Effects of Tropilaelaps mercedesae on midgut bacterial diversity of Apis mellifera Abstract Tropilaelaps mercedesae is an ectoparasite of Apis mellifera in Asia and is considered a major threat to honey bee health. Herein, we used the Illumina MiSeq platform 16S rDNA Amplicon Sequencing targeting the V3–V4 regions and analysed the effects on the midgut bacterial communities of honey bees infested with T. mercedesae. The overall bacterial community in honey bees infested with T. mercedesae were observed at different developmental stages. Honey bee core intestinal bacterial genera such as Gilliamella, Lactobacillus and Frischella were detected. Tropilaelapsmercedesae infestation changed the bacterial communities in the midgut of A. mellifera. Tropilaelapsmercedesae-infested pupae had greatly increased relative abundances of Micrococcus and Sphingomonas, whereas T. mercedesae-infested 15-day-old workers had significantly reduced relative abundance of non-core microbes: Corynebacterium, Sphingomonas, Acinetobacter and Enhydrobacter compared to T. mercedesae-infested newly emerged bees. The bacterial community was significantly changed at the various T. mercedesae-infested developmental stages of A. mellifera. Tropilaelapsmercedesae infestation also changed the non-core bacterial community from larvae to newly emerged honey bees. Bacterial communities were significantly different between T. mercedesa-infested and non-mite-infested 15-day-old workers. Lactobacillus was dominant in T. mercedesae-infested 15-day-old workers compared to non-mite-infested 15-day-old workers.

Choice test to determine potential attractants and repellents for the sheep scab mite, Psoroptes ovis (Acari: Psoroptidae) Abstract A choice test bioassay was devised to screen compounds as potential semiochemicals (e.g., kairomones or allomones that mediate aggregation, attraction or repellence) for the obligate parasitic mite, Psoroptes ovis. The choice test used filter paper discs in a 1:4 test:control ratio and was found to be a reliable, effective and efficient method. Four mammalian lipid components were assessed as potential attractants—linoleic acid, arachidonic acid, methyl myristate and squalene—, and the insect/tick repellent DEET for potential repellence. Linoleic acid was significantly attractive to P. ovis adult females and has the potential to act as an attractant. Identification of P. ovis semiochemicals, especially attractants, would be beneficial in the development of novel control methods and tools for this species. This is essential considering the increase in resistance to the limited prophylactic chemical treatments in the UK, and the high prevalence of scab infections.

Genetic diversity of Haemaphysalis longicornis from China and molecular detection of Rickettsia Abstract The tick Haemaphysalis longicornis (Neumann) (Acari, Ixodidae) is distributed throughout China and is the most notorious blood feeding ectoparasite of livestock. Haemaphysalis longicornis can transmit a large number of pathogens that cause human infectious diseases, such as Rickettsia spp. Here, we characterized the genetic structure of H. longicornis and tested for the presence of Rickettsia spp. from five regions in China. Analysis of the two mitochondrial marker sequences (16S rRNA and COI) and the nuclear sequence (ITS2) showed that the overall level of nucleotide diversity was low and the variability did not differ among the five regions. From the five locations, the infection rates of Rickettsia species ranged from 0 to 65%. The nucleotide diversities of the high-infected group were lower than those of the low- and uninfected group. And in neutrality tests for the high-infected group based on COI sequences, the Tajima’s D and Fu’s FS were coincidentally < 0 and significant, whereas they were closer to zero and non-significant in low- and uninfected groups.

Essential oils as tick repellents on clothing Abstract Essential oils show promise as natural alternatives to synthetic tick repellents, but few studies have investigated their repellent efficacy in vivo or under field conditions. Here, blanket-drags and standardised walks were employed to evaluate tick acquisition by 1 m2 cotton blankets or cotton trousers, respectively, in woodland edge habitats of known high tick abundance. Blankets and trousers had been treated with one of 5% oregano, rosemary, spearmint or thyme oils, 20% DEET (N,N-diethyl-3-methylbenzamide) (positive control) or ethanol excipient-only (negative control). The number of ticks present on the blankets or trousers differed significantly between treatments: spearmint oil treatments resulted in significantly fewer ticks than the negative controls for both blankets and trousers and significantly fewer ticks were present on the oregano oil treated blankets. For ticks that did attach to the trousers, the rate of drop off within 3 min was significantly higher for trousers treated with spearmint oil or thyme oil than ethanol, oregano oil and rosemary oil. No reduction in repellence was detected over a 24 h period between treatment and testing. The results suggest that 5% oregano and spearmint oils exhibit potential as natural clothing repellents, with an effective equivalence to 20% DEET.

Genetic diversity, piroplasms and trypanosomes in Rhipicephalus microplus and Hyalomma anatolicum collected from cattle in northern Pakistan Abstract The two most important tick species in Pakistan are Rhipicephalus microplus and Hyalomma anatolicum. When associated with cattle, these have one or three host life cycles, respectively, with potential implications for their population genetics and for their vector role in the transmission of pathogens. To compare the two tick species in this context with molecular-phylogenetic methods, during the present study 123 ticks were collected from cattle in northern Pakistan. Two mitochondrial markers of 36 ticks were molecularly analyzed. All 11 R. microplus specimens had identical cox1 haplotypes, whereas the 25 H. anatolicum specimens had nine cox1 haplotypes. The latter belonged to two distinct phylogenetic lineages with high support. However, in the 16S rRNA gene these differences were less evident. Among the 113 ticks molecularly analyzed for tick-borne protozoa, the sequence of Babesia occultans was successfully amplified from two specimens of H. anatolicum. Theileria annulata was present in both R. microplus (10.4%) and H. anatolicum (27.3%), with significantly higher prevalence rate in the latter species. Only one tick, a H. anatolicum female, was positive in the PCR detecting Trypanosoma spp. Sequencing revealed the presence of a new genotype, with the closest phylogenetic relationship to stercorarian trypanosomes (in particular, to a tick-associated Trypanosoma sp. from Japan). In conclusion, the above differences between R. microplus and H. anatolicum may be partly related to their life cycles involving one host or three hosts, respectively. Among the others, host switching (reducing chances of inbreeding) and shorter periods spent on-host (reducing gene flow between cattle herds) are supposed to be important drivers of cox1 gene diversification in case of H. anatolicum as a three host tick species. These results highlight the importance of studying differences in intraspecific genetic diversity and piroplasm burdens between one host and three host ticks in the local scale. In addition, a Trypanosoma sp. molecularly identified in H. anatolicum is reported here for the first time from South Asia, deserving further evaluation concerning its host and vector species.

Behavioral responses of Ixodes scapularis tick to natural products: development of novel repellents Abstract The spread of blacklegged ticks (Ixodes scapularis) and growing threat of Lyme disease transmission has increased demand for effective, safe and environmentally friendly repellent products. Plant-derived essential oils are natural products that exhibit insecticidal and repellant activities and represent a promising alternative to synthetic repellants. However, mechanisms by which ticks detect odor stimuli and how such stimuli may function as repellents are not well understood. We examined the repellent activity of selected essential oil components towards I. scapularis in short- and long-term dose–response trials. To determine the specific olfactory organs involved in detection of chemical stimuli, we tested tick behavioral response in repellency bioassays after removing appendages that house chemosensory sensilla (e.g., foretarsi or pedipalps). New prototype formulae were tested in longevity trials repelling up to 95% of tested ticks after 1 h post-application. This study provides new insight regarding tick olfaction and behavior, and innovative methods for selecting appropriate chemicals for development of novel plant-based repellent products for protection from ticks.

Ticks and accompanying pathogens of domestic and wild animals of Kerala, South India Abstract The objective of the present study was to detect the chosen nucleotide DNA or RNA sequences of the pathogens in ticks of domestic and wild animals of Kerala, South India based on molecular techniques. Among 602 ticks collected, 413 were from bovines (cattle and buffalo), 26 from goats, 101 from dogs and 62 from wild animals. Amblyomma integrum, Am. gervaisi, Dermacentor auratus, Haemaphysalis bispinosa, Ha. intermedia, Ha. shimoga, Ha. spinigera, Rhipicephalus annulatus, Rh. microplus, Rh. haemaphysaloides and Rh. sanguineus s.l. were identified from various domestic and wild animals of Kerala. The cDNA synthesized from the RNA isolated from fully or partially engorged adult female/nymphal ticks was used as template for the specific polymerase chain reactions (PCR). Out of 602 ticks examined, nucleotide sequences of pathogens were detected in 28 ticks (4.65%). The nucleotide sequences of tick-borne pathogens like Theileria orientalis, Babesia vogeli, Hepatozoon canis, Anaplasma marginale, An. bovis, Rickettsia sp. closely related to Ri. raoultii, Ri. massiliae, Ri. africae and Ri. slovaca were detected. The identification of the previously unreported nucleotide sequences of rickettsial pathogens from India is of particular interest due to their zoonotic significance. The phylogenetic analysis of the major piroplasm surface protein (MPSP) gene of T. orientalis amplified from Rh. annulatus ticks revealed that they were genetically close to type 7, which belong to the highly pathogenic Ikeda group.

Red imported fire ant ( Solenopsis invicta ) aggression influences the behavior of three hard tick species Abstract Few studies have documented the indirect effects of predators on tick behavior. We conducted behavioral assays in the laboratory to quantify the effects of a highly abundant predator, the red imported fire ant (Solenopsis invicta), on three species of ticks endemic to the southern USA: the lone star tick (Amblyomma americanum), the Gulf Coast tick (A. maculatum), and the Cayenne tick (A. mixtum). We documented ant aggression toward ticks (biting, carrying, and stinging) and determined the effects of ants on tick activity. Ticks were significantly less active in the presence of fire ants, and tick activity was negatively associated with ant aggression, but in many cases the effects of fire ants on ticks varied by tick species, stage, and engorgement status. For example, fire ants took half as long (~ 62 s) to become aggressive toward unfed A. americanum adults compared with unfed A. maculatum, and only ~ 8 s to become aggressive toward engorged A. maculatum nymphs. Correspondingly, the activity of unfed A. americanum adults and engorged A. maculatum nymphs was reduced by 67 and 93%, respectively, in the presence of fire ants. This reduction in tick activity translated to less questing by unfed ticks and less time spent walking by engorged nymphs. Our results suggest that fire ants may have important non-consumptive effects on ticks and demonstrate the importance of measuring the indirect effects of predators on tick behavior.

Oviposition by the vagrant eriophyoid mite Aculops allotrichus on leaves of black locust tree, Robinia pseudoacacia Abstract Leaf-dwelling mites often prefer to feed on young leaves and also are more likely to inhabit the abaxial leaf side. The aim of our study was to examine whether leaf age may affect production and distribution of eggs on black locust leaves by females of Aculops allotrichus. The eriophyoids were tested for 2.5 days on ‘trimmed’ compound leaves (with only two opposite leaflets left), which were maintained in vials filled with water. For the experiments we used leaves of three categories: (1) the ‘youngest’, in which both halves of the adaxial side of leaflets still adhered to each other (and usually remained folded for the next few hours), (2) ‘young’ with already unfolded leaflets, and (3) ‘mature’ with fully expanded leaflets. The tested females laid significantly more eggs on developing leaves than on ‘mature’ ones, although they deposited the highest number of eggs on the ‘young’ leaves. The distribution of eggs on adaxial or abaxial leaf sides also depended on leaf age. On the ‘youngest’ leaves, eriophyoids placed similar numbers of eggs on both sides of a blade. However, the older the leaf, the more willingly females deposited eggs on the abaxial side. Our biochemical and morphometrical analyses of black locust leaves indicated significant changes in the contents of nutrients and phenols within leaf tissue, and in the density of trichomes and thickness of the outer epidermal cell walls, correlated with leaf age. Their possible effects on the production and distribution of eggs on leaves by A. allotrichus are discussed.

Off-host survival of Eriophyoidea and remarks on their dispersal modes Abstract Dispersal of eriophyoid mites is crucial for the successful colonization of new plants. Literature suggests that their long-distance dispersal is through aerial transfer. During dispersal, eriophyoids might be captured in vapor or fine drops of water (perhaps most likely in clouds) where they might be protected against water loss and desiccation, but where they would have no food and be exposed to low temperatures and oxygen concentrations. Considerable resistance of these mites to these stressful environmental conditions is expected and has only partly been confirmed experimentally. The aim of the bioassays conducted here was to assess the survival of five eriophyoid species off their host plants, with poor oxygen availability under two temperature regimes. The bioassays were carried out on live mites dipped into two media used as microenvironments: (1) vaseline oil (used also as control treatment), and (2) water solution of Tween 80 (0.2%) and cycloheximide (50 mg/l). The bioassays were performed at 5 ± 1 and 25 ± 1 °C. The survival of mites was assessed weekly (5 °C) or daily (25 °C) by counting live and active specimens. The following species were subjected to the bioassays: Aceria caulobia (a stem gall mite), Aceria ficus (a vagrant mite), Cecidophyopsis hendersoni (a vagrant mite), Colomerus vitis (protogyne/male population and deutogyne morphs; a leaf gall mite) and Phytoptus avellanae (a bud gall mite). The survival rate of the mites was higher at 5 °C than at 25 °C under both experimental conditions. At 5 °C, the survival of almost all species was higher in the water solution (up to 6–7 weeks) than in vaseline oil (3–5 weeks). Longer survival was found for A. caulobia and P. avellanae (gall-making species) than for C. hendersoni and A. ficus (vagrant species). As expected, the deutogynes of C. vitis survived longer than its protogynes. The current results suggest that individuals of some of the tested species are well suited for withstanding cold, starvation and low oxygen rates, which could be found at higher atmospheric layers, within the clouds, allowing them an effective long-distance dispersal.