tropism in other life stages and function with regard to tick physiology is unknown. In order to determine the function of R. buchneri, it may be necessary to produce ticks free from this symbiont. We quantified the growth dynamics of R. buchneri naturally occurring in I. scapularis ticks throughout their life cycle and compared it with bacterial growth in ticks in which symbiont numbers were experimentally reduced or eliminated. To eliminate the bacteria, we exposed ticks to antibiotics through injection and artificial membrane feeding. Both injection and membrane feeding of the antibiotic ciprofloxacin were effective at eliminating R. buchneri from most offspring of exposed females. Because of its effectiveness and ease of use, we have determined that injection of ciprofloxacin into engorged female ticks is an efficient means of clearing R. buchneri from the majority of progeny.IMPORTANCE This paper describes the growth of symbiotic Rickettsia buchneri within Ixodes scapularis through the life cycle of the tick and provides methods to eliminate R. buchneri from I. scapularis ticks.

Interspecies bacterial communication produces a delicate balance between Vibrio cholerae and the chironomid egg mass microbiome.

The evolution of antimicrobial resistance in bacterial pathogens is considered by the World Health Organization to be one of the ten most concerning public health threats facing humanity (World Health Organization, 2020). Bacterial diseases previously controllable by antibiotics are resurging and treatment options are dwindling. Cholera is one such disease. Human pathogenic strains of Vibrio cholerae cause as many as 4 million cases of disease resulting in over 100,000 deaths each year (Ali et al. 2015) and multidrug-resistant V. cholerae is now established where pandemic cholera persists. Vibrio cholerae is fundamentally an aquatic species thriving in brackish and estuarial waters. Its environmental prevalence, together with both extracellular and intracellular infection of alternative arthropod and mollusc hosts, produces a highly complex ecological milieu that is not well understood. With the absence of reliable antibiotic-based treatment options, it is necessary to build a better understanding of V. cholerae biology and ecology in order to develop alternative methods for risk modelling and disease control. In this issue of Molecular Ecology, authors Sela, Hammer, and Halpern experimentally investigated a mechanism by which V. cholerae pathogenicity is affected by interspecies quorum sensing involving an array of bacterial species from the microbiome of an alternative arthropod host, the egg mass of a chironomid midge (Diptera:Chironomidae) (Sela et al. 2020). Quorum sensing is a mechanism whereby bacteria communicate with each other using autoinducers and is known to be important, for example, in shaping virulence in a variety of pathogenic bacteria. The innovative methodologies they used, both in molecular and protein biology and reductive investigative microbiomics, are helping to develop the tools needed for understanding this understudied ecological system and fighting cholera in a post-antibiotic world.

Ehrlichia Isolate from a Minnesota Tick: Characterization and Genetic Transformation.

Ehrlichia muris subsp. eauclairensis is recognized as the etiological agent of human ehrlichiosis in Minnesota and Wisconsin. We describe the culture isolation of this organism from a field-collected tick and detail its relationship to other species of Ehrlichia The isolate could be grown in a variety of cultured cell lines and was effectively transmitted between Ixodes scapularis ticks and rodents, with PCR and microscopy demonstrating a broad pattern of dissemination in arthropod and mammalian tissues. Conversely, Amblyomma americanum ticks were not susceptible to infection by the Ehrlichia Histologic sections further revealed that the wild-type isolate was highly virulent for mice and hamsters, causing severe systemic disease that was frequently lethal. A Himar1 transposase system was used to create mCherry- and mKate-expressing EmCRT mutants, which retained the ability to infect rodents and ticks.IMPORTANCE Ehrlichioses are zoonotic diseases caused by intracellular bacteria that are transmitted by ixodid ticks. Here we report the culture isolation of bacteria which are closely related to, or the same as the Ehrlichia muris subsp. eauclairensis, a recently recognized human pathogen. EmCRT, obtained from a tick removed from deer at Camp Ripley, MN, is the second isolate of this subspecies described and is distinctive in that it was cultured directly from a field-collected tick. The isolate's cellular tropism, pathogenic changes caused in rodent tissues, and tick transmission to and from rodents are detailed in this study. We also describe the genetic mutants created from the EmCRT isolate, which are valuable tools for the further study of this intracellular pathogen.

An O-Methyltransferase Is Required for Infection of Tick Cells by Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.

Infection of Immature Ixodes scapularis (Acari: Ixodidae) by Membrane Feeding.

A reduction in the use of animals in infectious disease research is desirable for animal welfare as well as for simplification and standardization of experiments. An artificial silicone-based membrane-feeding system was adapted for complete engorgement of adult and nymphal Ixodes scapularis Say (Acari: Ixodidae), and for infecting nymphs with pathogenic, tick-borne bacteria. Six wild-type and genetically transformed strains of four species of bacteria were inoculated into sterile bovine blood and fed to ticks. Pathogens were consistently detected in replete nymphs by polymerase chain reaction. Adult ticks that ingested bacteria as nymphs were evaluated for transstadial transmission. Borrelia burgdorferi and Ehrlichia muris-like agent showed high rates of transstadial transmission to adult ticks, whereas Anaplasma phagocytophilum and Rickettsia monacensis demonstrated low rates of transstadial transmission/maintenance. Artificial membrane feeding can be used to routinely maintain nymphal and adult I. scapularis, and infect nymphs with tick-borne pathogens.

Rickettsia buchneri sp. nov., a rickettsial endosymbiont of the blacklegged tick Ixodes scapularis.

We obtained a rickettsial isolate from the ovaries of the blacklegged tick, Ixodes scapularis. The isolate (ISO7(T)) was grown in the Ixodes ricinus embryonic cell line IRE11. We characterized the isolate by transmission electron microscopy and gene sequencing. Phylogenetic analysis of 11 housekeeping genes demonstrated that the isolate fulfils the criteria to be classified as a representative of a novel rickettsial species closely related to 'Rickettsia monacensis'. These rickettsiae form a clade separate from other species of rickettsiae. Gene sequences indicated that several genes important in rickettsial motility, invasiveness and temperature adaptation were mutated (e.g. sca2, rickA, hsp22, pldA and htrA). We propose the name Rickettsia buchneri sp. nov. for this bacterium that infects the ovaries of the tick I. scapularis to acknowledge the pioneering contributions of Professor Paul Buchner (1886-1978) to research on bacterial symbionts. The type strain of R. buchneri sp. nov. is strain ISO-7(T) ( = DSM 29016(T) = ATCC VR-1814(T)).

An Ixodes scapularis cell line with a predominantly neuron-like phenotype.

The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 ß-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells.

Motility characteristics are altered for Rickettsia bellii transformed to overexpress a heterologous rickA gene.

The rickettsial protein RickA activates host cell factors associated with the eukaryotic actin cytoskeleton and is likely involved with rickettsial host cell binding and infection and the actin-based motility of spotted fever group rickettsiae. The rickA gene sequence and protein vary substantially between Rickettsia species, as do observed motility-associated phenotypes. To help elucidate the function of RickA and determine the effects of species-specific RickA variations, we compared extracellular binding, intracellular motility, and intercellular spread phenotypes of three Rickettsia bellii variants. These included two shuttle vector-transformed R. bellii strains and the wild-type isolate from which they were derived, R. bellii RML 369C. Both plasmid shuttle vectors carried spectinomycin resistance and a GFPuv reporter; one contained Rickettsia monacensis-derived rickA, and the other lacked the rickA gene. Rickettsia bellii transformed to express R. monacensis rickA highly overexpressed this transcript in comparison to its native rickA. These rickettsiae also moved at higher velocities and followed a more curved path than the negative-control transformants. A lower proportion of R. monacensis rickA-expressing bacteria ever became motile, however, and they formed smaller plaques.

The role of autophagy in tick-endosymbiont interactions: insights from Ixodes scapularis and Rickettsia buchneri.

IMPORTANCE: Ticks are second only to mosquitoes in their importance as vectors of disease agents; however, tick-borne diseases (TBDs) account for the majority of all vector-borne disease cases in the United States (approximately 76.5%), according to Centers for Disease Control and Prevention reports. Newly discovered tick species and their associated disease-causing pathogens, and anthropogenic and demographic factors also contribute to the emergence and re-emergence of TBDs. Thus, incorporating different tick control approaches based on a thorough knowledge of tick biology has great potential to prevent and eliminate TBDs in the future. Here we demonstrate that replication of a transovarially transmitted rickettsial endosymbiont depends on the tick's autophagy machinery but not on apoptosis. Our findings improve our understanding of the role of symbionts in tick biology and the potential to discover tick control approaches to prevent or manage TBDs.

Environmental Drivers of Immature Ixodes scapularis in Minnesota's Metro Area.

Research on the public health significance of Ixodes scapularis ticks in the Midwest seldom focuses on extreme weather conditions that can modulate their population dynamics and ability to transmit pathogenic organisms. In this study, we assessed whether the distributional abundance of I. scapularis immatures is associated with current and time-lagged climatic determinants either directly or indirectly. We analyzed a 20-year longitudinal small mammal live-trapping dataset within a seven-county metropolitan area in Minnesota (1998-2016) using yearly tick counts at each site to assess whether inter- and intra-annual variation in immature I. scapularis counts is associated with climate and land-use conditions. We found that (1) immature I. scapularis ticks infesting mammals expanded southwesterly over the study period, (2) eastern chipmunks, Tamias striatus, supplied a substantial proportion of nymphal blood meals, (3) a suite of climatological variables are demonstrably associated with I. scapularis presence, and abundance across sites, most notably summer vapor pressure deficit, and (4) immature I. scapularis display an affinity for deciduous forests in metro areas. Our results suggest that climatic and land-type conditions may impact host-seeking I. scapularis ticks through numerous mechanistic avenues. These findings extend our understanding of the abiotic factors supporting I. scapularis populations in metro areas of the upper Midwest with strong implications for discerning future tick-borne pathogen risk.

Metagenomic surveillance for bacterial tick-borne pathogens using nanopore adaptive sampling.

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and genomic surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) strategy facilitates immediate mapping of individual nucleotide molecules to a given reference as each molecule is being sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence DNA of multiple bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis.

Nanopore adaptive sampling for targeted mitochondrial genome sequencing and bloodmeal identification in hematophagous insects.

BACKGROUND: Blood-feeding insects are important vectors for an array of zoonotic pathogens. While previous efforts toward generating molecular resources have largely focused on major vectors of global medical and veterinary importance, molecular data across a large number of hematophagous insect taxa remain limited. Advancements in long-read sequencing technologies and associated bioinformatic pipelines provide new opportunities for targeted sequencing of insect mitochondrial (mt) genomes. For engorged hematophagous insects, such technologies can be leveraged for both insect mitogenome genome assembly and identification of vertebrate blood-meal sources. METHODS: We used nanopore adaptive sampling (NAS) to sequence genomic DNA from four species of field-collected, blood-engorged mosquitoes (Aedes and Culex spp.) and one deer fly (Chrysops sp.). NAS was used for bioinformatical enrichment of mtDNA reads of hematophagous insects and potential vertebrate blood-meal hosts using publically available mt genomes as references. We also performed an experimental control to compare results of traditional non-NAS nanopore sequencing to the mt genome enrichment by the NAS method. RESULTS: Complete mitogenomes were assembled and annotated for all five species sequenced with NAS: Aedes trivittatus, Aedes vexans, Culex restuans, Culex territans and the deer fly, Chrysops niger. In comparison to data generated during our non-NAS control experiment, NAS yielded a substantially higher proportion of reference-mapped mtDNA reads, greatly streamlining downstream mitogenome assembly and annotation. The NAS-assembled mitogenomes ranged in length from 15,582 to 16,045 bp, contained between 78.1% and 79.0% A + T content and shared the anticipated arrangement of 13 protein-coding genes, two ribosomal RNAs, and 22 transfer RNAs. Maximum likelihood phylogenies were generated to further characterize each insect species. Additionally, vertebrate blood-meal analysis was successful in three samples sequenced, with mtDNA-based phylogenetic analyses revealing that blood-meal sources for Chrysops niger, Culex restuans and Aedes trivittatus were human, house sparrow (Passer domesticus) and eastern cottontail rabbit (Sylvilagus floridanus), respectively. CONCLUSIONS: Our findings show that NAS has dual utility to simultaneously molecularly identify hematophagous insects and their blood-meal hosts. Moreover, our data indicate NAS can facilitate a wide array of mitogenomic systematic studies through novel 'phylogenetic capture' methods. We conclude that the NAS approach has great potential for broadly improving genomic resources used to identify blood-feeding insects, answer phylogenetic questions and elucidate complex pathways for the transmission of vector-borne pathogens.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

Pathogens must adapt to disparate environments in permissive host species, a feat that is especially pronounced for vector-borne microbes, which transition between vertebrate hosts and arthropod vectors to complete their lifecycles. Most knowledge about arthropod-vectored bacterial pathogens centers on their life in the mammalian host, where disease occurs. However, disease outbreaks are driven by the arthropod vectors. Adapting to the arthropod is critical for obligate intracellular rickettsial pathogens, as they depend on eukaryotic cells for survival. To manipulate the intracellular environment, these bacteria use Type IV Secretion Systems (T4SS) to deliver effectors into the host cell. To date, few rickettsial T4SS translocated effectors have been identified and have only been examined in the context of mammalian infection. We identified an effector from the tick-borne rickettsial pathogen Anaplasma phagocytophilum , HGE1_02492, as critical for survival in tick cells and acquisition by ticks in vivo . Conversely, HGE1_02492 was dispensable during mammalian cell culture and murine infection. We show HGE1_02492 is translocatable in a T4SS-dependent manner to the host cell cytosol. In eukaryotic cells, the HGE1_02492 localized with cortical actin filaments, which is dependent on multiple sub-domains of the protein. HGE1_02492 is the first arthropod-vector specific T4SS translocated effector identified from a rickettsial pathogen. Moreover, the subcellular target of HGE1_02492 suggests that A. phagocytophilum is manipulating actin to enable arthropod colonization. Based on these findings, we propose the name AteA for Anaplasma ( phagocytophilum ) tick effector A. Altogether, we show that A. phagocytophilum uses distinct strategies to cycle between mammals and arthropods. Importance: Ticks are the number one vector of pathogens for livestock worldwide and for humans in the US. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, that is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affect tick transmission is critical to developing interventions.

An Anaplasma phagocytophilum T4SS effector, AteA, is essential for tick infection.

IMPORTANCE: Ticks are the number one vector of pathogens for livestock worldwide and for humans in the United States. The biology of tick transmission is an understudied area. Understanding this critical interaction could provide opportunities to affect the course of disease spread. In this study, we examined the zoonotic tick-borne agent Anaplasma phagocytophilum and identified a secreted protein, AteA, which is expressed in a tick-specific manner. These secreted proteins, termed effectors, are the first proteins to interact with the host environment. AteA is essential for survival in ticks and appears to interact with cortical actin. Most effector proteins are studied in the context of the mammalian host; however, understanding how this unique set of proteins affects tick transmission is critical to developing interventions.

Dome1-JAK-STAT signaling between parasite and host integrates vector immunity and development.

Ancestral signaling pathways serve critical roles in metazoan development, physiology, and immunity. We report an evolutionary interspecies communication pathway involving a central Ixodes scapularis tick receptor termed Dome1, which acquired a mammalian cytokine receptor motif exhibiting high affinity for interferon-gamma (IFN-γ). Host-derived IFN-γ facilitates Dome1-mediated activation of the Ixodes JAK-STAT pathway. This accelerates tick blood meal acquisition and development while upregulating antimicrobial components. The Dome1-JAK-STAT pathway, which exists in most Ixodid tick genomes, regulates the regeneration and proliferation of gut cells-including stem cells-and dictates metamorphosis through the Hedgehog and Notch-Delta networks, ultimately affecting Ixodes vectorial competence. We highlight the evolutionary dependence of I. scapularis on mammalian hosts through cross-species signaling mechanisms that dually influence arthropod immunity and development.

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy.

Hematophagous ectoparasites, such as ticks, rely on impaired wound healing for skin attachment and blood feeding. Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to arthropod-borne diseases. Here, we used orthogonal approaches combining single-cell RNA sequencing (scRNAseq), flow cytometry, murine genetics, and intravital microscopy to demonstrate how tick extracellular vesicles (EVs) disrupt networks involved in tissue repair. Impairment of EVs through silencing of the SNARE protein vamp33 negatively impacted ectoparasite feeding and survival in three medically relevant tick species, including Ixodes scapularis. Furthermore, I. scapularis EVs affected epidermal γδ T cell frequencies and co-receptor expression, which are essential for keratinocyte function. ScRNAseq analysis of the skin epidermis in wildtype animals exposed to vamp33-deficient ticks revealed a unique cluster of keratinocytes with an overrepresentation of pathways connected to wound healing. This biological circuit was further implicated in arthropod fitness when tick EVs inhibited epithelial proliferation through the disruption of phosphoinositide 3-kinase activity and keratinocyte growth factor levels. Collectively, we uncovered a tick-targeted impairment of tissue repair via the resident γδ T cell-keratinocyte axis, which contributes to ectoparasite feeding.

Tick Artificial Membrane Feeding for Ixodes scapularis.

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Epidemiology, Distribution and Identification of Ticks on Livestock in Pakistan.

Background: Ticks are ectoparasites that transmit a variety of pathogens that cause many diseases in livestock which can result in skin damage, weight loss, anemia, reduced production of meat and milk, and mortality. Aim: The aim of this study was to identify tick species and the distribution on livestock hosts (sheep, goat, dairy cattle, and buffalo) of Punjab, Khyber Pakhtunkhwa Province and Islamabad from October 2019 to November 2020. Materials and Methods: Surveillance was performed to calculate the prevalence of ticks on livestock. Tick prevalence data (area, host, breed, gender, age, and seasonal infestation rate) was recorded and analyzed. Results: A total of 2080 animals were examined from selected farms, and, of these, 1129 animals were tick-infested. A total of 1010 male tick samples were identified to species using published keys. Haemaphysalis punctata, Haemaphysalis sulcata, Hyalomma anatolicum, Hyalomma detritum, Hyalomma dromedarii, Hyalomma excavatum, Hyalomma marginatum, Hyalomma rufipes, Rhipicephalus decoloratus Rhipicephalus microplus, and Rhipicephalus sanguineus were collected from goats, sheep, buffalo, and cattle. The overall rates of tick infestation on livestock were 34.83% (buffalo), 57.11% (cattle), 51.97% (sheep) and 46.94% (goats). Within each species, different breeds demonstrated different proportions of infestation. For cattle breeds, infestation proportions were as follows: Dhanni (98.73%), Jersey (70.84%) and the Australian breed of cattle (81.81%). The Neeli Ravi breed (40%) of buffalo and the Beetal breed (57.35%) of goats were the most highly infested for these species. Seasonally, the highest prevalence of infestation (76.78%) was observed in summer followed by 70.76% in spring, 45.29% in autumn, and 20% in winter. The prevalence of tick infestation in animals also varied by animal age. In goats, animals aged 4-6 years showed the highest prevalence (90%), but in cattle, the prevalence of ticks was highest (68.75%) in 6 months-1-year-old animals. 1-3 years old buffalo (41.07%) and 6 months-1 year sheep (65.78%) had the highest prevalence rate. Females had significantly higher infestation rates (61.12%, 55.56% and 49.26%, respectively) in cattle, sheep, and goats. In buffalo, males showed a higher prevalence (38.46%) rate. Conclusions: This study showed tick diversity, infestation rate, and numerous factors (season, age, and gender of host) influencing tick infestation rate in different breeds of cattle, sheep, goats, and buffalo in Punjab Province, Khyber Pakhtunkhwa Province, and Islamabad, Pakistan. Higher tick burdens and rates of tick-borne disease reduce production and productivity in animals. Understanding tick species' prevalence and distribution will help to develop informed control measures.

Persistence of the invasive bird-parasitic fly Philornis downsi over the host interbreeding period in the Galapagos Islands.

Many parasites of seasonally available hosts must persist through times of the year when hosts are unavailable. In tropical environments, host availability is often linked to rainfall, and adaptations of parasites to dry periods remain understudied. The bird-parasitic fly Philornis downsi has invaded the Galapagos Islands and is causing high mortality of Darwin's finches and other bird species, and the mechanisms by which it was able to invade the islands are of great interest to conservationists. In the dry lowlands, this fly persists over a seven-month cool season when availability of hosts is very limited. We tested the hypothesis that adult flies could survive from one bird-breeding season until the next by using a pterin-based age-grading method to estimate the age of P. downsi captured during and between bird-breeding seasons. This study showed that significantly older flies were present towards the end of the cool season, with ~ 5% of captured females exhibiting estimated ages greater than seven months. However, younger flies also occurred during the cool season suggesting that some fly reproduction occurs when host availability is low. We discuss the possible ecological mechanisms that could allow for such a mixed strategy.