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Background Endosymbiotic bacteria inhabit a variety of arthropods including ticks and may have multiple effects on the host’s survival, reproduction or pathogen acquisition and transmission. Rhipicephalus haemaphysaloides is one of the most widely distributed tick species in China. The symbiotic bacteria composition and their impacts to R. haemaphysaloides ticks have not been studied. The present study investigated the composition of microbial community in R. haemaphysaloides ticks and then assessed the effects of endosymbionts on the host’s fecundity by antibiotic treatment experiments. Methods The microbial population of female and male R. haemaphysaloides ticks was analyzed using Illumina Miseq sequencing of 16S rRNA gene. Thirty engorged female ticks were then randomly divided into five groups and injected with ampicillin, ciprofloxacin, kanamycin, tetracycline, or phosphate-buffered solution (PBS), respectively. Effects of antibiotic treatments on maternal oviposition, egg hatching and density of endosymbionts were evaluated. Results Illumina Miseq sequencing showed that Coxiella and Rickettsia were the predominant bacterial genera inhabiting R. haemaphysaloides ticks. Antibiotic treatment experiments found that kanamycin reduced the density of Coxiella -like endosymbiont ( Coxiella -LE hereafter) in eggs, ciprofloxacin reduced the density of Rickettsia -like endosymbiont ( Rickettsia -LE), and tetracycline had effect on both endosymbionts, while ampicillin affected neither. Meanwhile hatching rates of eggs were observed to decrease greatly in the kanamycin or tetracycline-treated group but maintained in the ampicillin or ciprofloxacin-treated group. Furthermore, the reduced hatching rates were found to be associated with density of Coxiella -LE in eggs. Conclusions The findings indicate that Coxiella -LE is essential for the reproduction of R. haemaphysaloides ticks, and that kanamycin can be used to study the role of Coxiella -LE on ticks.

The cattle tick Rhipicephalus microplus is a hematophagous ectoparasite that causes important economic losses in livestock. Different species of ticks harbor a symbiont bacterium of the genus Coxiella . It was showed that a Coxiella endosymbiont from R . microplus (CERM) is a vertically transmitted mutualist symbiont, comprising 98% of the 16S rRNA sequences in both eggs and larvae. Sequencing of the bacterial genome revealed genes for biosynthetic pathways for several vitamins and key metabolic cofactors that may provide a nutritional complement to the tick host. The CERM was abundant in ovary and Malpighian tubule of fully engorged female. Tetracycline treatment of either the tick or the vertebrate host reduced levels of bacteria in progeny in 74% for eggs and 90% for larvae without major impact neither on the reproductive fitness of the adult female or on embryo development. However, CERM proved to be essential for the tick to reach the adult life stage, as under antibiotic treatment no tick was able to progress beyond the metanymph stage. Data presented here suggest that interference in the symbiotic CERM- R . microplus relationship may be useful to the development of alternative control methods, highlighting the interdependence between ticks and their endosymbionts.

Climate change ranks among the most important issues globally, affecting geographic distributions of vectors and pathogens, and inducing losses in livestock production among many other damaging effects. We characterized the potential geographic distribution of the ticks Rhipicephalus ( Boophilus ) microplus , an important vector of babesiosis and anaplasmosis globally. We evaluated potential geographic shifts in suitability patterns for this species in two periods (2050 and 2070) and under two emissions scenarios (RCPs 4.5 and 8.5). Our results anticipate increases in suitability worldwide, particularly in the highest production areas for cattle. The Indo-Malayan region resulted in the highest cattle exposure under both climate change projections (2050), with increases in suitability of > 30%. This study illustrates how ecological niche modeling can be used to explore probable effects of climate change on disease vectors, and the possible consequences on economic dimensions.

Ticks are widespread arthropods that transmit microorganisms of veterinary and medical significance to vertebrates, including humans. Rhipicephalus simus, an ixodid tick frequently infesting and feeding on humans, may play a crucial role in transmitting infectious agents across species. Despite the known association of many Rhipicephalus ticks with phleboviruses, information on R. simus is lacking. During a study in a riverine area in Lusaka Zambia, ten R. simus ticks were incidentally collected from the grass and bushes and subjected to metagenomic next generation sequencing (mNGS) in 2 pools of 5. Analysis detected a diverse microbial profile, including bacteria 82% (32/39), fungi 15.4% (6/39), and viruses 2.6% (1/39). Notably, viral sequence LSK-ZM-102022 exhibited similarity to tick phleboviruses, sharing 74.92% nucleotide identity in the RdRp gene and 72% in the NP gene with tick-borne phlebovirus (TBPV) from Greece and Romania, respectively. Its RNA-dependent RNA polymerase (RdRp) encoding region carried conserved RdRp and endonuclease domains characteristic of phenuiviridae viruses. Phylogenetic analysis positioned LSK-ZM-102022 in a distinct but lone lineage within tick phleboviruses basal to known species like brown dog tick phlebovirus and phlebovirus Antigone. Pair-wise genetic distance analysis revealed similar findings. This study emphasizes the urgency of further research on the ecology, transmission dynamics, and pathogenic potential of LSK-ZM-102022 and related TBPVs, crucial for local and global preparedness against emerging tick-borne diseases.

Ticks are regarded as the most relevant vectors of disease-causing pathogens in domestic and wild animals. The cattle tick, Rhipicephalus (Boophilus) microplus, hinders livestock production in tropical and subtropical parts of the world where it is endemic. Tick microbiomes remain largely unexplored. The objective of this study was to explore the R. microplus microbiome by applying the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to characterize its bacterial diversity. Pyrosequencing was performed on adult males and females, eggs, and gut and ovary tissues from adult females derived from samples of R. microplus collected during outbreaks in southern Texas. Raw data from bTEFAP were screened and trimmed based upon quality scores and binned into individual sample collections. Bacteria identified to the species level include Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, Staphylococcus sciuri, Serratia marcescens, Corynebacterium glutamicum, and Finegoldia magna. One hundred twenty-one bacterial genera were detected in all the life stages and tissues sampled. The total number of genera identified by tick sample comprised: 53 in adult males, 61 in adult females, 11 in gut tissue, 7 in ovarian tissue, and 54 in the eggs. Notable genera detected in the cattle tick include Wolbachia, Coxiella, and Borrelia. The molecular approach applied in this study allowed us to assess the relative abundance of the microbiota associated with R. microplus. This report represents the first survey of the bacteriome in the cattle tick using non-culture based molecular approaches. Comparisons of our results with previous bacterial surveys provide an indication of geographic variation in the assemblages of bacteria associated with R. microplus. Additional reports on the identification of new bacterial species maintained in nature by R. microplus that may be pathogenic to its vertebrate hosts are expected as our understanding of its microbiota expands. Increased awareness of the role R. microplus can play in the transmission of pathogenic bacteria will enhance our ability to mitigate its economic impact on animal agriculture globally. This recognition should be included as part of analyses to assess the risk for re-invasion of areas like the United States of America where R. microplus was eradicated.

Background Tick hemolymph is a sterile fluid that carries nutrients to maintain tick health. The hemolymph creates a hostile environment for invaders including the destruction of microorganisms by its circulating hemocytes. However, Babesia parasites escape and disseminate to other organs through the hemolymph to continue their transmission life cycle. Still, it is unknown how tick hemocytes respond to B. bovis or B. bigemina infection. In this study, we conducted a transcriptomic analysis of hemocytes from female Rhipicephalus microplus ticks infected with Babesia parasites to understand how gene expression changes during parasite infection. Methods During Babesia acute infection, female R. microplus ticks were fed on bovines to acquire parasites. Engorged females were collected and incubated to develop Babesia kinetes in tick hemolymph. The hemolymph was examined to identify ticks that were highly infected with Babesia kinetes. Hemocyte cells were collected from replete female ticks infected with Babesia bovis or Babesia bigemina to perform high-throughput RNA-sequencing (RNA-Seq) analysis. Results This study identified major changes in the gene profile of tick hemocytes during Babesia infection. The main groups of hemocyte genes that were altered during Babesia infection were associated with metabolism, immunity, and cytoskeletal rearrangement. Upregulated genes were mainly involved in defense mechanisms, while downregulated genes were related to cell proliferation and apoptosis. However, the expression of hemocyte genes varied among Babesia species’ infections, and it reflected the changes that occurred in the tick’s physiology, including growth, reproduction, and skeletal muscle development. Conclusions The differential gene expression of R. microplus hemocytes revealed that genes highly regulated upon Babesia infection were related to metabolism, tick immunity, cell growth, apoptosis, development, metabolism, and reproduction. Additional research is necessary to further define the genes that exhibited varying expression levels in hemocytes during the infection. The findings of this study will enhance our understanding on how Babesia parasites survive in the hostile environment of ticks and perpetuate their transmission cycle, ultimately contributing to the spread of bovine babesiosis. Graphical Abstract

The cattle tick, Rhipicephalus microplus (Acari: Ixodidae), is a multi-billion dollar ectoparasite of global importance affecting beef and milk production. Submerged cultures of cosmopolitan entomopathogenic fungal species of the genus Metarhizium typically produce microsclerotia that provide both long-term survival and environmental resistance. Microsclerotia hold great potential as an unconventional active propagule to control this tick under laboratory and semi-field conditions. However, heat stress caused especially by elevated temperatures poses a critical environmental constraint for the successful development and efficacy of microsclerotia under tropical conditions. First, we screened six strains of Metarhizium anisopliae, Metarhizium robertsii and Metarhizium humberi for their ability to produce microsclerotia by submerged liquid cultivation. In addition, we assessed the biological fitness and bioefficacy of dried microsclerotial pellets under amenable (27 °C) and heat-stressed (32 °C) incubation against engorged adult females of R. microplus. Microsclerotia in pelletized formulation prepared with carriers based on diatomaceous earth and microcrystalline cellulose exhibited conidial production at different extents according to the fungal strain and the incubation temperature, but most strains displayed reduced sporogenesis when exposed to 32 °C. Engorged tick females exposed to sporulated microsclerotia from pelletized M. anisopliae CG47 or IP 119 had fewer number of hatching larvae in comparison to the control group, irrespective of the incubation temperature tested. The minimum dosage of microsclerotial pellets that effectively reduced hatchability of tick larvae was estimated to be 2 mg per plate (equivalent to 6.0 kg per hectare). Metarhizium microsclerotial pellets exhibited significant tolerance to 32 °C and pronounced acaricidal activity against this economically important ectoparasite of cattle, even under simulated environmental heat stress.Key points• Heat stress affects conidial production by microsclerotia of most pelletized Metarhizium strains• Heat stress does not impair the acaricidal performance of pelletized microsclerotia• Pellet formulation of Metarhizium microsclerotia is a promising mycoacaricide

Background Vector-borne pathogens are the subject of several investigations due to the zoonotic concern of some of them. However, limited data are available about the simultaneous presence of these pathogens in cats and their ectoparasites. The aim of the present study was to define the species of ectoparasites found on cats as well as to investigate vector-borne pathogens in cats and their ectoparasites in southern Italy. Methods Blood from 42 cats and fleas or flea pools ( n  = 28) and ticks ( n  = 73) collected from them were investigated by quantitative PCR for the detection of vector-borne pathogens. Feline serum samples were tested by IFAT to detect IgG antibodies against Leishmania infantum, Bartonella henselae , Rickettsia conorii, Rickettsia felis, Rickettsia typhi , Babesia microti , Ehrlichia canis and Anaplasma phagocytophilum antigens. Results Only one flea species ( Ctenocephalides felis ) and four tick species belonging to the genera Rhipicephalus and Ixodes were identified on cats from southern Italy. Molecular evidence of Bartonella spp., Rickettsia spp., hemoplasmas, Babesia vogeli and L. infantum was found in ectoparasites (fleas and/or ticks) while DNA from Hepatozoon felis and Ehrlichia/Anaplasma spp. was not detected. Likewise, DNAs from Bartonella, hemoplasma and Leishmania were the only pathogens amplified from feline blood samples. Cats had also antibodies against all the investigated pathogens with the exception of Rickettsia typhi . Agreement between serological and molecular results in individual cats and their ectoparasites was not found. The only exception was for Bartonella with a fair to moderate agreement between individual cats and their ectoparasites. Bartonella clarridgeiae was the species most frequently found in cats and their fleas followed by B. henselae . Conclusions In conclusion, cats harboring ticks and fleas are frequently exposed to vector-borne pathogens. Furthermore, ticks and fleas harbored by cats frequently carry pathogens of zoonotic concern therefore appropriate feline ectoparasiticide preventative treatments should be used in cats.

Background Metabolic responses to infection differ based on arthropod and pathogen. Increased metabolic rates can result in faster depletion of energetic resources, and decreases may allow for energy conservation. Babesia bovis is a protozoan pathogen transmitted by the cattle fever tick, Rhipicephalus microplus . Adult female ticks acquire B. bovis by feeding on an infected animal. Babesia bovis undergoes development and invades the ovaries where it is transmitted transovarially to tick offspring. The effects of infection on R. microplus metabolic rate are not well studied. Methods We tested the hypothesis that R. microplus infected with B. bovis would have altered metabolic rates (volume of carbon dioxide [VCO 2 ]) across life stages using flow-through respirometry. Replete females from either an infected or naïve calf were measured across 3 days to determine differences in VCO 2 . Hemolymph smears were used to categorize the number of B. bovis kinetes present in the hemolymph of replete females during egg oviposition. The VCO 2 for groups of their offspring were measured twice as eggs and once as larvae. The number of individuals and successfully hatched larvae in each group were enumerated at the end of the experiment to determine the average VCO 2 per individual. Results Infected replete females have decreased VCO 2 while their offspring have increased VCO 2 at the egg and larval stages. Interestingly, replete females had a 25% reduction in body mass compared to uninfected female tick controls. Uninfected larvae were twice as likely to hatch than larvae from infected replete female ticks. Conclusions VCO 2 varied between control and infected ticks depending on life stage. Infected replete females had lower VCO 2 and body mass while their offspring had higher VCO 2 than their control counterparts. Higher larval VCO 2 may promote earlier questing and a shorter lifespan. Changes in metabolic and hatch rates have implications that may promote disease spread. Graphical Abstract

The current study investigates the presence and prevalence of Anaplasma species in dogs from the Galapagos Islands, focusing on the potential vectorial role of Rhipicephalus linnaei in the transmission of these pathogens. Blood samples were collected from 1221 dogs across four islands, with tick collections for morphological and genetic identification. The results revealed a significant molecular prevalence of Anaplasma phagocytophilum (20.3%), predominantly in Santa Cruz (35.16%) and Isabela (18.9%), while A. platys was identified in 2.9% of samples. Genetic analysis identified the presence of A. phagocytophilum ecotype I, aligning more closely with European strains. Furthermore, R. linnaei was confirmed as the only tick species associated with dogs, suggesting its role as a vector for both A. phagocytophilum and A. platys . This study marks the first molecular confirmation of these pathogens in the Galapagos, contributing with important insights into the epidemiology of tick‐borne diseases in this ecosystem. The findings highlight the need for improved surveillance and control to reduce the risk and further spread of these tick‐borne diseases.