Establishment and application of bioassay- and molecular marker-based methods for monitoring fluvalinate resistance of Varroa mites.

The Varroa mite, Varroa destructor, is an ectoparasite that infests honey bees. The extensive use of acaricides, including fluvalinate, has led to the emergence of resistance in Varroa mite populations worldwide. This study's objective is to monitor fluvalinate resistance in field populations of Varroa mites in Korea through both bioassay-based and molecular marker-based methods. To achieve this, a residual contact vial (RCV) bioassay was established for on-site resistance monitoring. A diagnostic dose of 200 ppm was determined based on the bioassay using a putative susceptible population. In the RCV bioassay, early mortality evaluation was effective for accurately discriminating mites with the knockdown resistance (kdr) genotype, while late evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 indicated potential resistance development in four populations. As an alternative approach, quantitative sequencing was employed to assess the frequency of the L925I/M mutation in the voltage-gated sodium channel (VGSC), associated with fluvalinate kdr trait. While the mutation was absent in 2020 Varroa mite populations, it emerged in 2021, increased in frequency in 2022, and became nearly widespread across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation further underscores the urgent need to replace fluvalinate with alternative acaricides. A few novel VGSC mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Seasonal distribution of Haemaphysalis longicornis (Acari: Ixodidae) and detection of SFTS virus in Gyeongbuk Province, Republic of Korea, 2018.

The seasonal distribution of hard ticks was investigated in 2018 in Gyeongbuk Province, Republic of Korea. Ticks were assayed for severe fever with thrombocytopenia syndrome virus (SFTSV). Ticks were collected monthly using CO2-baited traps from April to November in four habitats (grasslands, grave sites, hiking trails, and mixed forests). Based on morphological and molecular identification, Haemaphysalis longicornis was the most commonly collected species, followed by H. flava and Ixodes nipponensis. Ticks were more commonly collected in grassland habitats, followed by the grave sites, hiking trails, and mixed forests. Peak numbers of nymphs and adults of H. longicornis occurred in May and June, respectively, and Haemaphysalis larvae were collected from August to October. A total of 9/187 (4.8%) pools were positive for SFTSV between June and October in 2018. Phylogenetic analysis of partial fragments of the SFTSV obtained in this study showed that all positive virus samples clustered into genotype B.

Reference gene selection for qRT-PCR analysis of season- and tissue-specific gene expression profiles in the honey bee Apis mellifera.

Honey bees are both important pollinators and model insects due to their highly developed sociality and colony management. To better understand the molecular mechanisms underlying honey bee colony management, it is important to investigate the expression of genes putatively involved in colony physiology. Although quantitative real-time PCR (qRT-PCR) can be used to quantify the relative expression of target genes, internal reference genes (which are stably expressed across different conditions) must first be identified to ensure accurate normalisation of target genes. To identify reliable reference genes in honey bee (Apis mellifera) colonies, therefore, we evaluated seven candidate genes (ACT, EIF, EF1, RPN2, RPS5, RPS18 and GAPDH) in samples collected from three honey bee tissue types (head, thorax and abdomen) across all four seasons using three analysis programmes (NormFinder, BestKeeper and geNorm). Subsequently, we validated various normalisation methods using each of the seven reference genes and a combination of multiple genes by calculating the expression of catalase (CAT). Although the genes ranked as the most stable gene were slightly different on conditions and analysis methods, our results suggest that RPS5, RPS18 and GAPDH represent optimal honey bee reference genes for target gene normalisation in qRT-PCR analysis of various honey bee tissue samples collected across seasons.

Characterization of overwintering sites of Haemaphysalis longicornis (Acari: Ixodidae) and tick infection rate with severe fever with thrombocytopenia syndrome virus from eight provinces in South Korea.

Haemaphysalis longicornis (Acari: Ixodidae) is an important vector of pathogens causing tick-borne diseases such as severe fever with thrombocytopenia syndrome (SFTS) in eastern Asia. Although an understanding of the overwintering ecology of ticks is fundamental to management of this vector, its winter biology remains unclear. Therefore, we conducted a field survey from eight provinces in South Korea to characterize overwintering sites of H. longicornis and investigate their SFTS virus infection rates. First, we conducted flagging which consists of horizontal sweeping of a 1 m2 cloth back-and-forth to collect ticks that may exhibit questing behaviors in four different landscapes: grassland, shrub, coniferous forest, and deciduous forest. From 640 sweeps of flagging (where each sweep covered 3.8 m2), we collected five unfed ixodid ticks. However, H. longicornis was not found. After the flagging, to locate overwintering ticks, we inspected a total of 679 samples consisting of three different structures: ground (leaf litter, soil surface, and topsoil layer), rocks, and dead trees. From the samples inspected, 85 unfed overwintering ixodid ticks were found. Haemaphysalis longicornis was the dominant species (88 %), and mostly nymphs were collected (94 %). This species was collected from ground samples, especially from the topsoil layer. Most H. longicornis were found in herbaceous landscapes such as grassland (46 %) and shrub (52 %). SFTS virus was found in 3 out of 38 pools of unfed nymphs (minimal infection rate: 4 %). Our results can serve as baseline information for the development of vector management programs.

Validation of quantitative real-time PCR reference genes for the determination of seasonal and labor-specific gene expression profiles in the head of Western honey bee, Apis mellifera.

Honey bee is not only considered an important pollinator in agriculture, but is also widely used as a model insect in biological sciences, thanks to its highly evolved sociality, specialization of labor division, and flexibility of colony management. For an intensive investigation of the seasonal and labor-dependent expression patterns of its genes, accurate quantification of the target gene transcription level is a fundamental step. To date, quantitative real-time PCR (qRT-PCR) has been widely used for rapid quantification of gene transcripts, with reliable reference gene(s) for normalization. To this end, in an attempt to search for reliable reference genes, the amplification efficiencies of six candidate reference genes (rp49, rpL32, rpS18, tbp, tub, and gapdh) were determined. Subsequently, four genes (rpL32, rpS18, tbp, and gapdh) with PCR efficiencies of 90% to 110% were evaluated for their expression stabilities with three programs (geNorm, NormFinder, and BestKeeper) and used for normalization of seasonal expression patterns of target genes in the forager and nurse heads. Although the three programs revealed slightly different results, two genes, rpS18 and gapdh, were suggested to be the optimal reference genes for qRT-PCR-based determination of seasonal and labor-specific gene expression profiles. Furthermore, the combined use of these two genes yielded a more accurate normalization, compared with the use of a single gene in the head of honey bee. The validated reference genes can be widely used for quantification of target gene expression in honey bee head although it is still remained to be elucidated the expression levels of the selected reference genes in specific tissues in head.

Regulation of retinal axon growth by secreted Vax1 homeodomain protein.

Retinal ganglion cell (RGC) axons of binocular animals cross the midline at the optic chiasm (OC) to grow toward their synaptic targets in the contralateral brain. Ventral anterior homeobox 1 (Vax1) plays an essential role in the development of the OC by regulating RGC axon growth in a non-cell autonomous manner. In this study, we identify an unexpected function of Vax1 that is secreted from ventral hypothalamic cells and diffuses to RGC axons, where it promotes axonal growth independent of its transcription factor activity. We demonstrate that Vax1 binds to extracellular sugar groups of the heparan sulfate proteoglycans (HSPGs) located in RGC axons. Both Vax1 binding to HSPGs and subsequent penetration into the axoplasm, where Vax1 activates local protein synthesis, are required for RGC axonal growth. Together, our findings demonstrate that Vax1 possesses a novel RGC axon growth factor activity that is critical for the development of the mammalian binocular visual system.