Rhipicephalus linnaei is a widespread tick species infesting dogs and capable of transmitting pathogens of veterinary and zoonotic concern. However, its associated bacterial communities remain poorly described in Malaysia. This study profiles the bacterial microbiome of R. linnaei collected from stray dogs in Kampar, Perak, using 16S rRNA gene amplicon sequencing targeting the V3-V4 region. A total of 360 ticks were collected from 13 dogs, of which 290 were pooled according to life stages and sex for microbial profiling. Shannon diversity indices indicated the mixed adult/nymph pool exhibited the highest richness and evenness (H’=5.4), whereas engorged adult females displayed the lowest diversity (H’=2.35), dominated by Gammaproteobacteria. Principal coordinate analysis revealed distinct microbial assemblages among pools, explaining 72% of total variance. Among 137 detected genera, Coxiella (0.6–34%), Staphylococcus (0.4–29%), Stenotrophomonas (0.3–5%), and Streptococcus (0.02–6%) were consistently found across all pools. Low-abundance but clinically relevant genera, including Ehrlichia (0.64%) and Nocardia (<0.01%), were detected in adult males. The consistent presence of Coxiella-like endosymbionts across all stages suggests a likely symbiotic role in nutrient provisioning and reproduction. To our knowledge, this is the first 16S rRNA gene-based profiling of the bacterial communities associated with R. linnaei collected from stray dogs in Malaysia. This study highlights variation across pooled tick categories and contributes to improved understanding of tick-borne pathogen ecology within a One Health framework.

Mosquito-borne arboviral diseases such as dengue and chikungunya have continuously been a major global public health concern. Introducing the intracellular bacterial endosymbiont Wolbachia into mosquito populations has been proven to reduce dengue virus transmission, and its broader efficacy against other arboviruses has also been investigated. Several Wolbachia strains have been successfully propagated in insect cell lines, highlighting the utility of in vitro systems for studying microbial-host interactions under controlled conditions. This study investigated the initial establishment and replication kinetics of a flea-derived Wolbachia strain (wCfe), which was originally isolated from Malaysian Ctenocephalides felis and maintained in the Ixodes scapularis tick-derived cell line (IDE8), and then transferred into an Aedes albopictus-derived cell line (C6/36). The wCfe strain was semi-purified from IDE8 cultures and inoculated into C6/36 cells in 24-well plates. Replication dynamics were monitored by quantitative real-time PCR targeting the Wolbachia pipientis 16S rRNA gene. Following infection, a lag phase was observed at day 0 to 5 days post-infection (d.p.i.), followed by exponential growth from 6 d.p.i. after which Wolbachia levels remained relatively stable until the end of the observation period at 12 d.p.i. Overall, a 25.30-fold increase in Wolbachia density was detected relative to 0 d.p.i. Across replicates, the estimated generation time of wCfe in C6/36 cells ranged from 1.7 to 2.5 days. These results demonstrate successful initial establishment and replication of the flea-derived Wolbachia strain in the Aedes mosquito cell line. However, longer-term in vivo studies will be necessary to determine the persistence of wCfe infection in C6/36 cells and within the mosquito host.

Simulium (Gomphostilbia) okinawense Takaoka and S. (G.) tokarense Takaoka, both from the Nansei Islands, Japan, were morphologically reexamined and genetically analysed by using the COI gene sequences. The female, male, pupa and mature larva of the two species are redescribed. Morphological reexamination shows that both species are more similar to species in the S. asakoae species-group than to those in the S. ceylonicum species-group, by having a medium-long female sensory vesicle, yellow tuft hairs (S. (G.) okinawense) or yellow tuft hairs mixed with a few to several dark hairs (S. (G.) tokarense) at the base of the radial vein in the female and male, and medium-long larval postgenal cleft. However, the body of the male ventral plate (viewed ventrally) is parallel-sided (S. (G.) okinawense) or parallelsided or slightly narrowed (S. (G.) tokarense) and not emarginated basally, differing from those of most species in the S. asakoae species-group. Our genetic analysis shows that S. (G.) tokarense is in the S. asakoae species-group, and S. (G.) okinawense formed a separate sister clade with other members of the S. asakoae species-group with high bootstrap support. From the results of morphological and genetic analysis combined, S. (G.) okinawense and S. (G.) tokarense are transferred from the S. ceylonicum species-group to the S. asakoae species-group.

Simulium takahasii (Rubtsov), which was originally described from Japan, and recorded from Korea and China, is the first among the 19 species of the subgenus Wilhelmia Enderlein recorded from East Asia. It is striking in mating, blood-feeding and ovipositing in captivity and in experimentally transmitting Dirofilaria immitis (Leidy) and Brugia pahangi (Buckley & Edeson), and it is a severe biter of cattle and horses, rarely of humans. Nevertheless, updated information about its morphological characteristics was lacking, making comparisons with related species described from China difficult, since species of the subgenus Wilhelmia are almost indistinguishable from one another, in particular, in their female terminalia, male genitalia and most of larval features. In this study, as many morphological characteristics as possible of S. takahasii based on specimens from Japan are redescribed. New information about many features of this species including the length of the female sensory vesicle against the third palpal segment, number of male upper-eye (large) facets, arrangement of the eight pupal gill filaments, presence or absence of tiny dark setae on the dorsum of the larval abdomen and the number of rows and hooklets of the larval posterior circlet will be useful in evaluating the species status of several Wilhelmia species in China including the species regarded as S. takahasii.

Simulium (Gomphostilbia) ogatai (Rubtsov) from Japan, the only Palaearctic species in the S. ceylonicum species-group, is morphologically revised. Its female is described for the first time and its male and pupa are redescribed based on specimens from Gifu, Honshu. It is characterized in the female and male by the base of the radius with a tuft of yellow hairs, in the female by the head slightly wider than the thorax, 5.89 times as wide as the greatest width of the frons, and short sensory vesicle 0.21–0.24 times as long as the third palpal segment; in the male by the number of upper-eye (large) facets in 15 vertical columns and 16 horizontal rows, and hind basitarsus spindle-shaped, enlarged, 3.79 times as long as wide, and 0.83 and 1.00 times as wide as the greatest width of the hind tibia and femur, respectively; and in the pupa by the terminal hooks simple and cone-like, and the cocoon with an anterodorsal projection. Taxonomic notes for this species are given relevant to several related species in the Oriental Region, and its assignment to the S. ceylonicum species-group is confirmed.

Simulium (Simulium) rufibasis Brunetti originally described from India was once considered a geographic generalist widely distributed in the Oriental Region. In this study, the species previously regarded as S. (S.) rufibasis in Taiwan was morphologically re-evaluated and found to be distinguished in the male and pupa from true S. (S.) rufibasis from India. This new species is described as S. (S.) hehuanense sp. nov. based on a female, a male and their pupal exuviae in Taiwan. This new species is placed in the S. rufibasis subgroup of the S. tuberosum species-group, and is similar to S. (S.) yamatoense Takaoka, Adler & Fukuda from Japan and Korea, but it is barely distinguished by the slenderer forebasitarsi of the female and male. Another new, related species, S. (S.) xiulinense sp. nov., is described based on a male and its pupal exuviae. This new species is distinguished from S. (S.) hehuanense sp. nov. by the number of male upper-eye (large) facets in 20 or 21 vertical columns and 22 horizontal rows (19 vertical columns and 19 horizontal rows in the latter species), and presence of a pair of dorsolateral shiny spots on male abdominal segment 5 (absence in the latter species). Both new species inhabit streams at high elevations (ca, 2,600 m). They differ from S. (S.) sp. (probably S. (S.) arisanum Shiraki) from Taiwan, of the same subgroup, which breeds in streams at low elevations (433–685 m), by the pupal head and thorax covered with tubercles (bare in the latter species).

A new black fly species, Simulium yukawai, is described on the basis of one female and its pupal exuviae from Vietnam. This new species is placed in the S. crocinum species-group of Simulium (Simulium) (Diptera: Simuliidae). It is characterized in the female by the paraproct covered with 48–50 distinct hairs on its ventral and lateral surfaces, and in the pupa by the frons and most of the thorax bare, gill with six dark filaments in three pairs closely arising from the base, of which the two outer filaments of the dorsal and middle pairs are much longer than other filaments, abdomen with distinct spine-combs on the dorsal surface of segments 7–9, and cocoon wall-pocket shaped, with a large anterolateral window on each side. Taxonomic notes are given to separate this new species from related species. This species is the fourth member of the S. crocinum species-group known from Vietnam.