Initial study and phylogenetic analysis of hard ticks (Acari: Ixodidae) in Nantong, China along the route of avian migration.

The growing concern about migratory birds potentially spreading ticks due to global warming has become a significant issue. The city of Nantong in this study is situated along the East Asia-Australasian Flyway (EAAF), with numerous wetlands serving as roosting sites for migratory birds. We conducted an investigation of hard ticks and determined the phylogenetic characteristics of tick species in this city. We utilized three different genes for our study: the mitochondrial cytochrome oxidase subunit 1 (COX1) gene, the second internal transcribed spacer (ITS2), and the mitochondrial small subunit rRNA (12 S rRNA) gene. The predominant tick species were Haemaphysalis flava (H. flava) and Haemaphysalis longicornis (H. longicornis). Additionally, specimens of Haemaphysalis campanulata (H. campanulata) and Rhipicephalus sanguineus (R. sanguineus) were collected. The H. flava specimens in this study showed a close genetic relationship with those from inland provinces of China, as well as South Korea and Japan. Furthermore, samples of H. longicornis exhibited a close genetic relationship with those from South Korea, Japan, Australia, and the USA, as well as specific provinces in China. Furthermore, R. sanguineus specimens captured in Nantong showed genetic similarities with specimens from Egypt, Nigeria, and Argentina.

Analysis of High Molecular Mass Compounds from the Spider Pamphobeteus verdolaga Venom Gland. A Transcriptomic and MS ID Approach.

Nowadays, spider venom research focuses on the neurotoxic activity of small peptides. In this study, we investigated high-molecular-mass compounds that have either enzymatic activity or housekeeping functions present in either the venom gland or venom of Pamphobeteus verdolaga. We used proteomic and transcriptomic-assisted approaches to recognize the proteins sequences related to high-molecular-mass compounds present in either venom gland or venom. We report the amino acid sequences (partial or complete) of 45 high-molecular-mass compounds detected by transcriptomics showing similarity to other proteins with either enzymatic activity (i.e., phospholipases A2, kunitz-type, hyaluronidases, and sphingomyelinase D) or housekeeping functions involved in the signaling process, glucanotransferase function, and beta-N-acetylglucosaminidase activity. MS/MS analysis showed fragments exhibiting a resemblance similarity with different sequences detected by transcriptomics corresponding to sphingomyelinase D, hyaluronidase, lycotoxins, cysteine-rich secretory proteins, and kunitz-type serine protease inhibitors, among others. Additionally, we report a probably new protein sequence corresponding to the lycotoxin family detected by transcriptomics. The phylogeny analysis suggested that P. verdolaga includes a basal protein that underwent a duplication event that gave origin to the lycotoxin proteins reported for Lycosa sp. This approach allows proposing an evolutionary relationship of high-molecular-mass proteins among P. verdolaga and other spider species.

Single-cell RNA-seq analysis reveals penaeid shrimp hemocyte subpopulations and cell differentiation process.

Crustacean aquaculture is expected to be a major source of fishery commodities in the near future. Hemocytes are key players of the immune system in shrimps; however, their classification, maturation, and differentiation are still under debate. To date, only discrete and inconsistent information on the classification of shrimp hemocytes has been reported, showing that the morphological characteristics are not sufficient to resolve their actual roles. Our present study using single-cell RNA sequencing revealed six types of hemocytes of Marsupenaeus japonicus based on their transcriptional profiles. We identified markers of each subpopulation and predicted the differentiation pathways involved in their maturation. We also predicted cell growth factors that might play crucial roles in hemocyte differentiation. Different immune roles among these subpopulations were suggested from the analysis of differentially expressed immune-related genes. These results provide a unified classification of shrimp hemocytes, which improves the understanding of its immune system.

Large lipid transfer proteins in hepatopancreas of the mud crab Scylla paramamosain.

Large lipid transfer proteins (LLTPs) are extensively involved in various physiological processes. In the present study, five LLTP sequences encoding apolipocrustacein 1 (apoCr 1), apoCr 2, precursor of the large discoidal lipoprotein (dLp) and high density lipoprotein/ß-glucan binding protein (HDL-BGBP) (dLp-BGBP), microsomal triglyceride transfer protein (MTP) and clotting protein (CP) were identified in the hepatopancreas of Scylla paramamosain. Of these, apoCr 2, dLp-BGBP, and MTP were newly identified in this species, and the former two proteins were classified into the APO family while the later into the MTP family in phylogenetic trees. The apoCr 1 expression level was dramatically increased in the hepatopancreas towards ovarian maturation, which was extremely greater than that in the ovaries concurrently, likely to meet the considerable requirements of yolk protein and lipids for embryo development. The dLp-BGBP expression level in male crabs was comparable to that in female crabs, supporting HDL-BGBP acts as a major circulatory lipid carrier. The close phylogenetic relationship between dLp-BGBP and the scaffolding protein of lipid transfer particle implied dLp might facilitate lipid transfer between the hepatopancreas and HDL-BGBP-containing lipoproteins. The MTP expression level was positively related to ovarian development in both the hepatopancreas and ovaries, indicating MTP may be involved in lipoprotein assembly in the hepatopancreas and lipid droplet maturation in the ovaries. CP may play a crucial role in embryo development based on high expression level observed in the testes of mature crabs. Our findings provide novel insights into LLTP superfamily members and their functions in decapods.

Functional characterization of the insulin signaling pathway in the hard tick Ixodes ricinus.

Ticks are blood-feeding arachnids transmitting a variety of pathogens to humans and animals. A unique trait in tick physiology is their ability to engorge and digest large amounts of host blood, ensuring their high reproductive potential. Activation of the blood digestive machinery in the tick gut, as well as processes controlling maturation of ovaries, are triggered upon blood meal uptake by still largely unknown mechanisms. Sensing of the nutritional status in metazoan organisms is facilitated by the evolutionarily conserved Insulin Signaling Pathway (ISP) and the interlinked Target of Rapamycin (TOR) pathway. Recently, we have identified three components of these pathways in the hard tick Ixodes ricinus midgut transcriptome, namely a putative insulin receptor (InR), and the downstream intracellular serine/threonine kinases AKT and TOR. In this study, we primarily focus on the molecular and functional characterization of the I. ricinus insulin receptor (IrInR), the first InR characterized in Chelicerates. A phylogenetic analysis across the major Arthropod lineages demonstrated that ticks possess only one gene encoding an InR-related molecule. Tissue expression profiling by quantitative PCR in semi-engorged I. ricinus females revealed that the IrInR, as well as AKT (IrAKT) and TOR (IrTOR) are expressed in various organs, with the highest expression being detected in ovaries. We have further evaluated the impact of RNAi-mediated knock-down (KD) of IrInR, IrAKT, and IrTOR on tick blood-feeding and reproductive capacity. Weights of engorged IrInR KD females and laid egg clutches were reduced compared to the control group, and these quantitative parameters clearly correlated with the efficiency of RNAi-KD achieved in individual ticks. The most striking phenotype was observed for IrAKT KD that impaired tick feeding and completely aborted egg production. A recombinant extracellular fragment of the IrInR α-subunit was used to produce antibodies in experimental rabbits to assess its potential as a protective antigen against tick feeding and reproduction. Our data clearly indicate the functionality of the ISP in ticks and demonstrate the need for further investigation of specific roles played by the endogenous insulin-like peptides in tick physiological processes.

Targeted Top-Down Mass Spectrometry for the Characterization and Tissue-Specific Functional Discovery of Crustacean Hyperglycemic Hormones (CHH) and CHH Precursor-Related Peptides in Response to Low pH Stress.

Crustacean hyperglycemic hormones (CHHs) are a family of neuropeptides that were discovered in multiple tissues in crustaceans, but the function of most isoforms remains unclear. Functional discovery often requires comprehensive qualitative profiling and quantitative analysis. The conventional enzymatic digestion method has several limitations, such as missing post-translational modification (PTM) information, homology interference, and incomplete sequence coverage. Herein, by using a targeted top-down method, facilitated by higher sensitivity instruments and hybrid fragmentation modes, we achieved the characterization of two CHH isoforms from the sinus glands (SG-CHH) and the pericardial organs (PO-CHH) from the Atlantic blue crab, Callinectes sapidus, with improved sequence coverage compared to earlier studies. In this study, both label-free and isotopic labeling approaches were adopted to monitor the response of CHHs and CHH precursor-related peptide (CPRP) under low pH stress. The identical trends of CPRP and CHH expression indicated that CPRP could serve as an ideal probe in tracking the CHH expression level changes, which would greatly simplify the quantitative analysis of large peptides. Furthermore, the distinct patterns of changes in the expression of CHHs in the SG and the PO suggested their tissue-specific functions in the regulation of low pH stress. Ion mobility-mass spectrometry (IM-MS) was also employed in this study to provide conformation analysis of both CHHs and CPRPs from different tissues.

A standardized protocol for measuring phenoloxidase in captive and wild Murray crayfish Euastacus armatus.

Innate immunity in arthropods is achieved largely through melanization which is in turn the result of the prophenoloxidase (ProPO) activation cascade; a series of biochemical reactions triggered by the immune identification of pathogen-recognition proteins (PRPs). Within this activation cascade, inactive proPO is cleaved to form the reactive enzyme phenoloxidase (PO). Methods of detecting PO are used to assess an arthropod's ability to respond to immune challenges. These detection assays have been described for some arthropods, especially those of commercial value, but none are available for Euastacus, a genus within the superfamily Parastacoidea. This study is the first step in developing a standardized protocol for the detection and quantification of PO activity in wild or captive Murray crayfish Euastacus armatus. Hemolymph extracts from 49 crayfish were assessed for PO activity using an assay measuring the conversion of l-dopa (3,4-dihydroxy-l-phenylalanine) into dopachrome. Short periods (up to 15 min) out of water did not cause any measurable change in PO activity. Phenoloxidase activity was detected in captive (n = 24, stressed) and wild (n = 25, healthy) crayfish with captive crayfish showing lower levels of PO possibly indicating immunosuppression. The proven protocol is the first of its kind to propose a standardized methodology for the detection and quantification of PO activity in Murray crayfish hemolymph as a means of determining stress.

The sialotranscriptome of the gopher-tortoise tick, Amblyomma tuberculatum.

The gopher tortoise tick, Amblyomma tuberculatum, is known to parasitize keystone ectotherm reptile species. The biological success of ticks requires precise mechanisms to evade host hemostatic and immune responses. Acquisition of a full blood meal requires attachment, establishment of the blood pool, and engorgement of the tick. Tick saliva contains molecules which counter the host responses to allow uninterrupted feeding on the host. RNASeq of the salivary glands of Amblyomma tuberculatum ticks were sequenced resulting in 138,030 pyrosequencing reads which were assembled into 29,991 contigs. A total of 1875 coding sequences were deduced from the transcriptome assembly, including 602 putative secretory and 982 putative housekeeping proteins. The annotated data sets are available as a hyperlinked spreadsheet. The sialotranscriptome assembled for this tick species made available a valuable resource for mining novel pharmacological activities and comparative analysis.

Proteotranscriptomic Insights into the Venom Composition of the Wolf Spider Lycosa tarantula.

Spider venoms represent an original source of novel compounds with therapeutic and agrochemical potential. Whereas most of the research efforts have focused on large mygalomorph spiders, araneomorph spiders are equally promising but require more sensitive and sophisticated approaches given their limited size and reduced venom yield. Belonging to the latter group, the genus Lycosa ("wolf spiders") contains many species widely distributed throughout the world. These spiders are ambush predators that do not build webs but instead rely strongly on their venom for prey capture. Lycosa tarantula is one of the largest species of wolf spider, but its venom composition is unknown. Using a combination of RNA sequencing of the venom glands and venom proteomics, we provide the first overview of the peptides and proteins produced by this iconic Mediterranean spider. Beside the typical small disulfide rich neurotoxins, several families of proteins were also identified, including cysteine-rich secretory proteins (CRISP) and Hyaluronidases. Proteomic analysis of the electrically stimulated venom validated 30 of these transcriptomic sequences, including nine putative neurotoxins and eight venom proteins. Interestingly, LC-MS venom profiles of manual versus electric stimulation, as well as female versus male, showed some marked differences in mass distribution. Finally, we also present some preliminary data on the biological activity of L. tarantula crude venom.

Exploring the prevalence and diversity of bovine ticks in five agro-ecological zones of Pakistan using phenetic and genetic tools.

Tick infestation is a leading cause of tick-worry and tick-borne diseases in livestock and associated economic losses in tropical and subtropical regions of the world. The cattle and buffalo populations in Pakistan are exposed to tick infestation throughout the year, but very little is known about the biology, diversity and distribution of tick species across different agro-ecological zones (AEZ) of the country. The present study aimed to investigate the prevalence (number of bovines infested with ticks out of the investigated population) and diversity of hard ticks infesting bovines in 30 villages located in five distinct AEZs (i.e. Arid, Indus delta, Northern irrigated plain, Sandy desert and Southern irrigated plain). We collected a total of 774 ticks (adult and nymphs) from cattle (n = 116) and water buffaloes (n = 88) on small-holder dairy farms (with <10 bovids per establishment) from September to November 2017. The overall tick prevalence was 46.1% (cattle: 47.9%; buffaloes: 44%), which varied significantly from 22.2% in the Indus delta to 70.5% in the Sandy desert. Tick prevalence was slightly higher in female (46.5%) than male animals (45%), and higher in calves (i.e. ≤ 1 year of age) (55%) than in young animals (i.e. up to 3 years of age) (39%) and adults (48%). Five tick species - Hyalomma anatolicum, Hyalomma hussaini, Hyalomma scupense, Rhipicephalus microplus and Rhipicephalus annulatus - were identified morphologically and then genetically. Genetic identification, achieved using the sequences of two mitochondrial (cytochrome c oxidase subunit 1 and 16S) and one nuclear ribosomal (second internal transcribed spacer) regions, was consistent with the morphological findings. Phylogenetic analyses of the DNA sequence data sets showed that the five species of tick identified here were closely related to the same species or closely related species from within and outside of Pakistan. Of five presently recognised taxa within the R. microplus complex, two were identified herein, including the R. microplus clade C and R. annulatus. This investigation provides the first genetic evidence of the occurrence of R. annulatus in Pakistan as well as Hy. hussaini and Hy. scupense in bovines specifically in the provinces of Sindh and Punjab, respectively. The present findings emphasise the importance of combining morphological and molecular approaches to study the diversity of ticks. Further longitudinal studies are required to establish seasonal variations in the prevalence and distribution of bovine ticks in different AEZs of Pakistan.

Molecular and Morphological Diversity of Heterodesmus Brady and Its Phylogenetic Position within Cypridinidae (Ostracoda).

Ostracod genus Heterodesmus Brady, 1866 is known thus far to contain only three species: H. adamsii Brady, 1866; H. apriculus Hiruta, 1992; and H. naviformis (Poulsen, 1962). This genus has been recorded from the Sea of Japan, and the coastal areas of Thailand and Vietnam. The main generic character is the presence of antero-dorsal and postero-dorsal tube-like processes on the rostrum on both valves. The three species mostly differ in the shell lateral projections. Their relationship and the position of Heterodesmus within family Cypridinidae are poorly understood, partly due to the lack of publication of DNA data so far. We study Heterodesmus collected from several localities in the Northwest Pacific, namely Tsushima and Iki Islands in Japan and Maemul Island in Korea. Besides morphological characters, we also use two mitochondrial markers (16S rRNA and mtCOI) and three nuclear regions (18S rRNA, 28S rRNA, and internal transcribed spacer - ITS) in the samples to detect the biodiversity of this genus. Our phylogenetic tree based on molecular data coupled with morphology reveals the presence of two species, H. adamsii and H. apriculus. We report on their morphological variability, molecular diversity, and phylogenetic position within Cypridinidae based on 16S, 28S and 18S rRNAs, and provide a taxonomic key for all living genera of this family. For the first time, we give an overview of the intrageneric and intrafamily DNA distances of the above markers for the entire subclass Myodocopa.

Fatty acid bioconversion in harpacticoid copepods in a changing environment: a transcriptomic approach.

By 2100, global warming is predicted to significantly reduce the capacity of marine primary producers for long-chain polyunsaturated fatty acid (LC-PUFA) synthesis. Primary consumers such as harpacticoid copepods (Crustacea) might mitigate the resulting adverse effects on the food web by increased LC-PUFA bioconversion. Here, we present a high-quality de novo transcriptome assembly of the copepod Platychelipus littoralis, exposed to changes in both temperature (+3°C) and dietary LC-PUFA availability. Using this transcriptome, we detected multiple transcripts putatively coding for LC-PUFA-bioconverting front-end fatty acid (FA) desaturases and elongases, and performed phylogenetic analyses to identify their relationship with sequences of other (crustacean) taxa. While temperature affected the absolute FA concentrations in copepods, LC-PUFA levels remained unaltered even when copepods were fed an LC-PUFA-deficient diet. While this suggests plasticity of LC-PUFA bioconversion within P. littoralis, none of the putative front-end desaturase or elongase transcripts was differentially expressed under the applied treatments. Nevertheless, the transcriptome presented here provides a sound basis for future ecophysiological research on harpacticoid copepods. This article is part of the theme issue 'The next horizons for lipids as 'trophic biomarkers': evidence and significance of consumer modification of dietary fatty acids'.

Venomics Approach Reveals a High Proportion of Lactrodectus-Like Toxins in the Venom of the Noble False Widow Spider Steatoda nobilis.

The noble false widow spider Steatoda nobilis originates from the Macaronesian archipelago and has expanded its range globally. Outside of its natural range, it may have a negative impact on native wildlife, and in temperate regions it lives in synanthropic environments where it frequently encounters humans, subsequently leading to envenomations. S. nobilis is the only medically significant spider in Ireland and the UK, and envenomations have resulted in local and systemic neurotoxic symptoms similar to true black widows (genus Latrodectus). S. nobilis is a sister group to Latrodectus which possesses the highly potent neurotoxins called α-latrotoxins that can induce neuromuscular paralysis and is responsible for human fatalities. However, and despite this close relationship, the venom composition of S. nobilis has never been investigated. In this context, a combination of transcriptomic and proteomic cutting-edge approaches has been used to deeply characterise S. nobilis venom. Mining of transcriptome data for the peptides identified by proteomics revealed 240 annotated sequences, of which 118 are related to toxins, 37 as enzymes, 43 as proteins involved in various biological functions, and 42 proteins without any identified function to date. Among the toxins, the most represented in numbers are α-latrotoxins (61), δ-latroinsectotoxins (44) and latrodectins (6), all of which were first characterised from black widow venoms. Transcriptomics alone provided a similar representation to proteomics, thus demonstrating that our approach is highly sensitive and accurate. More precisely, a relative quantification approach revealed that latrodectins are the most concentrated toxin (28%), followed by α-latrotoxins (11%), δ-latroinsectotoxins (11%) and α-latrocrustotoxins (11%). Approximately two-thirds of the venom is composed of Latrodectus-like toxins. Such toxins are highly potent towards the nervous system of vertebrates and likely responsible for the array of symptoms occurring after envenomation by black widows and false widows. Thus, caution should be taken in dismissing S. nobilis as harmless. This work paves the way towards a better understanding of the competitiveness of S. nobilis and its potential medical importance.

Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus.

In the present study we show that hemocytes in the freshwater crayfish Pacifastacus leniusculus express two different transglutaminases. We describe the sequence of a previously unknown TGase (Pl_TGase1) and named this as Pl_TGase2 and compared this sequence with similar sequences from other crustaceans. The catalytic core domain is similar to the previously described TGase in P. leniusculus, but Pl_TGase2 has significant differences in the N-terminal and C-terminal domains. Further, we show conclusive evidences that these different transglutaminases are specific for different hemocyte types so that Pl_TGase1 is expressed in the hematopoietic tissue and in the cytoplasm of semigranular hemocytes, while Pl_TGase2 is expressed in vesicles in the granular hemocytes. By in situ hybridization we show that both Pl_TGase1 and Pl_TGase2 mRNA are present only in a subset of the respective hemocyte population. This observation indicates that there may be different subtypes of semigranular as well as granular hemocytes which may have different specific functions.

Integrative taxonomy of Pseudolepeophtheirus longicauda (Crustacea: Copepoda: Caligidae) parasitic on Platichthys stellatus (Actinopterygii: Pleuronectidae).

Based on specimens from the gill cavities of one Platichthys stellatus individual collected in the Sea of Japan, we investigated the taxonomic status of the enigmatic caligid genus Pseudolepeophtheirus and its type species, Pseudolepeophtheirus longicauda. In a maximum likelihood (ML) tree based on 18S rRNA gene sequences, the sequence from our sample was nested in a well-supported Lepeophtheirus clade, along with the type species, confirming that Pseudolepeophtheirus should be considered a junior synonym of Lepeophtheirus; our morphological data support this synonymy. Although a previous study had synonymized Pseudolepeophtheirus longicauda with Lepeophtheirus parvicruris, we found that the former differs morphologically from the latter in having a short leg-4 exopod, with the articulation between the first and second segments not evident (the shape of the posterior striated membrane on the leg-2 intercoxal sclerite also differs between two species), and detected slight differences in 18S rRNA sequences between two taxa. We thus concluded that this synonymy is invalid, and reinstate Lepeophtheirus longicauda as a valid species. A ML analysis of COI sequences from Pl. stellatus (the host fish for both L. longicauda and L. parvicruris) showed the host species to comprise distinct northwestern- and northeastern-Pacific clades. Lepeophtheirus longicauda is distributed in the northwestern Pacific and L. parvicruris in the northeastern Pacific, indicating co-divergence of the two copepod species with the host lineages.

Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene.

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

Residual vitellus and energetic state of wolf spiderlings Pardosa saltans after emergence from egg-sac until first predation.

The aim of this study was to evaluate energetic source used by juveniles of a terrestrial oviparous invertebrate during the earliest periods of their life. Growth, behavioural activities and energy contents of Pardosa saltans spiderlings' residual vitellus were monitored during 8 days after their emergence from their egg-sac until they disperse autonomously. The life-cycle of juvenile after emergence can be divided into three periods: a gregarious while juveniles are aggregated on their mother, dismounting off their mother's back and dispersion. We present the first biochemical study of residual vitellus and energy expenditure during these three periods. At emergence, the mean weight of juveniles was 0.59 mg and energy stock from residual vitellus averaged 51 cal/g wet mass. During gregarious period, the weight of the juveniles aggregated on their mother did not vary significantly and juveniles utilized only 1 cal/day from their residual vitellus. During the period from dismounting until their first exogenous feed, juveniles lost weight and used 30% of their residual vitellus stock. Proteins from the residual vitellus contributed principally to their energy expenditure during this period: 1.5 µg protein/day. Juveniles' first exogenous feeding was observed 7-8 days after emergence, when 70% of residual vitellus energy had been utilized. Juveniles dispersed after eating, reconstituting an energy stock comparable to that observed at emergence from egg-sac (50 cal/g wet mass). This new energy stock contains mainly lipids unlike the energy stock from the residual vitellus.

First records and molecular-phylogenetic analyses of three tick species (Ixodes kaiseri, Hyalomma lusitanicum and Ornithodoros coniceps) from Malta.

The Maltese islands are situated south of mainland Europe and north of Africa, therefore are expected to share tick species and tick-borne pathogens with both continents. This situation highlights the importance of studying ticks in this country. Nevertheless, the tick fauna of Malta appears to be a seldom investigated issue, with hitherto only five tick species reported in the country. Here, as part of a tick collection campaign continuing since 2016 in Malta, three tick species new to the country are reported and analyzed in comparison with GenBank data. Ixodes kaiseri (collected from North African hedgehog in Malta) had unique cytochrome c oxidase subunit I (cox1) and 16S rRNA gene haplotypes (with 98.1-99.3 % sequence identity to I. kaiseri from Europe and China). Phylogenetically, these haplotypes from Malta clustered separately from other, mainland-associated haplotypes, with high support. On the other hand, Ornithodoros coniceps (collected from domestic chicken in Malta) had identical or nearly identical cox1 and 16S rRNA gene haplotypes with soft ticks reported from France, northern Africa and western African islands, similarly to Hyalomma lusitanicum (collected from rabbit and cat in Malta) in comparison with conspecific ticks in Spain and Portugal. These results are most likely related to differences in host associations and corresponding translocality of these three tick species. Taken together, results of the present study add three new tick species to those five already known to be present in Malta.

The prevalence and molecular characterization of Acarapis woodi and Varroa destructor mites in honeybees in the Tohoku region of Japan.

Honeybee acarapiosis and vorrosis were designated as Notifiable Infectious Diseases in the Act on Domestic Animal Infectious Diseases Control by the Minister of Agriculture, Forestry and Fisheries of Japan in 1997. However, the prevalences of A. woodi and V. destructor in Japan, especially in the Tohoku region, have not been sufficiently elucidated. This study was designed to clarify the prevalence of A. woodi and V. destructor mites in Apis cerana japonica and Apis mellifera in the Tohoku region and the characteristics of their mitochondrial cytochrome c oxidase I (COI) DNA. Acarapis woodi mites were detected from 13.5% of A. c. japonica and 0% of A. mellifera. Aomori prefecture, Japan is a new distribution locality for A. woodi. None of the honeybees examined showed infection by V. destructor mites. The COI sequences (1638 bp) of A. woodi were identical and phylogenetically closely related to those of A. woodi from Japan and the UK, suggesting that the mite would have been introduced into Japan with A. mellifera during the last 150 years and spread throughout the country.