Inference of selection pressures that drive insecticide resistance in Anopheles and Culex mosquitoes in Korea.

Pyrethroids are primarily used for mosquito control in Korea. However, high frequencies of mutations conferring resistance to not only pyrethroids but also to other insecticides have been found in mosquito populations. This study aimed to examine the hypothesis that insecticides used outside of public health may play a role in selection. Briefly, the resistance mutation frequencies to three insecticide groups (pyrethroids, organophosphates, and cyclodienes) were estimated in two representative groups of mosquito species (Anopheles Hyrcanus Group and Culex pipiens complex). The relationship between these frequencies and the land-use status of the collection sites was investigated through multiple regression analysis. In the Anopheles Hyrcanus Group, the frequencies of both ace1 (organophosphate resistance) and rdl (cyclodiene resistance) mutations were positively correlated with 'proximity to golf course', possibly be due to the insecticides used for turf maintenance. They also showed positive correlations with field area and rice paddy area, respectively, suggesting the role of agricultural insecticides in the selection of these resistance traits. For the Cx. pipiens complex, the kdr (pyrethroid resistance), ace1, and rdl mutations were positively correlated with the residential area, field, and rice paddy, respectively. Therefore, pyrethroids used for public health could serve as a direct source of resistance selection pressure against kdr, whereas non-public health insecticides may pose primary selection pressure against the ace1 and rdl traits. The current findings suggest that the insecticides used in agriculture and the golf industry play a significant role in mosquito selection, despite variations in the extent of indirect selection pressure according to the mosquito groups and insecticide classes.

Multiplex PCR assay for the identification of eight Anopheles species belonging to the Hyrcanus, Barbirostris and Lindesayi groups.

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.

Regional and seasonal detection of resistance mutation frequencies in field populations of Anopheles Hyrcanus Group and Culex pipiens complex in Korea.

Pyrethroid (PYR) and organophosphate (OP) insecticides have been extensively used for mosquito control for several decades in South Korea, and has resulted in the rapid development of resistance in the field. In this study, quantitative sequencing (QS) protocols were developed for the frequency prediction of insecticide resistance alleles [e.g., the L1014F/C mutation on the voltage sensitive sodium channel as a PYR resistance allele and the G119S mutation on the acetylcholinesterase 1 as OP resistance alleles] in four regional populations of Anopheles Hyrcanus Group and Culex pipiens complex. Both of the L1014F/C and G119S mutations were observed in all examined regional populations of An. Hyrcanus Group, suggesting a wide distribution of both PYR and OP resistance. In contrast, populations of the Cx. pipiens complex were determined to possess almost no G119S mutation, but relatively higher frequencies of the L1014F mutation, showing differential resistance patterns between different mosquito groups. The mutation frequencies were also monitored throughout a mosquito season (May-October) at one collection site to determine the seasonal changes of resistance mutation frequency in mosquito populations. Dramatic decreases of both L1014F/C and G119S mutation frequencies were observed in the An. Hyrcanus Group toward the fall, with no mutations observed in the early spring, suggesting a connection between the fitness costs of overwintering and insecticide resistance. However, no apparent trends were detectable in the Cx. pipiens complex populations due to low or zero mutation frequencies.

Placental growth factor (PlGF) is linked to inflammation and metabolic disorders in mice with diet-induced obesity.

Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) sub-family, plays a major role in angiogenesis and vasculogenesis. Previous study demonstrated that PlGF-overexpressing transgenic (Tg) mice had gestational loss. In addition, PlGF secretion was up-regulated in isolated T lymphocytes (T-cell) upon CD3/CD28 stimulation, suggesting that PlGF could be a regulator of T-cell differentiation and development. T-cells are well known to play a critical role in obesity-induced inflammation. Therefore, to verify the possible link of diet-induced obesity (DIO) with inflammation and related metabolic disorders, such as insulin resistance, we fed high-fat diet (HFD) to Tg mice for 16 weeks. Adiposity and glucose intolerance significantly increase in Tg mice fed a HFD (Tg HFD) compared to wild-type (WT) mice fed HFD (WT HFD). In addition, macrophage infiltrations were significantly higher in the epididymal white adipose tissue (EWAT), liver, and pancreatic islets of Tg HFD mice compared to WT HFD mice. In the in vitro study, we showed that isolated CD4+ T-cells from Tg mice further differentiate into type 1 (Th1) and type 17 (Th17) helper T-cells via CD3/CD28 stimulation. Furthermore, we observed that the pro-inflammatory cytokines IL-6, IL-17, and TNFα, are remarkably increased in Tg mice compared to WT mice. These findings demonstrate that PlGF overexpression in T-cells might lead to inflammatory T-cell differentiation and accumulation in adipose tissue (AT) or metabolism-related tissues, contributing to the development of systemic metabolic disorders. Thus, PlGF may provide an effective therapeutic target in the management of obesity-induced inflammation and related metabolic disorders.

Non-Destructive Analysis of the Internal Anatomical Structures of Mosquito Specimens Using Optical Coherence Tomography.

The study of mosquitoes and analysis of their behavior are of crucial importance in the on-going efforts to control the alarming increase in mosquito-borne diseases. Furthermore, a non-destructive and real-time imaging technique to study the anatomical features of mosquito specimens can greatly aid the study of mosquitoes. In this study, we demonstrate the three-dimensional imaging capabilities of optical coherence tomography (OCT) for structural analysis of Anopheles sinensis mosquitoes. The anatomical features of An. sinensis head, thorax, and abdominal regions, along with the morphology of internal structures, such as foregut, midgut, and hindgut, were studied using OCT imaging. Two-dimensional and three-dimensional OCT images, used in conjunction with histological images, proved useful for anatomical analysis of mosquito specimens. By presenting this work as an initial study, we demonstrate the applicability of OCT for future mosquito-related entomological research, and also in identifying changes in mosquito anatomical structure.

Methionine sulfoxide reductase A, B1 and B2 are likely to be involved in the protection against oxidative stress in the inner ear.

The methionine sulfoxide reductase (Msr) family of proteins is a class of repair enzymes that reduce methionine-S (MsrA) or methionine-R (MsrB) sulfoxide to methionine. Recent studies have reported that mutations in the MSRB3 gene cause autosomal recessive hearing loss in humans, and in mice MsrB3 deficiency leads to profound hearing loss due to hair cell apoptosis and stereocilia degeneration. However, apart from MsrB3, studies on Msr proteins in the inner ear have not yet been reported. In this study, we identified and characterized Msr expression in the cochlea and vestibule. First, we confirmed RNA expression levels of Msr family members in the cochlea and vestibule using reverse transcription PCR and detected Msr family members in both tissues. We also conducted immunohistochemical staining to localize Msr family members within the cochlea and vestibule. In the cochlea, MsrA was detected in supporting cells, spiral ligament, spiral limbus, Reissner's membrane and the spiral ganglion. MsrB1 was specifically expressed in hair cells and the spiral ganglion. MsrB2 was noted in the spiral ganglion, tectorial membrane and stria vascularis. In the vestibule, MsrA and MsrB1 were detected in hair cells and the vestibular ganglion, while MsrB2 was restricted to the vestibular ganglion. In this study, we identified distinct distributions of Msr family members in the organ of Corti and hypothesized that MsrA, MsrB1 and MsrB2 protect proteins in the organ of Corti from oxidative stress.

Effect of salinity on the oviposition and growth of Ochlerotatus togoi.

Ochlerotatus togoi is a salt-tolerant euryhaline mosquito that lays its eggs in rock pools. Although it is a pest that can transmit flaviviruses and filarial worms to humans, ecological studies have not been previously conducted because of its limited habitat. However, rising sea levels have created a more favorable environment for Oc. togoi, increasing the risk of Oc. togoi-borne diseases. We examined the oviposition and growth rates of Oc. togoi at 0-35 psu to obtain ecological data. It exhibited the highest oviposition preference at 0 psu; however, the hatching rate was highest at 10 psu, the pupation rate was highest at 25 psu, and the emergence rate was highest at 5 psu. Oc. togoi showed the highest rate of growth into adults at 25 psu. The results were assessed using Mann-Whitney U and Kruskal-Wallis H tests (post hoc test: Bonferroni), and a regression equation was generated for the incidence of adult Oc. togoi based on the change in salinity (y = -14.318 + 9.821x; y = adult incidence rate; x = salinity). The oviposition habits and developmental conditions of Oc. togoi were confirmed, and the incidence of Oc. togoi based on changes in sea level and ocean salinity was predicted. The results of this study will be useful for controlling salt-tolerant vectors and responding to vector-borne diseases.

Distribution and frequency of ace-1 and kdr mutations of Culex pipiens subgroup in the Republic of Korea.

Mosquitoes in the Culex pipiens subgroup are the primary vectors of the West Nile virus. Two members, Culex pallens and Culex pipiens f. molestus, are present in the Republic of Korea (ROK). Because the Culex pipiens subgroup occurs in large amounts, often near human habitation, it is frequently exposed to various insecticides, which is probably responsible for the rapid evolution of insecticide resistance traits. Experiments related to insecticide resistance in the Culex pipiens subgroup conducted in the ROK have been performed without discrimination below the species level. This study categorized Culex pipiens mosquitoes subgroup from 13 regions in the ROK into Culex pallens and Culex pipiens f. molestus, and target site genotypes for acetylcholinesterase-1 (ace-1) and voltage-gated sodium channel (vgsc) genes were identified for each taxon. Screening for ace-1 did not identify a resistant allele (G119S) in Cx. pipiens f. molestus, and heterozygous resistance (AGC/GGC) was identified in one Cx. pallens collected in Mokpo. In vgsc, knockdown resistance (kdr) mutations [TTT(L1014F) and TCA(L1014S)] were present in both taxa, with Cx. pipiens f. molestus having homozygous resistance (TTT/TTT): 44%, heterozygous resistance (TTT/TTA): 28%, and homozygous susceptibility (TTA/TTA): 28%, whereas Cx. pallens showed homozygous resistance (TTT/TTT or TCA/TCA): 26%, heterozygous resistance (TTT/TTA, TTT/TCA, or TCA/TTA): 26%, and homozygous susceptibility (TTA/TTA): 48%. Furthermore, the unique vgsc allele was present in both Cx. pipiens f. molestus and Cx. pallens. This was the first experiment to analyze the Culex pipiens subgroup living in the ROK below the species level, and its results could be used in the future for more detailed mosquito control.

Wing geometric morphometrics and COI barcoding of Culex pipiens subgroup in the Republic of Korea.

Two members of the Culex pipiens subgroup, Culex pallens and Culex pipiens f. molestus, are known to occur in the Republic of Korea (ROK). These species exhibit morphologically similar features and are challenging to distinguish below the species level. Therefore, this study utilized wing geometric morphometrics (GM) on the right wing of the Culex pipiens subgroup, alongside sequencing of the cytochrome c oxidase subunit I (COI) region. Mosquitoes were collected from 11 locations between June and October (2020-2022) to minimize regional and seasonal variations. Additionally, Culex pipiens f. pipiens, which is not native to the ROK, was included in the analysis. Culex tritaeniorhynchus, Aedes albopictus, and Anopheles sinensis, the primary vectors in the ROK, were used as outgroups for comparison. All three taxa in the Culex pipiens subgroup could be identified with an 82.4%-97.0% accuracy using GM. However, a comparison of the COI regions of the Culex pipiens subgroup revealed no clear differences between the taxa. These data can be used for accurate identification, contributing to effective mosquito control, in addition to providing a foundation for evolutionary and ecological studies on wing shape differences.

Analysis of geometric morphometrics and molecular phylogeny for Anopheles species in the Republic of Korea.

Human malaria, transmitted by Anopheles mosquitoes, is the most predominant mosquito-borne disease that is responsible for hundreds of thousands of deaths worldwide each year. In the Republic of Korea (ROK), there are currently several hundred malaria cases annually, mostly near the demilitarized zone (DMZ). Eight species of Anopheles mosquitoes are currently known to be present in the ROK. Similar to other major malaria vectors in Africa and India, it is very challenging to morphologically differentiate Anopheles mosquitoes in the ROK due to their extremely similar morphology. In this study, wing geometric morphometrics (WGM) were used to differentiate the eight Anopheles species collected at six locations near the DMZ, Seoul and Pyeongtaek from April-October 2021. Phylogenetic analysis was also performed using cytochrome c oxidase subunit 1 (COI), internal transcribed spacer 2 (ITS2), and tyrosine hydroxylase (TH) genes for comparison with WGM analysis and to infer evolutionary relationships. The results of cross-validation (overall accuracy = 74.8%) demonstrated that species identification using WGM alone was not possible with a high accuracy for all eight species. While phylogenetic analyses based on the COI region could not clearly distinguish some species, the analysis based on ITS2 and TH was more useful for resolving the phylogenetic correlation of the eight species. Our results may improve Anopheles species identification strategies for effective identification and control of malaria vectors in the ROK.

Insect fauna including unrecorded species in Ulleungdo, South Korea.

Background: Ulleungdo harbours a unique ecosystem owing to its isolation from the mainland alongside its maritime climate. The island, formed via volcanic activity, is the largest island in the East Sea of Korea and retains a primeval forest. The ecosystems are being destroyed owing to increasing human activity on the island. Therefore, through the investigation of the insect fauna of Ulleungdo, we tried to provide information that can be the basis for understanding the island ecology of Ulleungdo. This survey was conducted four times between April and October in 2020 at Seonginbong. New information: The findings of the survey regarding insect fauna at Seonginbong, Ulleungdo included 10 orders, 105 families, 216 genera and 212 species, of which 12 families, two subfamilies, 13 genera and 74 species were previously unrecorded. The data have been registered in the Global Biodiversity Information Facility (GBIF; www.GBIF.org).

Larvicidal Activity of Methyl Benzoate, a Volatile Organic Compound, Against the Mosquitoes Aedes albopictus and Culex pipiens (Diptera: Culicidae).

Methyl benzoate (MBe) is a volatile organic molecule found in various plants; it is used as an insect semiochemical. MBe also has a biorational insecticidal effect against various agricultural and urban arthropod pests. The present study was the first to assess the larvicidal potential of MBe against fourth-instar larvae of the mosquitoes Aedes albopictus (Skuse) and Culex pipiens (L.). A positive association was observed between MBe concentrations and larval mortality in both the species. The highest mortality recorded was 100% for Ae. albopictus and 56% for Cx. pipiens after 24 h of exposure to 200 ppm MBe. The lethal median concentration (LC50) values of MBe against fourth-instar larvae of Ae. albopictus and Cx. pipiens were 61 ppm and 185 ppm, respectively. These results suggest that MBe has great potential for use as an environmentally friendly larvicidal agent for mosquito control.

Species Diversity of Anopheles Mosquitoes and Plasmodium vivax Infection Rates, Gyeonggi Province, Republic of Korea During 2020.

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.

The Insect database in Dokdo, Korea: An updated version in 2020.

BACKGROUND: Dokdo, a group of islands near the East Coast of South Korea, comprises 89 small islands. These volcanic islands were created by an eruption that also led to the formation of the Ulleungdo Islands (located in the East Sea), which are approximately 87.525 km away from Dokdo. Dokdo is important for geopolitical reasons; however, because of certain barriers to investigation, such as weather and time constraints, knowledge of its insect fauna is limited compared to that of Ulleungdo. Until 2017, insect fauna on Dokdo included 10 orders, 74 families, 165 species and 23 undetermined species; subsequently, from 2018 to 2019, we discovered 23 previously unrecorded species and three undetermined species via an insect survey. NEW INFORMATION: As per our recent study, the database of insect species on Dokdo has been expanded to 10 orders, 81 families, 188 species and 23 undetermined species. This database has been registered in the Global Biodiversity Information Facility (GBIF; www.GBIF.org) and is the first record for insect fauna on Dokdo.

A multiplex PCR assay for six Aedini species, including Aedes albopictus.

BACKGROUND: Mosquitoes, as vectors of various human pathogens, are significant drivers of serious human illness. In particular, those species in the Aedini tribe, which typically transmit dengue virus, Chikungunya fever virus, and Zika virus, are increasing their range because of climate change and international commerce. In order to evaluate the risk of disease transmission, accurate mosquito species identification and monitoring are needed. The goal of this work was to develop a rapid and simple molecular diagnostic method for six morphologically similar Aedini species (Aedes flavopictus, Aedes albopictus, Ochlerotatus koreicus, Ochlerotatus japonicus, Ochlerotatus togoi and Ochlerotatus hatorii) in Korea. METHODS: A total of 109 samples were assayed in this study. The internal transcribed spacer 2 (ITS2) regions from all six species were amplified, sequenced and analyzed using Mega 6. Following the identification of regions that were consistently different in terms of sequence between all six species, multiplex primers were designed to amplify these regions to generate species-specific fragments distinguishable by their size. RESULTS: Uniquely sized fragments were generated in Ae. flavopictus (495 bp), Ae. albopictus (438 bp), Oc. koreicus (361 bp), Oc. togoi (283 bp), Oc. hatorii (220 bp) and Oc. japonicus (160 bp). Pairwise distance analysis showed that the difference was 35.0 ± 1.5% between Aedes spp. and Ochlerotatus spp., 17.4 ± 0.2% between Ae. albopictus and Ae. flavopictus and 11.1 ± 0.3% between Oc. koreicus and Oc. japonicus. CONCLUSIONS: In this study, a multiplex PCR assay for six species of the Aedini tribe was developed. This assay is more accurate than morphological identification and will be useful for monitoring and controlling these vector mosquitoes.

Simultaneous identification of the Anopheles funestus group and Anopheles longipalpis type C by PCR-RFLP.

BACKGROUND: Anopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage although it is very different at the larval stage. Despite the development of the species-specific multiplex PCR assay for the An. funestus group, the genomic DNA of Anopheles longipalpis type C specimens can be amplified with the Anopheles vaneedeni and Anopheles parensis primers from this assay. The standard, species-specific An. funestus group PCR, results in the amplification of two fragments when An. longipalpis type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between An. vaneedeni and An. parensis. Anopheles longipalpis type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the An. funestus group, such as An. funestus, An. funestus-like and An. parensis, are included. The present study provides a multiplex assay that will identify An. longipalpis along with other common members of the African An. funestus group, including Anopheles leesoni. METHODS: A total of 70 specimens from six species (An. funestus, An. funestus-like, An. parensis, Anopheles rivulorum, An. vaneedeni and An. leesoni) in the An. funestus group and An. longipalpis type C from Malawi, Mozambique, South Africa and Zambia were used for the study. A restriction fragment length polymorphism (RFLP) assay was designed based on the DNA sequence information in the GenBank database. RESULTS: The enzyme, EcoRI digested only An. longipalpis type C and An. funestus-like after the species-specific An. funestus group PCR assay. The An. longipalpis and An. funestus-like digestion profiles were characterized by three fragments, 376 bp, 252 bp and 211 bp for An. longipalpis type C and two fragments, 375 bp and 15 bp for An. funestus-like. CONCLUSIONS: An RFLP method for the group was developed that is more accurate and efficient than those used before. Hence, this assay would be useful for field-collected adult specimens to be identified routinely in malaria vector research and control studies.

A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance (kdr) mutations in Anopheles gambiae from the Republic of the Congo.

BACKGROUND: Knockdown resistance (kdr) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the kdr mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the kdr mutations in An. gambiae s.s. from the Republic of the Congo were compared with DNA sequence analysis. METHODS: A total of 52 pyrethroid and DDT resistant An. gambiae from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two kdr mutations (kdr-e and kdr-w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population. RESULTS: Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the kdr-e and kdr-w resistance mutations, seven specimens (13.5%) were homozygous for the kdr-e mutation, and 14 specimens (26.9%) were homozygous for the kdr-w mutation. A single individual was genotyped as heterozygous kdr-e mutation (1.9%) only and another as heterozygous kdr-w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous kdr-w, six specimens (11.5%) homozygous for the kdr-e mutation, one specimen (1.9%) heterozygous for the kdr-w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous kdr-e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis. CONCLUSION: Both the kdr-e and kdr-w mutations were abundant in An. gambiae S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both kdr-e and kdr-w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of kdr is still held as the gold standard assay, this paper highlights the importance of kdr mutation confirmation via sequencing especially in regions where kdr frequency has never been reported before or where both the kdr-e and kdr-w mutations are present simultaneously.

Crystal Structure of Histidine Triad Nucleotide-Binding Protein from the Pathogenic Fungus Candida albicans.

Histidine triad nucleotide-binding protein (HINT) is a member of the histidine triad (HIT) superfamily, which has hydrolase activity owing to a histidine triad motif. The HIT superfamily can be divided to five classes with functions in galactose metabolism, DNA repair, and tumor suppression. HINTs are highly conserved from archaea to humans and function as tumor suppressors, translation regulators, and neuropathy inhibitors. Although the structures of HINT proteins from various species have been reported, limited structural information is available for fungal species. Here, to elucidate the structural features and functional diversity of HINTs, we determined the crystal structure of HINT from the pathogenic fungus Candida albicans (CaHINT) in complex with zinc ions at a resolution of 2.5 Å. Based on structural comparisons, the monomer of CaHINT overlaid best with HINT protein from the protozoal species Leishmania major. Additionally, structural comparisons with human HINT revealed an additional helix at the C-terminus of CaHINT. Interestingly, the extended C-terminal helix interacted with the N-terminal loop (α1-ß1) and with the α3 helix, which appeared to stabilize the dimerization of CaHINT. In the C-terminal region, structural and sequence comparisons showed strong relationships among 19 diverse species from archea to humans, suggesting early separation in the course of evolution. Further studies are required to address the functional significance of variations in the C-terminal region. This structural analysis of CaHINT provided important insights into the molecular aspects of evolution within the HIT superfamily.

Protection of nigral dopaminergic neurons by AAV1 transduction with Rheb(S16H) against neurotoxic inflammation in vivo.

We recently reported that adeno-associated virus serotype 1 (AAV1) transduction of murine nigral dopaminergic (DA) neurons with constitutively active ras homolog enriched in brain with a mutation of serine to histidine at position 16 [Rheb(S16H)] induced the production of neurotrophic factors, resulting in neuroprotective effects on the nigrostriatal DA system in animal models of Parkinson's disease (PD). To further investigate whether AAV1-Rheb(S16H) transduction has neuroprotective potential against neurotoxic inflammation, which is known to be a potential event related to PD pathogenesis, we examined the effects of Rheb(S16H) expression in nigral DA neurons under a neurotoxic inflammatory environment induced by the endogenous microglial activator prothrombin kringle-2 (pKr-2). Our observations showed that Rheb(S16H) transduction played a role in the neuroprotection of the nigrostriatal DA system against pKr-2-induced neurotoxic inflammation, even though there were similar levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1ß), in the AAV1-Rheb(S16H)-treated substantia nigra (SN) compared to the SN treated with pKr-2 alone; the neuroprotective effects may be mediated by the activation of neurotrophic signaling pathways following Rheb(S16H) transduction of nigral DA neurons. We conclude that AAV1-Rheb(S16H) transduction of neuronal populations to activate the production of neurotrophic factors and intracellular neurotrophic signaling pathways may offer promise for protecting adult neurons from extracellular neurotoxic inflammation.

Comparison of Adult Mosquito Black-Light and Light-Emitting Diode Traps at Three Cowsheds Located in Malaria-Endemic Areas of the Republic of Korea.

Adult mosquito surveillance and field trials evaluated selected commercially available ultraviolet black-light (BL) and light-emitting diode (LED) traps at three sites where vivax malaria is endemic from May to October 2015 in northwestern Republic of Korea. Collections totaled 283,929 adult mosquitoes (280,355 [98.74%] females and 3,574 [1.26%] males) comprising 17 species (including six members of the Anopheles Hyrcanus Group) belonging to six genera. The four most predominant female species collected were Aedes vexans nipponii (Theobald) (83.84%), followed by Anopheles Hyrcanus Group (13.66%), Culex pipiens Group (1.67%), and Culex tritaeniorhynchus Giles (0.54%). Overall, LED traps (188,125) collected significantly more female mosquitoes compared with BL traps (92,230; P = 0.0001, P < 0.05). Results from these field evaluations significantly enhance vector and disease surveillance efforts, especially for the primary vectors of malaria (Anopheles Hyrcanus Group) and Japanese encephalitis (Cx. tritaeniorhynchus).