Diversity study of Beauveria bassiana species for finding the most virulent strain to manage Bemisia tabaci in cotton.

Beauveria bassiana (Bal.-Criv.) is an important entomopathogenic fungus being used for the management of various agricultural pests worldwide. However, all strains of B. bassiana may not be effective against whitefly, Bemisia tabaci, or other pests, and strains show diversity in their growth, sporulation, virulence features, and overall bioefficacy. Thus, to select the most effective strain, a comprehensive way needs to be devised. We studied the diversity among the 102 strains of B. bassiana isolated from 19 insect species based on their physiological features, virulence, and molecular phylogeny, to identify promising ones for the management of B. tabaci. Strains showed diversity in mycelial growth, conidial production, and their virulence against B. tabaci nymphs. The highest nymphal mortality (2nd and 3rd instar) was recorded with MTCC-4511 (95.1%), MTCC-6289 (93.8%), and MTCC-4565 (89.9%) at a concentration of 1 × 106 conidia ml-1 under polyhouse conditions. The highest bioefficacy index (BI) was in MTCC-4511 (78.3%), MTCC-4565 (68.2%), and MTCC-4543 (62.1%). MTCC-4511, MTCC-4565, and MTCC-4543 clustered with positive loading of eigenvalues for the first two principal components and the cluster analysis also corresponded well with PCA (principal component analysis) (nymphal mortality and BI). The molecular phylogeny could not draw any distinct relationship between physiological features, the virulence of B. bassiana strains with the host and location. The BI, PCA, and square Euclidean distance cluster were found the most useful tools for selecting potential entomopathogenic strains. The selected strains could be utilized for the management of the B. tabaci nymphal population in the field through the development of effective formulations. KEY POINTS: • 102 B. bassiana strains showed diversity in growth and virulence against B. tabaci. • Bioefficacy index, PCA, and SED group are efficient tools for selecting potential strains. • MTCC-4511, 4565, and 4543 chosen as the most virulent strains to kill whitefly nymphs.

Effect of environmental variables on the abundance of Amblyomma ticks, potential vectors of Rickettsia parkeri in central Brazil.

Amblyomma ticks are vectors of both Rickettsia rickettsii and R. parkeri in the Americas, where capybaras (Hydrochoerus hydrochaeris) are the main hosts in urban areas, thus contributing to the transmission of spotted fever. Herein, we studied: (i) the seasonal dynamics and abundance of ticks in areas where capybaras live, (ii) the effect of environmental variables on tick abundance, and (iii) the presence of Rickettsia-infected ticks. Between September 2021 and September 2022, we sampled ticks using cloth-dragging at 194 sites on the shore of Lake Paranoá in Brasília, Brazil. We measured environmental data (season, vegetation type, canopy density, temperature, humidity, and presence or vestige of capybara) at each site. Nymphs and adults were morphologically identified to the species level, and a selected tick sample including larvae was subjected to genotypic identification. We investigated Rickettsia-infected ticks by PCR (gltA, htrA, ompB, and ompA genes) and associations between tick abundance and environmental variables using Generalized Linear Models. A total of 30,334 ticks (96% larvae) were captured. Ticks were identified as Amblyomma, with A. sculptum comprising 97% of the adult/nymphs. Genotype identification of a larval sample confirmed that 95% belonged to A. dubitatum. Seasonal variables showed significant effects on tick abundance. Most larvae and nymphs were captured during the early dry season, while the adults were more abundant during the wet season. Vegetation variables and the presence of capybaras showed no association with tick abundance. Rickettsia parkeri group and R. bellii were identified in A. dubitatum, while A. sculptum presented R. bellii. We conclude that: (i) Amblyomma ticks are widely distributed in Lake Paranoá throughout the year, especially larvae at the dry season, (ii) the abundance of Amblyomma ticks is explained more by climatic factors than by vegetation or presence of capybaras, and (iii) A. dubitatum ticks are potential vectors of R. parkeri in Brasília.

Ticks (Acari: Ixodidae) on resident and migratory wild birds in Orinoquia region, Colombia.

Several species of hard ticks, including those of the genera Ixodes, Haemaphysalis, Amblyomma, and Rhipicephalus, are of medical and veterinary importance and have been reported in association with Neotropical wild birds. Colombia, known for its great bird diversity, has 57 confirmed tick species. However, there are few studies on the association between wild birds and ticks in Colombia. The Orinoquia region, a migratory center in Colombia, provides a unique opportunity to study wild bird-tick associations and their implications for tick-borne disease dynamics. Our study, conducted between October and December 2021, aimed to identify hard ticks infesting resident and migratory wild birds in the department of Arauca and to assess the presence of bacteria from the genera Anaplasma, Borrelia, Ehrlichia, Rickettsia, and piroplasms. A total of 383 birds were examined, of which 21 were infested. We collected 147 ticks, including Amblyomma dissimile (larvae), Amblyomma longirostre (nymphs), Amblyomma mixtum (adults), and Amblyomma nodosum (larvae and nymphs). We did not detect bacterial DNA in the tested ticks; however, piroplasm DNA was detected in ticks from three of the infested birds. Of the 21 bird-tick associations, six are new to the Americas, and interesting documentation of piroplasm DNA in A. longirostre, A. nodosum, and A. dissimile ticks from wild birds in the region. This study provides valuable insights into the ticks associated with wild birds and their role in the dispersal of ticks and pathogens in Colombia, enhancing our understanding of tick life cycles and tick-borne disease dynamics.

Sequences Related to Chimay Rhabdovirus Are Widely Distributed in Ixodes ricinus Ticks across England and Wales.

Ticks are the main arthropod vector of pathogens to humans and livestock in the British Isles. Despite their role as a vector of disease, many aspects of tick biology, ecology, and microbial association are poorly understood. To address this, we investigated the composition of the microbiome of adult and nymphal Ixodes ricinus ticks. The ticks were collected on a dairy farm in Southwest England and RNA extracted for whole genome sequencing. Sequences were detected from a range of microorganisms, particularly tick-associated viruses, bacteria, and nematodes. A majority of the viruses were attributed to phlebo-like and nairo-like virus groups, demonstrating a high degree of homology with the sequences present in I. ricinus from mainland Europe. A virus sharing a high sequence identity with Chimay rhabdovirus, previously identified in ticks from Belgium, was detected. Further investigations of I. ricinus ticks collected from additional sites in England and Wales also identified Chimay rhabdovirus viral RNA with varying prevalence in all tick populations. This suggests that Chimay rhabdovirus has a wide distribution and highlights the need for an extended exploration of the tick microbiome in the United Kingdom (UK).

Density and behavior of capybara (Hydrochoerus hydrochaeris) ticks (Acari: Ixodidae) Amblyomma sculptum and Amblyomma dubitatum with notes on Rickettsia bellii infection: Assessing human exposure risk.

In several urban and peri­urban areas of Brazil, populations of Amblyomma sculptum and Amblyomma dubitatum ticks are maintained by capybaras (Hydrochoerus hydrochaeris). In some of these areas, this host and these tick species are associated with Brazilian spotted fever (BSF), a lethal human disease caused by the bacterium Rickettsia rickettsii. In this work, we evaluated the risk of human exposure to these tick species using four collection techniques to discern host-seeking behavior. The study was carried out in 10 urban sites inhabited by capybaras in Uberlândia, a BSF-free municipality in southeastern Brazil. Ticks were collected in areas of 400 m2 at each site and at three seasons. Within the same municipality, the distance and speed of A. sculptum nymphs moving towards the CO2 traps were evaluated. In a sample of ticks Rickettsia DNA was investigated. During the study period, 52,953 ticks were collected. Among these, 83.4 % were A. sculptum (1,523 adults, 10,545 nymphs and 32,104 larvae) and 16.6 % were A. dubitatum (464 adults, 2,153 nymphs and 6,164 larvae). An average annual questing tick density of 4.4/m² was observed, with the highest density recorded at one site in autumn (31.8/m²) and the lowest in summer at another site (0.03/m²). The visual search yielded the highest proportion of A. sculptum larvae, constituting 47 % of the total and 63.6 % of all A. sculptum larvae. In contrast, CO2 traps collected a greater proportion of nymphs and adults of A. sculptum ticks. In the case of A. dubitatum, the CO2 trap was the most efficient technique with 57.7 % of captures of this species, especially of nymphs (94.5 % of captures) and adults (97.8 % of captures). Ticks' ambush height on vegetation (9 to 77 cm), observed by visual search 30 times, yielded a total of 20,771 ticks. Of these, 28 (93 %) were A. sculptum ticks, with only two (7 %) identified as A. dubitatum ticks. Among A. sculptum ticks, the nymph was the most attracted stage to humans and larva in the case of A. dubitatum. Amblyomma sculptum adults and nymphs were significantly more attracted to humans than those of A. dubitatum, but A. dubitatum larvae were significantly more attracted than the same stage of A. sculptum. The maximum distance and speed of horizontal displacement for A. sculptum nymphs were five meters and 2.0 m/h, respectively. The only species of Rickettsia detected in ticks, exclusively in A. dubitatum, was R. bellii. Importantly, it was observed that the higher the proportion of A. sculptum in the community of ticks, the lower the rate of infection of A. dubitatum by R. bellii. In conclusion, host-seeking behavior differed between the two tick species, as well as between stages of the same species. A greater restriction of A. dubitatum ticks to the soil was observed, while larvae and nymphs of A. sculptum dispersed higher in the vegetation. The behavior presented by A. sculptum provides greater opportunities for contact with the hosts, while A. dubitatum depends more on an active search for a host, the hunter behavior. Taken together, these observations show that a human being crossing an area infested with A. sculptum and A. dubitatum ticks will have almost exclusive contact with A. sculptum larvae and/or nymphs. Humans in a stationary position (sitting, lying or immobile) are exposed to both tick species, but they are more attractive to adults and mainly nymphs of A. sculptum compared to the corresponding stages of the tick A. dubitatum. The negative effect of A. sculptum on A. dubitatum infection by R. bellii deserves further studies.

New hard tick (Acari: Ixodidae) reports and detection of Rickettsia in ticks from Sierra Nevada de Santa Marta, Colombia.

The Sierra Nevada de Santa Marta (SNSM), located in northern Colombia, is considered a geographical island with high levels of biodiversity and endemism. However, little is known about tick species and their associated microorganisms at the SNSM. In this study we sampled host-seeking ticks in areas of the town of Minca within the SNSM. We collected 47 ticks identified as Amblyomma pacae, Amblyomma longirostre, Amblyomma ovale, Amblyomma mixtum, Haemaphysalis juxtakochi, Ixodes sp. cf. Ixodes affinis and Ixodes sp. Of these ticks, we tested for Rickettsia spp. by amplifying the gltA, SCA1, and 16S rRNA genes via PCR. Rickettsia amblyommatis was detected in one pool of 3 larvae and in a female of A. pacae. Additonally, we isolated Rickettsia sp. belonging to the group of spotted fevers in larvae of A. longirostre. This study reports new findings of six species of ticks and two species of Rickettsia within the SNSM.

Parasitism on domestic cats by Amblyomma auricularium and serological evidence of exposure to Rickettsia amblyommatis.

The domestic cat is not considered a primary host for any specific tick species; however, it can be affected by some Ixodidae species, such as Rhipicephalus sanguineus sensu lato and Amblyomma spp. The study reports parasitism by Amblyomma auricularium and the detection of anti-Rickettsia spp. antibodies in domestic cats from a rural property in the Afrânio municipality, Pernambuco, Brazil. Amblyomma auricularium (24 nymphs, six females, and four males) and Amblyomma sp. (42 larvae) parasitized three cats, and 73 free-living ticks were captured in armadillo burrows: A. auricularium (36 nymphs, six females, five males) and Amblyomma sp. (26 larvae). Blood samples from cats were collected and the obtained plasma were subjected to indirect immunofluorescence assay (IFA) to detect antibodies against Rickettsia antigens. Thus, anti-Rickettsia spp. antibodies were determined (titers ranging from 128 to 512) and showed a predominant antibody response to Rickettsia amblyommatis or a very closely related genotype. This study reports the first infestation of nymphs and adults of A. auricularium on cats in a new area of occurrence in the semi-arid region of Northeastern Brazil and reports for the first time the presence of anti-Ricketsia antibodies in cats in the region, with R. amblyommatis as the probable infectious agent.

Blood meal analysis reveals sources of tick-borne pathogens and differences in host utilization of juvenile Ixodes ricinus across urban and sylvatic habitats.

AIMS: Urban green spaces are locations of maximal human activity, forming areas of enhanced risk for tick-borne disease (TBD) transmission. Being also limited in spatial scale, green spaces form prime targets for control schemes aiming to reduce TBD risk. However, for effective control, the key species maintaining local tick and tick-borne pathogen (TBP) populations must be identified. To determine how patterns of host utilization vary spatially, we utilized blood meal analysis to study the contributions of voles, shrews, squirrels, leporids and cervids towards blood meals and the acquisition of TBPs of juvenile Ixodes ricinus in urban and sylvatic areas in Finland. METHODS AND RESULTS: A total of 1084 nymphs were collected from the capital city of Finland, Helsinki and from a sylvatic island in southwestern Finland, and subjected to qPCR analysis to identify DNA remnants of the previous host. We found significant differences in host contributions between urban and sylvatic environments. Specifically, squirrels and leporids were more common hosts in urban habitats, whereas cervids and voles were more common in sylvatic habitats. In addition to providing 18.4% of larval blood meals in urban habitats, red squirrels were identified as the source of 28.6% (n = 48) of Borrelia afzelii detections and 58.1% (n = 18) of Borrelia burgdorferi sensu stricto detections, indicating an important role for local enzootic cycles. CONCLUSIONS: Our study highlights that the key hosts maintaining tick and TBP populations may be different in urban and sylvatic habitats. Likewise, hosts generally perceived as important for upkeep may have limited importance in urban environments. Consequently, targeting control schemes based on off-site data of host importance may lead to suboptimal results.

Activity of natural occurring entomopathogenic fungi on nymphal and adult stages of Philaenus spumarius.

The spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the predominant vector of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) in Apulia, Italy and the rest of Europe. Current control strategies of the insect vector rely on mechanical management of nymphal stages and insecticide application against adult populations. Entomopathogenic fungi (EPF) are biological control agents naturally attacking spittlebugs and may effectively reduce population levels of host species. Different experimental trials in controlled conditions have been performed to i) identify naturally occurring EPF on P, spumarius in Northwestern Italy, and ii) evaluate the potential for biocontrol of the isolated strains on both nymphal and adult stages of the spittlebug. Four EPF species were isolated from dead P. spumarius collected in semi-field conditions: Beauveria bassiana, Conidiobolus coronatus, Fusarium equiseti and Lecanicillium aphanocladii. All the fungal isolates showed entomopathogenic potential against nymphal stages of P. spumarius (≈ 45 % mortality), except for F. equiseti, in preliminary trials. No induced mortality was observed on adult stage. Lecanicillium aphanocladii was the most promising fungus and its pathogenicity against spittlebug nymphs was further tested in different formulations (conidia vs blastospores) and with natural adjuvants. Blastospore formulation was the most effective in killing nymphal instars and reducing the emergence rate of P, spumarius adults, reaching mortality levels (90%) similar to those of the commercial product Naturalis®, while no or adverse effect of natural adjuvants was recorded. The encouraging results of this study pave way for testing EPF isolates against P, spumarius in field conditions and find new environmentally friendly control strategies against insect vectors of X. fastidiosa.

Survey of Rickettsia spp. in ticks (Acari: Ixodidae) infesting opossums (Didelphis albiventris) and capybaras (Hydrochoerus hydrochaeris) from an urban park in southeastern Brazil.

Ticks are hematophagous arthropods and, during feeding, may transmit pathogens to vertebrate hosts, including humans. This study aimed to investigate the presence of Rickettsia spp. in ticks collected between 2010 and 2013 from free-ranging capybaras (Hydrochoerus hydrochaeris) and opossums (Didelphis albiventris) that inhabit Sabiá Park in Uberlândia, Brazil. Overall, 1,860 ticks were collected: 1,272 (68.4%) from capybaras (487 of the species Amblyomma sculptum, 475 adults and 12 nymphs; 778 Amblyomma dubitatum, 727 adults and 51 nymphs; and seven larva clusters of the genus Amblyomma); and 588 (31.6%) from opossums (21 A. sculptum, one adult and 20 nymphs; 79 A. dubitatum, all nymphs; 15 Ixodes loricatus, 12 adults and three nymphs; 457 Amblyomma sp. larva clusters; 15 Ixodes sp. larva clusters; and one Argasidae larva cluster). Out of 201 DNA samples tested for the presence of Rickettsia spp. DNA using polymerase chain reaction (PCR) 12 showed amplification of a gtlA gene segment that was specific to Rickettsia bellii, a bacterium non-pathogenic to humans. As there has been a report showing serological evidence of infections caused by Rickettsia species of the spotted fever group (SFG) in capybaras and opossums in the park, including Rickettsia rickettsii, the etiological agent of Brazilian spotted fever, and considering the presence of A. sculptum ticks, which are aggressive to humans, as well as these vertebrate hosts, which are amplifiers of R. rickettsii, it is important to monitor the presence of SFG rickettsiae in the Sabiá Park, which is visited daily by thousands of people.

Optimization of artificial membrane feeding system for lone star ticks, Amblyomma americanum (Acari: Ixodidae), and experimental infection with Rickettsia amblyommatis (Rickettsiales: Rickettsiaceae).

With the introduction of siliconized artificial membranes, various artificial feeding systems (AFS) for hard ticks (Ixodidae) have been developed over the last decades. Most AFS utilize similar core components but employ diverse approaches, materials, and experimental conditions. Published work describes different combinations of the core components without experimental optimizations for the artificial feeding of different tick species. Amblyomma americanum L., (Acari: Ixodidae) (lone star tick) is a known vector and reservoir for diverse tick-borne pathogens, such as Rickettsia amblyommatis and Ehrlichia chaffeensis. Ongoing environmental changes have supported the expansion of A. americanum into new habitats, contributing to increased tick-borne diseases in endemic areas. However, a significant knowledge gap exists in understanding the underlying mechanisms involved in A. americanum interactions with tick-borne pathogens. Here, we performed a systematic analysis and developed an optimized AFS for nymphal lone star ticks. Our results demonstrate that Goldbeater's membranes, rabbit hair, hair extract, and adult lone star ticks significantly improved the attachment rate of nymphal ticks, whereas tick frass and frass extract did not. With the optimized conditions, we achieved an attachment rate of 46 ±â€…3% and a success rate of 100% (i.e., one or more attached ticks) in each feeding experiment for nymphal lone star ticks. When fed on sheep blood spiked with R. amblyommatis, both nymphal and adult lone star ticks acquired and maintained R. amblyommatis, demonstrating the feasibility of studying A. americanum-pathogen interactions using AFS. Our study can serve as a roadmap to optimize and improve AFS for other medically relevant tick species.

Acquisition of Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) by Haemaphysalis longicornis (Acari: Ixodidae) through co-feeding with infected Dermacentor variabilis (Acari: Ixodidae) in the laboratory.

Haemaphysalis longicornis (Neumann) is an invasive ixodid tick originating from eastern Asia which recently has become established in the United States. In its native range, this tick can transmit several pathogens to animals and humans, but little is known about its ability to acquire and transmit pathogens native to the United States. Geographic overlap with ticks such as Dermacentor variabilis (Say), a known vector of Rickettsia rickettsii, makes investigation into the interactions between H. longicornis and D. variabilis of interest to the public health community. Previous studies have shown that H. longicornis can serve as a competent vector of R. rickettsii under laboratory settings, but there is little information on its ability to acquire this pathogen via other biologically relevant routes, such as co-feeding. Here, we assess the ability of H. longicornis nymphs to acquire R. rickettsii through co-feeding with infected D. variabilis adults on a vertebrate animal model under laboratory conditions. The median infection prevalence in engorged H. longicornis nymphs across 8 cohorts was 0% with an interquartile range (IQR) of 4.13%. Following transstadial transmission, the median infection prevalence in flat females was 0.7% (IQR = 2.4%). Our results show that co-feeding transmission occurs at low levels in the laboratory between these 2 species. However, based on the relatively low transmission rates, this may not be a likely mechanism of R. rickettsii introduction to H. longicornis.

Cross-alteration of murine skin and tick microbiome concomitant with pathogen transmission after Ixodes ricinus bite.

BACKGROUND: Ticks are major vectors of diseases affecting humans such as Lyme disease or domestic animals such as anaplasmosis. Cross-alteration of the vertebrate host skin microbiome and the tick microbiome may be essential during the process of tick feeding and for the mechanism of pathogen transmission. However, it has been poorly investigated. METHODS: We used mice bitten by field-collected ticks (nymphs and adult ticks) in different experimental conditions to investigate, by 16S rRNA gene metabarcoding, the impact of blood feeding on both the mouse skin microbiome and the tick microbiome. We also investigated by PCR and 16S rRNA gene metabarcoding, the diversity of microorganisms transmitted to the host during the process of tick bite at the skin interface and the dissemination of the pathogen in host tissues (blood, heart, and spleen). RESULTS: Most of the commensal bacteria present in the skin of control mice were replaced during the blood-feeding process by bacteria originating from the ticks. The microbiome of the ticks was also impacted by the blood feeding. Several pathogens including tick-borne pathogens (Borrelia/Borreliella, Anaplasma, Neoehrlichia, Rickettsia) and opportunistic bacteria (Williamsia) were transmitted to the skin microbiome and some of them disseminated to the blood or spleen of the mice. In the different experiments of this study, skin microbiome alteration and Borrelia/Borreliella transmission were different depending on the tick stages (nymphs or adult female ticks). CONCLUSIONS: Host skin microbiome at the bite site was deeply impacted by the tick bite, to an extent which suggests a role in the tick feeding, in the pathogen transmission, and a potentially important impact on the skin physiopathology. The diversified taxonomic profiles of the tick microbiome were also modified by the blood feeding. Video Abstract.

Seasonal dynamics of Amblyomma cajennense (Fabricius, 1787) sensu stricto in a degraded area of the Amazon biome, with notes on Rickettsia amblyommatis infection.

BACKGROUND: The tick Amblyomma cajennense sensu stricto (A. cajennense s.s.) frequently parasitizes animals and humans in the Amazon biome, in addition to being a vector of Rickettsia amblyommatis. In the present study, we evaluated both the population dynamics of A. cajennense s.s. in a degraded area of the Amazon biome and the presence of rickettsial organisms in this tick population. METHODS: The study was carried out in a rural area of the Santa Inês municipality (altitude: 24 m a.s.l.), Maranhão state, Brazil. Ticks were collected from the environment for 24 consecutive months, from June 2021 to May 2023. The region is characterized by two warm seasons: a rainy season (November-May) and a dry season (June-October). We characterized the temporal activity of A. cajennense s.s. on the vegetation by examining questing activity for each life stage (larvae, nymphs, adults [males and females]) in relation to the dry and rainy season. Ticks collected in this study were randomly selected and individually tested by a TaqMan real-time PCR assay that targeted a 147-bp fragment of the rickettsial gltA gene. RESULTS: Overall, 1843 (62.4%) adults (52.6% females, 47.4% males), 1110 (37.6%) nymphs and 398 larval clusters were collected. All adult females and nymphs were morphologically identified as A. cajennense s.s. Larval activity was observed from April to December, with a peak from June to September (dry season); nymph abundance peaked from September to November (transition period between dry and rainy seasons); and adult ticks were abundant from October to May (spring/summer/early autumn). The infection rate by R. amblyommatis in A. cajennense s.s. ticks was at least 7% (7/99). CONCLUSION: Our data suggest a 1-year generation pattern for A. cajennense s.s., with a well-defined seasonality of larvae, nymphs and adults in the Amazon biome. Larvae predominate during the dry season, nymphs are most abundant in the dry-rainy season transition and adults are most abundant in the rainy season. The presence of R. amblyommatis in adult ticks suggests that animals and humans in the study region are at risk of infection by this species belonging to the spotted fever group of Rickettsia.

Deer management generally reduces densities of nymphal Ixodes scapularis, but not prevalence of infection with Borrelia burgdorferi sensu stricto.

Human Lyme disease-primarily caused by the bacterium Borrelia burgdorferi sensu stricto (s.s.) in North America-is the most common vector-borne disease in the United States. Research on risk mitigation strategies during the last three decades has emphasized methods to reduce densities of the primary vector in eastern North America, the blacklegged tick (Ixodes scapularis). Controlling white-tailed deer populations has been considered a potential method for reducing tick densities, as white-tailed deer are important hosts for blacklegged tick reproduction. However, the feasibility and efficacy of white-tailed deer management to impact acarological risk of encountering infected ticks (namely, density of host-seeking infected nymphs; DIN) is unclear. We investigated the effect of white-tailed deer density and management on the density of host-seeking nymphs and B. burgdorferi s.s. infection prevalence using surveillance data from eight national parks and park regions in the eastern United States from 2014-2022. We found that deer density was significantly positively correlated with the density of nymphs (nymph density increased by 49% with a 1 standard deviation increase in deer density) but was not strongly correlated with the prevalence of B. burgdorferi s.s. infection in nymphal ticks. Further, while white-tailed deer reduction efforts were followed by a decrease in the density of I. scapularis nymphs in parks, deer removal had variable effects on B. burgdorferi s.s. infection prevalence, with some parks experiencing slight declines and others slight increases in prevalence. Our findings suggest that managing white-tailed deer densities alone may not be effective in reducing DIN in all situations but may be a useful tool when implemented in integrated management regimes.

Exposure of dogs and cats to Borrelia miyamotoi infected Ixodes ricinus ticks in urban areas of the city of Poznan, west-central Poland.

Borrelia miyamotoi is an emerging human pathogen that causes a relapsing fever-like disease named B. miyamotoi disease. The bacterium belongs to the relapsing fever borreliae, and similar to spirochetes of the Borrelia burgdorferi sensu lato group, it is transmitted only by hard ticks of the Ixodes ricinus complex. To date, B. miyamotoi has not been demonstrated to cause illness in dogs or cats, and is poorly documented in veterinary medicine. The aim of this study was to determine the B. miyamotoi presence in (i) host-seeking ticks and (ii) engorged Ixodes sp. ticks collected from dogs and cats during their inspection in veterinary clinics of the city of Poznan, west-central Poland. Host-seeking ticks were sampled in dog walking areas localized in urban forested recreational sites of the city. In this study, 1,059 host-seeking and 837 engorged I. ricinus ticks collected from 680 tick-infested animals (567 dogs and 113 cats) were screened. Additionally, 31 I. hexagonus ticks (one larva, 13 nymphs, and 17 females) were collected from three cats; one larva and one nymph were collected from two dogs; and one dog was infested with a single Dermacentor reticulatus female. Borrelia DNA was identified by the amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene and flaB gene fragments. DNA of B. miyamotoi was detected in 22 (2.1%) of the host-seeking ticks (in all developmental tick stages and in all study areas). In addition, the engorged I. ricinus ticks exhibited a similar B. miyamotoi presence (1.8%). Fifteen I. ricinus ticks collected from animals tested positive for the presence of B. miyamotoi DNA, and the DNA of B. miyamotoi was observed in three (9.1%; one female and two nymphs) I. hexagonus ticks. The single D. reticulatus female collected from a dog tested PCR-negative for the bacterium. The results of this study demonstrated the establishment and broad presence of the bacterium in tick populations from different urban ecosystems of the city of Poznan. The lack of difference in the mean infection presence of animal-derived and host-seeking I. ricinus ticks suggests that the systematic surveillance of pets may be useful for the evaluation of human exposure to B. miyamotoi infected ticks in urban areas. Additional studies are required to further elucidate the role of domestic and wild carnivores in the epidemiology of B. miyamotoi, which remains unknown.

Voles, shrews and red squirrels as sources of tick blood meals and tick-borne pathogens on an island in southwestern Finland.

Molecular identification of the previous blood meal source of a questing tick (Acari: Ixodidae) from blood meal fragments was proposed a few decades ago. Following this, several blood meal assays have been developed and published, but none of them have been taken into widespread use. Recently, novel retrotransposon-based qPCR assays designed for detecting blood meal fragments of North American host species were published. We wanted to assess their function with host species present in Finland. Questing ticks were collected by cloth dragging in August-September 2021 from an island in southwestern Finland. DNA was extracted from Ixodes ricinus nymphs (n=438) and qPCR assays applied to identify larval blood meal sources (voles, shrews and red squirrels) and screen for several tick-borne human pathogens and other microbes with pathogenic potential [Borrelia spp. (including specific assays for Borrelia afzelii, Borrelia garinii, Borrelia valaisiana), Anaplasma phagocytophilum, Babesia spp., Rickettsia spp., and Neoehrlichia mikurensis]. The probability of a nymph having fed as larva on either a vole, shrew or red squirrel was 0.34 (0.30 - 0.38; 95% confidence interval). Bacteria of the genus Borrelia were the most common pathogens detected, with host-specific probabilities of carrying Borrelia of 0.30 (0.18 - 0.44) for nymphs that had fed on voles, 0.23 (0.14 - 0.35) for nymphs that had fed on shrews, and 0.42 (0.28 - 0.58) for nymphs that had fed on red squirrels. Other microbes were rarely acquired from these hosts, apart from N. mikurensis from voles. This study highlights that shrews and red squirrels may equal voles as blood meal sources for I. ricinus larvae. Overall, variation in proportions of blood meals provided by these animals may be high across even proximate study areas. All studied host species appeared to be important sources for particularly Borrelia afzelii, and voles also for N. mikurensis.

Tick transmission of Borrelia burgdorferi to the murine host is not influenced by environmentally acquired midgut microbiota.

BACKGROUND: Ixodes scapularis is the predominant tick vector of Borrelia burgdorferi, the agent of Lyme disease, in the USA. Molecular interactions between the tick and B. burgdorferi orchestrate the migration of spirochetes from the midgut to the salivary glands-critical steps that precede transmission to the vertebrate host. Over the last decade, research efforts have invoked a potential role for the tick microbiome in modulating tick-pathogen interactions. RESULTS: Using multiple strategies to perturb the microbiome composition of B. burgdorferi-infected nymphal ticks, we observe that changes in the microbiome composition do not significantly influence B. burgdorferi migration from the midgut, invasion of salivary glands, or transmission to the murine host. We also show that within 24 and 48 h of the onset of tick feeding, B. burgdorferi spirochetes are within the peritrophic matrix and epithelial cells of the midgut in preparation for exit from the midgut. CONCLUSIONS: This study highlights two aspects of tick-spirochete interactions: (1) environmental bacteria associated with the tick do not influence spirochete transmission to the mammalian host and (2) the spirochete may utilize an intracellular exit route during migration from the midgut to the salivary glands, a strategy that may allow the spirochete to distance itself from microbiota in the midgut lumen effectively. This may explain in part, the inability of environment-acquired midgut microbiota to significantly influence spirochete transmission. Unraveling a molecular understanding of this exit strategy will be critical to gain new insights into the biology of the spirochete and the tick. Video Abstract.

Tick microbial associations at the crossroad of horizontal and vertical transmission pathways.

BACKGROUND: Microbial communities can affect disease risk by interfering with the transmission or maintenance of pathogens in blood-feeding arthropods. Here, we investigated whether bacterial communities vary between Ixodes ricinus nymphs which were or were not infected with horizontally transmitted human pathogens. METHODS: Ticks from eight forest sites were tested for the presence of Borrelia burgdorferi sensu lato, Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis by quantitative polymerase chain reaction (qPCR), and their microbiomes were determined by 16S rRNA amplicon sequencing. Tick bacterial communities clustered poorly by pathogen infection status but better by geography. As a second approach, we analysed variation in tick microorganism community structure (in terms of species co-infection) across space using hierarchical modelling of species communities. For that, we analysed almost 14,000 nymphs, which were tested for the presence of horizontally transmitted pathogens B. burgdorferi s.l., A. phagocytophilum, and N. mikurensis, and the vertically transmitted tick symbionts Rickettsia helvetica, Rickettsiella spp., Spiroplasma ixodetis, and Candidatus Midichloria mitochondrii. RESULTS: With the exception of Rickettsiella spp., all microorganisms had either significant negative (R. helvetica and A. phagocytophilum) or positive (S. ixodetis, N. mikurensis, and B. burgdorferi s.l.) associations with M. mitochondrii. Two tick symbionts, R. helvetica and S. ixodetis, were negatively associated with each other. As expected, both B. burgdorferi s.l. and N. mikurensis had a significant positive association with each other and a negative association with A. phagocytophilum. Although these few specific associations do not appear to have a large effect on the entire microbiome composition, they can still be relevant for tick-borne pathogen dynamics. CONCLUSIONS: Based on our results, we propose that M. mitochondrii alters the propensity of ticks to acquire or maintain horizontally acquired pathogens. The underlying mechanisms for some of these remarkable interactions are discussed herein and merit further investigation. Positive and negative associations between and within horizontally and vertically transmitted symbionts.

Mpp51Aa1 toxicity to Diaphorina citri nymphs demonstrated using a new, long-term bioassay method.

While pesticidal proteins from Bacillus thuringiensis have provided for effective management of several insect pests of agricultural importance, few with toxicity to hemipteran species have been identified. The Asian citrus psyllid, Diaphorina citri transmits Candidatus Liberibacter asiaticus (CLas), the presumed bacterial causative agent of the devastating disease citrus greening. Despite the critical role of D. citri nymphs in the acquisition and inoculation of CLas, the lack of a long-term feeding method impedes the screening of Bt proteins for toxicity against nymphs, which play a key role in CLas transmission. Here, we developed a long-term nymph bioassay and determined the toxicity of the Bt pesticidal protein Mpp51Aa1. The new bioassay method allows nymphs to survive for up to six days when maintained on treated folded wipes. The standard hemipteran membrane feeding assay was used to assess Mpp51Aa1 toxicity against D. citri adults. Mpp51Aa1 was toxic to D. citri nymphs with a median lethal concentration (LC50) of 56.5 µg/ml in wipe feeding assays, and to D. citri adults with an LC50 of 110.4 µg/ml in membrane feeding assays. These results demonstrate the utility of this long-term nymph bioassay method and suggest that Mpp51Aa1 has potential for sustainable use in D. citri management toward mitigation of citrus greening disease.