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A nationwide survey of chigger mites causing scrub typhus and an investigation of epidemiologic factors for chigger mites was conducted at 16 localities in 8 provinces in Korea during autumn 2009, 2012, and 2013. A total of 233 Apodemus agrarius were captured, and all were infested with chigger mites. The chigger index was highest in Chungcheongbuk-do in 2009 (358.3) and 2012 (290.1) and Chungcheongnam-do in 2013 (294.4). The predominant chigger mite species was Leptotrombidium pallidum in the northern and central parts and L. scutellare in the southern and western parts, Korea. L. pallidum was not found in Jellanam-do and Gyeongsangnam-do and the distribution of L. scutellare had been expanded in the northern parts of Korea. The chigger index of L. pallidum was positively correlated with temperature and negatively correlated with humidity. The incidence of scrub typhus is dependent on L. scutellare index. These findings could be helpful to monitor the distribution of chigger mites and to develop a preventive measures for scrub typhus in Korea.

Paramyxoviruses harbored by multiple natural reservoirs pose a potential threat to public health. Jeilongvirus has been proposed as a novel paramyxovirus genus found in rodents, bats, and cats. Paramyxovirus RNA was detected in 108/824 (13.1%) Apodemus agrarius captured at 14 trapping sites in the Republic of Korea. We first present two genetically distinct novel paramyxoviruses, Paju Apodemus paramyxovirus 1 (PAPV-1) and 2 (PAPV-2). The disparity between PAPV-1 (19,716 nucleotides) and -2 (17,475 nucleotides) revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogeny of PAPV-1 and -2 belonged to distinct genetic lineages of Jeilongvirus, respectively, even though these viruses were originated from A. agrarius. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and pro-inflammatory cytokines. Therefore, this study provides insights into the molecular epidemiology, genetic diversity, and virus-host interactions of novel rodent-borne paramyxoviruses.

Tick surveillance was conducted annually from February–November 2019–2021 at US Army Garrison (USAG) Humphreys, Pyeongtaek, Republic of Korea (ROK). Three general habitats, cut grasses, uncut grasses + herbaceous vegetation, and young mixed (deciduous and conifer) forest groves, were surveyed using tick drags. Ticks also were collected from Korean water deer, Hydropotes inermis argyropus, killed by vehicles at USAG Humphreys. A total of 53,428 ticks (18,004, 16,921, and 18,503 during 2019–2021, respectively) belonging to two genera and five species were collected by tick drag. Haemaphysalis longicornis (48,880; 91.49%) was the most frequently collected tick, followed by H. flava (2,501; 4.68%), Ixodes nipponensis (2,019; 3.78%), H. phasiana (27; 0.05%), and H. japonica (1

As part of a tick-borne disease surveillance program, ticks were collected using tick drags and by making incidental collections from humans and animals in six provinces (Gyeonggi, Gangwon, Chungnam, Gyeongbuk, Gyeongnam, and Jeonbuk) of the Republic of Korea (ROK) during 2020–2021. A total of 57,751 Haemaphysalis longicornis, 2,020 H. flava, 1,861 Ixodes nipponensis, 14 H. phasiana, 9 H. japonica, and 9 Amblyomma testudinarium were collected. Cases of ectromely were observed in two H. longicornis with six legs (one female and one nymph), and six H. longicornis (two females and four nymphs), two female Haemaphysalis flava and one male Ixodes nipponensis with seven legs. Additionally, two female H. longicornis were found with extra legs (tarsus), and two female H. longicornis and one male H. japonica were observed with atrophied legs.

The extended distribution and potential introduction of exotic ticks and associated tick-borne pathogens along the northern and southern routes of migratory birds pose zoonotic tick-borne disease r...

Tick-borne disease surveillance was conducted by tick drag among uncut grasses/herbaceous vegetation and mixed forest habitats from 2018–2020 at US Army Garrison Humphreys, Pyeongtaek, the Republic of Korea (ROK). While identifying ticks collected in the spring of 2020, teratological anomalies were observed in nymphs of Haemaphysalis longicornis (in one specimen, partial twinning of the posterior idiosoma, resulting in 2 anuses; in a second, asymmetry of the idiosoma) and Ixodes nipponensis (7 legs). These teratological observations in H. longicornis and I. nipponensis are reported for the first time in the ROK.

There are currently >300 malaria cases reported annually in the Republic of Korea (ROK), with most cases attributed to exposure in northern Gangwon and Gyeonggi provinces near the demilitarized zone (DMZ). The species diversity and malaria infection rate were determined for a sample of Anopheles mosquitoes collected from May to early November 2020 for six sites in a malaria high-risk area in/near the DMZ and two malaria low-risk areas in southern Gyeonggi province using Mosquito Magnet traps in the ROK. A total of 1864 Anopheles spp. were identified to species by PCR. Overall, An. kleini (31.4%, 510/1622) was the most frequently species assayed, followed by An. pullus (25.5%, 413/1622), An. sineroides (23.9%, 387/1622), and An. sinensis (10.2%, 165/1622), while the other four species only accunted for 9.1% (147/1622) collected in/near the DMZ. Only three species, An. pullus, An. sinensis, and An. sineroides were collected at Humphreys US Army Garrison (USAG) (235 individuals), while only An. sinensis was collected at Yongsan USAG (7 individuals). A total of 36 Anopheles specimens belonging to five species collected in/near the DMZ were positive for Plasmodium vivax by PCR. Anopheles kleini (9) was the most frequent species positive for P. vivax, followed by An. belenrae (8), An. pullus (8), An. sinensis (5), An. sineroides (5), and a member of the Anopheles Lindesayi Complex in the ROK (1). This is the first report of P. vivax in a member of the An. Lindesayi Complex in the ROK. These findings can assist in guiding future malaria vector management in the ROK.

Deer and horse flies, Family Tabanidae, are biological, mechanical, and potential vectors of approximately 27 viral, bacterial, and helminthic pathogens of veterinary and medical importance. Biting fly surveillance was conducted using New Jersey light traps and Mosquito Magnet® traps from May-October during 2015–2018 at eight sites (villages and US military installations and training areas) near/in the Demilitarized Zone and at a US military installation in southern Gyeonggi province, Republic of Korea. A total of 9985 deer and horse flies, comprising 4 genera and 17 species, were collected. The predominant species collected was Chrysops mlokosiewiczi (95.39%), followed by Chrysops suavis (1.48%) and Haematopota koryoensis (1.07%), while the remaining species accounted for only 2.06% of the specimens collected. A bimodal peak was observed for C. mlokosiewiczi in June and late-July, but not for the other two predominant species during the whole of the study period. This study provides and insight into ecological behavior and seasonal abundance of deer and horse flies in Gyeonggi province, Republic of Korea.

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (Cimex lectularius) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

Paramyxoviruses, negative-sense single-stranded RNA viruses, pose a critical threat to human public health. Currently, 78 species, 17 genera, and 4 subfamilies of paramyxoviruses are harbored by multiple natural reservoirs, including rodents, bats, birds, reptiles, and fish. Henipaviruses are critical zoonotic pathogens that cause severe acute respiratory distress and neurological diseases in humans. Using reverse transcription-polymerase chain reaction, 115 Crocidura species individuals were examined for the prevalence of paramyxovirus infections. Paramyxovirus RNA was observed in 26 (22.6%) shrews collected at five trapping sites, Republic of Korea. Herein, we report two genetically distinct novel paramyxoviruses (genus: Henipavirus): Gamak virus (GAKV) and Daeryong virus (DARV) isolated from C. lasiura and C. shantungensis, respectively. Two GAKVs and one DARV were nearly completely sequenced using next-generation sequencing. GAKV and DARV contain six genes (3′-N-P-M-F-G-L-5′) with genome sizes of 18,460 nucleotides and 19,471 nucleotides, respectively. The phylogenetic inference demonstrated that GAKV and DARV form independent genetic lineages of Henipavirus in Crocidura species. GAKV-infected human lung epithelial cells elicited the induction of type I/III interferons, interferon-stimulated genes, and proinflammatory cytokines. In conclusion, this study contributes further understandings of the molecular prevalence, genetic characteristics and diversity, and zoonotic potential of novel paramyxoviruses in shrews.

There are eight Anopheles spp. present in the Republic of Korea (ROK), including five members of the Anopheles Hyrcanus Group that cannot be identified using current morphological methods. The vector competence of only Anopheles sinensis s.s., An. lesteri, and An. kleini have been investigated. As the geographical distribution of Anopheles spp. varies in the ROK, determining the relative vector competence of the Anopheles spp. provides a basis for delineating malaria risks to Korean populations and U.S. military/civilian populations deployed to the ROK. Anopheles belenrae and An. pullus, collected from a malaria high-risk area in the ROK, were evaluated for vector competence of P. vivax. A total of 1,000 each of An. dirus (Thai strain), and Korean strains of An. pullus and An. belenrae were fed on P. vivax infected blood collected from Thai patients via artificial membrane feeding. The overall oocyst infection rates for An. dirus, An. pullus, and An. belenrae dissected on days 8–9 postfeed were 64.1, 12.0, and 11.6%, respectively. The overall sporozoite infection rates for those species dissected on days 14–15 postfeed were 84.5, 3.4, and 5.1% respectively. The salivary gland sporozoite indices for positive females with +4 (>1,000 sporozoites) were observed in An. dirus (72.8%), but not observed for either An. pullus or An. belenrae. Most sporozoite-positive An. pullus (83.3%) and An. belenrae (71.4%) females were observed with only +1 (1–10 sporozoites) salivary glands. These data indicate that both An. belenrae and An. pullus are very poor vectors of P. vivax.

Recent outbreaks of emerging and re-emerging viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There are rising concerns that emergent JEV genotype V (GV) is circulating in Asia, against which current vaccines may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools of Culex tritaeniorhynchus and Culex bitaeniorhynchus mosquitoes (6540 specimens) collected at Camp Humphreys, Republic of Korea (ROK) in 2018. Interrogation of our data revealed a highly abundant and diverse virosphere that contained sequences from 122 distinct virus species. Our statistical and hierarchical analysis uncovered correlates of potential health, virological, and ecological relevance. Furthermore, we obtained evidence that JEV GV was circulating in Pyeongtaek and, retrospectively, in Seoul in 2016 and placed these findings within the context of human and fowl reservoir activity. Sequence-based analysis of JEV GV showed a divergent genotype that is the most distant from the GIII-derived live attenuated SA14-14-2 vaccine strain and indicated regions probably responsible for reduced antibody affinity. These results emphasize recent concerns of shifting JEV genotype in East Asia and highlight the critical need for a vaccine proven efficacious against this re-emergent virus. Together, our one-health approach to Culex viral metagenomics uncovered novel insights into virus ecology and human health.

Tick-borne pathogens are contributing factors for the increased incidence of vector-borne diseases throughout the world, including Lyme borreliosis, one of the most prevalent spirochetes belonging to the Borrelia burgdorferi sensu lato group. The present study focused on the detection of Borrelia species from hard ticks collected at U.S. Army Garrison Humphreys, Republic of Korea (ROK), using molecular and genotypic analyses. Tick-borne disease surveillance was conducted from January to December, 2018–2019. A total of 24,281 ticks (2 genera and 5 species) were collected from road-killed Korean Water deer (KWD) and by tick drag. Haemaphysalis longicornis (92.0%) was the most commonly collected species, followed by Haemaphysalis flava (4.9%), Ixodes nipponensis (3.1%), Haemaphysalis phasiana (0.07%), and Haemaphysalis japonica (

When free-ranging birds are accidentally killed or die, there may be greater potential for their associated ticks to detach, seek alternate hosts, and become established. We examined 711 carcasses of 95 avian species for ticks at a stopover island of migratory birds in the Republic of Korea where only Ixodes nipponensis and I. persulcatus were previously reported from local mammals and vegetation. A total of 16 ticks, I. turdus and Haemaphysalis flava, were collected from 8 fresh carcasses belonging to 5 avian species. Despite their known abundance on migratory birds and mainland Korea, these species had not colonized the isolated insular ecosystem possibly due to the low abundance and diversity of local hosts. The results imply that increasing human impact, such as the anthropogenic mortality of migratory birds and the introduction of non-native mammalian hosts, will increase the potential invasion and colonization risk of ticks. This finding also suggests that tick surveillance consisting of fresh carcasses of dead migratory birds may provide additional information, often ignored in surveillance of ticks on live birds, for the potential introduction of non-native ticks and associated pathogens affecting animal and human health.

Background Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria re-emerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species (Anopheles sinensis, Anopheles pullus, Anopheles belenrae, Anopheles lesteri, Anopheles kleini, Anopheles sineroides, Anopheles koreicus, Anopheles lindesayi) are distributed throughout Korea. Members of the Anopheles Hyrcanus group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK. Methods Anopheles spp. used in this study were collected near/in the demilitarized zone in ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analysed for molecular identification. Results DNA from a total of 165 Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Each species was clearly distinguished by electrophoresis as follows: 1,112 bp for An. sinensis; 925 bp for An. koreicus; 650 bp for An. lindesayi; 527 bp for An. kleini; 436 bp for An. lesteri; 315 bp for An. sineroides; 260 bp for An. belenrae; and, 157 bp for An. pullus. Conclusion A multiplex PCR assay was developed to identify Anopheles spp. distributed in ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

Whole-genome sequencing of infectious agents enables the identification and characterization of emerging viruses. The MinION device is a portable sequencer that allows real-time sequencing in fields or hospitals. Hantaan orthohantavirus (Hantaan virus, HTNV), harbored by Apodemus agrarius, causes hemorrhagic fever with renal syndrome (HFRS) and poses a critical public health threat worldwide. In this study, we aimed to evaluate the feasibility of using nanopore sequencing for whole-genome sequencing of HTNV from samples having different viral copy numbers. Amplicon-based next-generation sequencing was performed in A. agrarius lung tissues collected from the Republic of Korea. Genomic sequences of HTNV were analyzed based on the viral RNA copy numbers. Amplicon-based nanopore sequencing provided nearly full-length genomic sequences of HTNV and showed sufficient read depth for phylogenetic analysis after 8 h of sequencing. The average identity of the HTNV genome sequences for the nanopore sequencer compared to those of generated from Illumina MiSeq revealed 99.8% (L and M segments) and 99.7% (S segment) identities, respectively. This study highlights the potential of the portable nanopore sequencer for rapid generation of accurate genomic sequences of HTNV for quicker decision making in point-of-care testing of HFRS patients during a hantavirus outbreak.

Ticks are important vectors of various pathogens that result in clinical illnesses in humans and domestic and wild animals. Information regarding tick infestations and pathogens transmitted by ticks is important for the identification and prevention of disease. This study was a large-scale investigation of ticks collected from dogs and their associated environments in the Republic of Korea (ROK). It included detecting six prevalent tick-borne pathogens (Anaplasma spp., A. platys, Borrelia spp., Babesia gibsoni, Ehrlichia canis, and E. chaffeensis). A total of 2293 ticks (1110 pools) were collected. Haemaphysalis longicornis (98.60%) was the most frequently collected tick species, followed by Ixodes nipponensis (0.96%) and H. flava (0.44%). Anaplasma spp. (24/1110 tick pools, 2.16%) and Borrelia spp. (4/1110 tick pools, 0.36%) were detected. The phylogenetic analyses using 16S rRNA genes revealed that the Anaplasma spp. detected in this study were closely associated with A. phagocytophilum reported in humans and rodents in the ROK. Borrelia spp. showed phylogenetic relationships with B. theileri and B. miyamotoi in ticks and humans in Mali and Russia. These results demonstrate the importance of tick-borne disease surveillance and control in dogs in the ROK.

This study was done to characterize distribution of Rickettsia spp. in ticks in the northwestern and southwestern provinces in the Republic of Korea. A total of 2,814 ticks were collected between May and September 2009. After pooling, 284 tick DNA samples were screened for a gene of Rickettsia-specific 17-kDa protein using nested PCR (nPCR), and produced 88 nPCR positive samples. Of these positives, 75% contained 190-kDa outer membrane protein gene (ompA), 50% 120-kDa outer membrane protein gene (ompB), and 64.7% gene D (sca4). The nPCR products of ompA, ompB, and sca4 genes revealed close relatedness to Rickettsia japonica, R. heilongjiangensis, and R. monacensis. Most Rickettsia species were detected in Haemaphysalis longicornis. This tick was found a dominant vector of rickettsiae in the study regions in the Republic of Korea.

Background Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. Methods Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction–based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. Results The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. Conclusions This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

The Anopheles Hyrcanus Group in the Republic of Korea (ROK) consists of 5 morphologically indistinct species that can only be identified with certainty by polymerase chain reaction (PCR). A total of 86 bloodfed Anopheles spp. were collected from a cow barn located in the village of Tongilchon near the demilitarized zone in the ROK on June 13, 2016, and sent to the Armed Forces Research Institute of Medical Sciences in Bangkok, Thailand, where they were identified to species by PCR. The 1st shipment contained 15 An. belenrae and 37 An. pullus females that were used to start the colonies. Parent females that oviposited were identified by PCR for colonization. A higher proportion of F1–F4 females of An. belenrae than An. pullus bloodfed when provided both blood meals on human arms and using a membrane feeding system with human blood. Following blood meals, the females were forced mated for colony maintenance. The mean numbers of eggs oviposited per female for An. belenrae was 127.7 ± 19.3 and for An. pullus was 136 ± 23.6. On average, at 25°C (±2°C) An. belenrae and An. pullus took 15.1 and 16.1 days to develop from egg to adult, respectively. A 2nd group of bloodfed Anopheles spp. was collected at the same location in the ROK on June 24, 2017. This group contained 13 An. belenrae and 27 An. pullus. Similarly, eggs were obtained and adults identified by PCR and then reared to adults and subsequent generations forced mated to members of each of the existing colonies to increase genetic diversity. The colonies were established to evaluate their susceptibility to vector vivax malaria, which is essential to better understand the epidemiology of malaria transmission in Korea. This is the 1st report of colonization of both An. belenrae and An. pullus.

Recent outbreaks of emerging and re-emerging viruses such as Zika, West Nile and Japanese encephalitis (JEV) viruses have shown that timely detection of novel arboviruses with epidemic potential is essential to mitigate human health risks. There have been rising concerns that an emergent JEV genotype (genotype V, GV) is circulating in Asia, against which the current US-FDA-approved JEV vaccine may not be efficacious. To ascertain if JEV GV and other arboviruses are circulating in East Asia, we conducted next-generation sequencing on 260 pools of Culex tritaeniorhynchus and Culex bitaeniorhynchus mosquitoes (6,540 specimens) collected at Camp Humphreys, Republic of Korea (ROK), from mid-May - October 2018. Metagenomic analysis demonstrated a highly abundant and diverse virome with correlates of health and ecological relevance. Additionally, two complete JEV GV genome sequences were obtained from separate mosquito pools, indicating that JEV GV is circulating in the Pyeongtaek area near Seoul, ROK. Retrospective sample and sequence analyses showed that JEV GV was also present in 2016 mosquito pools collected in Seoul, ROK. Sequence-based analysis of JEV GV indicates a divergent genotype that is the most distant from the GIII derived live attenuated SA14-14-2 vaccine strain. A GV E protein investigation and 3D modeling in context to SA14-14-2 indicated likely regions responsible for reduced antibody affinity, including clusters of significant amino acid changes at externally exposed domains. These data highlight the critical need for continued mosquito surveillance as a means of detecting and identifying emerging and re-emerging arboviruses of public health relevance. Importantly, our results emphasize recent concerns that there may be a possible shift in the circulating JEV genotype in East Asia and highlights the critical need for a vaccine proven to be efficacious against this re-emergent virus.

Biting midges (Culicoides: Ceratopogonidae) were collected using New Jersey light traps at Yongsan US Army Garrison (USAG;urban), Seoul Metropolitan city and Camp Humphreys USAG (rural), Pyeongtaek, Gyeonggi-do (province), Republic of Korea , from May-October 2010-2013 and 2015-2017, to determine species composition and seasonal distribution patterns in urban and rural habitats. A total of 9,958 female (53.85%) and 8,533 male (46.15%) Culicoides comprising 16 species were collected. Overall, the most commonly collected species was Culicoides arakawae (74.3%), followed by C. circumscriptus (16.2%), C. kibunensis (2.5%), C. nasuensis (2.2%), C. clavipalpis (1.4%), and C. pallidulus (1.3%), while the remaining 10 species accounted for

Background Genus Anopheles mosquitoes are the primary vectors of human malaria, which is a serious threat to public health worldwide. To reduce the spread of malaria and identify the malaria infection rates in mosquitoes, accurate species identification is needed. Malaria reemerged in 1993 in the Republic of Korea (ROK), with numbers peaking in 2004 before decreasing to current levels. Eight Anopheles species (Anopheles sinensis, An. pullus, An. belenrae, An. lesteri, An. kleini, An. sineroides, An. koreicus, and An. lindesayi) are distributed throughout Korea. Members of the Anopheles Hyrcanus Group currently cannot be identified morphologically. The other species of Anopheles can be identified morphologically, except when specimens are damaged in traps. The purpose of this study was to develop a rapid and accurate method for simultaneous molecular identification of the eight Anopheles species present in the ROK. Methods Anopheles spp. used in this study were collected near/in the demilitarized zone in the ROK, where most malaria cases are reported. DNA from 165 of the Anopheles specimens was used to develop a multiplex PCR assay. The internal transcribed spacer 2 (ITS2) region of each species was sequenced and analyzed for molecular identification. Results DNA from a total of 165 Anopheles specimens was identified to species using a multiplex diagnostic system. These included: 20 An. sinensis, 21 An. koreicus, 17 An. lindesayi, 25 An. kleini, 11 An. lesteri, 22 An. sineroides, 23 An. belenrae, and 26 An. pullus. Molecular weights determined by electrophoresis were 1112 bp for An. sinensis, 925 bp for An. koreicus, 650 bp for An. lindesayi, 527 bp for An. kleini, 436 bp for An. lesteri, 315 bp for An. sineroides, 260 bp for An. belenrae, and 157 bp for An. pullus. Conclusion A multiplex PCR assay was developed to identify Anopheles spp. distributed in the ROK. This method can be used to accurately identify Anopheles species that are difficult to identify morphologically to determine species distributions and malaria infection rates.

The extended distribution and potential introduction of exotic ticks and associated tick-borne pathogens along the northern and southern routes of migratory birds pose zoonotic tick-borne disease risks to wild and domestic animals and incidentally to humans. A knowledge of bird migratory patterns, species of attached ticks, and associated pathogens during their migrations to and from their feeding and nesting grounds is central to understanding associated tick-borne disease risks. Tick-borne disease surveillance was conducted from 2010 to 2011 and 2016 at Hong-do (do = island), Heuksan-do, and Nan-do, major stopovers for migratory birds in Republic of Korea (ROK), as part of the Migratory Birds Research Center bird-banding program for studying bird migration patterns in the ROK. A total of 877 ticks belonging to three genera and nine species were collected

In a follow-up to the investigations of soft ticks identified from seabird nest soil and litter collected from coastal islands of the Republic of Korea (ROK), Ornithodoros sawaii and Ornithodoros capensis were assessed for the presence and identification of rickettsiae. Ticks collected from samples of 50–100 g of nest litter and soil from seabird nests were identified individually by morphological techniques, and species confirmed by sequencing of the mt-rrs gene. Subsequently, tick DNA preparations were screened for the presence of rickettsiae using a genus-specific nested PCR (nPCR) assay targeting the 17 kDa antigen gene. The amplicons from the 17 kDa assay and two additional nPCR assays targeting the gltA and ompB gene fragments were sequenced and used to identify the rickettsiae. A total of 134 soft ticks belonging to two species, O. sawaii Kitaoka & Suzuki 1973 (n = 125) and O. capensis Neumann 1901 (n = 9), were collected. Rickettsia lusitaniae DNA was detected and identified among O. sawaii ticks (n = 11, 8.8%) collected from nest litter and soil of the Japanese murrelet (Synthliboramphus wumizusume Temminck 1836) at Gugul Island along the western coastal area of the ROK. This study confirmed for the first time the presence of R. lusitaniae associated with O. sawaii collected from migratory seabird nests in the ROK.

Wing lengths of parous (P) and nulliparous (NP) PCR-identified female Anopheles belenrae, An. kleini, An. pullus, and An. sinensis were determined from weekly trap collections at Camp Humphreys (CH), Ganghwa Island (GH), and Warrior Base (WB), Republic of Korea (ROK) during Jun-Oct, 2009. Wing length was greatest at the beginning and end of the study period. Wing length of NPs tended to be less than that of Ps before the period of maximum greening (Jul-Aug) but greater thereafter. Larger specimens tended to be Ps, and weekly wing length of Ps appeared less variable than NPs, possibly due to selection. A bimodal wing length frequency distribution of An. sinensis suggested two forms comprising small- (≤4.5 mm, SW) and large-winged females (>4.5 mm, LW). LW comprised the majority of peaks in abundance, however %SW, while still a minority, often increased during these times suggesting a density-dependent effect. At WB and GH, a two to three-week periodicity in %SW was obvious for An. sinensis and An. kleini. Analyses of weather station and satellite data showed that smaller-winged An. sinensis were associated with warmer, more humid, and greener times of the year. SW and LW specimens possibly result from agricultural practices that are common across large areas; regular synchronous peaks of SW and LW were observed from different sites. Peaks in SW Ps followed peaks in NPs in a 'ripple effect' one to two weeks apart, suggesting that wing length combined with parity could be used to follow the emergence and survival of mosquito cohorts.

Two new species of crane flies belonging to the genus Atypophthalmus Brunetti, 1911, A. (Atypophthalmus) kleini Podenas, sp. nov. and A. (Microlimonia) jeju Podenas & Podeniene, sp. nov. collected in South Korea in 2017 and 2019 are described and illustrated. Larvae and female pupa for A. (Microlimonia) jeju sp. nov. are described. The pupa for the subgenus A. (Microlimonia) is described for the first time. An updated list and key for adults of all Korean Atypophthalmus are presented.

Haemaphysalis longicornis, the cattle tick or bush tick, has an extended distribution throughout Asia and the Pacific region, including China, Russia, the Republic of Korea (ROK), Japan, Australia, New Zealand, and the South Pacific islands. It is an obligate ectoparasite found commonly on medium to large sized wild and domestic animals, with humans as an accidental host. Haemaphysalis longicornis transmits a number of pathogens, including severe fever with thrombocytopenia syndrome and tick-borne encephalitis viruses, bacteria, helminths, and protozoans, that impact on veterinary (wild and domestic animals) and human health. Surveys of rickettsial pathogens associated with H. longicornis from China, the ROK, and Japan have resulted in the discovery of more than 35 incompletely characterized molecular isolates of Rickettsia. In response to the increased global threat of tick-borne rickettsial diseases, H. longicornis collected in the ROK and China were assessed in our laboratory and two additional Rickettsia spp. isolates (ROK-HL727 and XinXian HL9) were identified. These agents were fully characterized by multilocus sequence typing using partial gene fragment sequences of rrs, gltA, ompA, ompB, and sca4. Phylogenetic comparisons of these Rickettsia isolates with known Rickettsia species and other molecular isolates identified from H. longicornis were performed to better understand their interrelationships. Phylogenetic analysis of the sequences from these 5 gene fragments showed that ROK-HL727 was closely related to rickettsial isolates of H. longicornis previously reported from China, the ROK and Japan, but distinct from any currently recognized Rickettsia species. It therefore qualifies genetically as a new species, introduced herein as Candidatus Rickettsia longicornii. The XinXian-HL9 isolate detected from China was determined to be genetically similar to the human pathogen Rickettsia heilongjiangensis. People living and working in areas where H. longicornis is endemic should be aware of the potential for rickettsial diseases.