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.

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.

Identifying the origin and fate of dissolved U in the Boeun aquifer based on microbial signatures and C, O, Fe, S, and U isotopes.

The uranium inventory in the Boeun aquifer is situated near an artificial reservoir (40-70 m apart) intended to supply water to nearby cities. However, toxic radionuclides can enter the reservoir. To determine the U mobility in the system, we analyzed groundwater and fracture-filling materials (FFMs) for environmental tracers, including microbial signatures, redox-sensitive elements and isotopes. In the site, U mass flux ranged from only 9.59 × 10-7 µg/L/y to 1.70 × 10-4 µg/L/y. The δ18O-H2O and 14C signatures showed that groundwater originated mainly from upland recharges and was not influenced by oxic surface water. We observed U accumulations (∼157 mg/kg) in shallow FFMs and Fe enrichments (∼226798 mg/kg) and anomalies in the 230Th/238U activity ratio (AR), 230Th/234U AR, δ56Fe and δ57Fe isotopes, suggesting that low U mobility in shallow depths is associated with a Fe-rich environment. At shallow depths, anaerobic Fe-oxidizers, Gallionella was prevalent in the groundwater, while Acidovorax was abundant near the U ore deposit depth. The Fe-rich environment at shallow depths was formed by sulfide dissolution, as demonstrated by δ34S-SO4 and δ18O-SO4 distribution. Overall, the Fe-rich aquifer including abundant sulfide minerals immobilizes dissolved U through biotic and abiotic processes, without significant leaching into nearby reservoirs.

Potential of indigenous bacteria driven U(VI) reduction under relevant deep geological repository (DGR) conditions.

Understanding the biogeochemical U redox processes is crucial for controlling U mobility and toxicity under conditions relevant to deep geological repositories (DGRs). In this study, we examined the microbial reduction of aqueous hexavalent uranium U(VI) [U(VI)aq] by indigenous bacteria in U-contaminated groundwater. Three indigenous bacteria obtained from granitic groundwater at depths of 44-60 m (S1), 92-116 m (S2), and 234-244 m (S3) were used in U(VI)aq bioreduction experiments. The concentration of U(VI)aq was monitored to evaluate its removal efficiency for 24 weeks under anaerobic conditions with the addition of 20 mM sodium acetate. During the anaerobic reaction, U(VI)aq was precipitated in the form of U(IV)-silicate with a particle size >100 nm. The final U(VI)aq removal efficiencies were 37.7%, 43.1%, and 57.8% in S1, S2, and S3 sample, respectively. Incomplete U(VI)aq removal was attributed to the presence of a thermodynamically stable calcium uranyl carbonate complex in the U-contaminated groundwater. High-throughput 16S rRNA gene sequencing analysis revealed the differences in indigenous bacterial communities in response to the depth, which affected to the U(VI)aq removal efficiency. Pseudomonas peli was found to be a common bacterium related to U(VI)aq bioreduction in S1 and S2 samples, while two SRB species, Thermodesulfovibrio yellowstonii and Desulfatirhabdium butyrativorans, played key roles in the bioreduction of U(VI)aq in S3 sample. These results indicate that remediation of U(VI)aq is possible by stimulating the activity of indigenous bacteria in the DGR environment.

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.

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.

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.

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.

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.

Uranium sequestration in fracture filling materials from fractured granite aquifers.

The migration of the uranium (U) in high-level radioactive waste that is held in deep geological repositories via fractures in deep granite aquifers is a serious safety concern, thus, this study investigates the effect of fracture filling materials designed to mitigate these concerns. Geochemical analysis was conducted on granite rock core and groundwater samples collected from boreholes located in granite areas. Sequential extraction tests on fracture filling material (FFM) samples were also conducted. The rock core samples were classified as two-mica granite that had uranium (U) content ranging from 1900 to 22,100 µg/kg with an arithmetic mean of 8500 µg/kg. The total U concentration in the FFM samples was found to range from 790 to 80,781 µg/kg. The U in the FFM samples was mainly associated with a carbonate phase that made up from 29.9 to 100% of the total U in the FFM. The U fraction of carbonate phase was closely correlated with the Ca fraction. U associated with crystalline inorganic FFM constituents (e.g, clay minerals and metal oxyhydroxides) was also found in FFM samples in fractions ranging from 21.1 to 70.1%. U in FFM is mainly incorporated via Ca-carbonate, which might have not been formed in modern groundwater, but the time and temperature during formation are unknown. In addition, the Fe, Si, Al, Ca, K, and U levels were found to be well correlated with each other, suggesting that U can also become geochemically associated with crystalline clay minerals or Fe-oxyhydroxides.

Chlorite alteration in aqueous solutions and uranium removal by altered chlorite.

Chlorite alteration and the U removal capacity of altered chlorite were investigated. Batch kinetic dissolution tests using clinochlore CCa-2 were conducted for 60days in aqueous solutions of various pHs and ionic strengths. Batch sorption tests using these altered chlorite samples were conducted for 48h with natural groundwater containing 3.06×10-6M U. Chlorite dissolution was influenced more by pHo than by the ionic strength of the solution. TEM analysis revealed Fe(oxy)hydroxide aggregates in the solid residue from the batch dissolution test with 0.1M NaClO4 solution at pHo=10. The U removal capacity of the reacted chlorite samples at pHo=6-10 was higher than that of the reacted chlorite samples at pHo=3. The degree of dissolution of chlorite samples reacted at pHo=3-8 was inversely proportional to the U removal capacity, but that of chlorite samples reacted at pHo=10 was proportional to the U removal capacity. The positive correlation between the U removal capacity and degree of chlorite dissolution at pHo=10 might be due to the formation of Fe-containing secondary minerals and changes in the reactive sites.

A microfluidic approach to water-rock interactions using thin rock sections: Pb and U sorption onto thin shale and granite sections.

The feasibility of using microfluidic tests to investigate water-rock (mineral) interactions in fractures regarding sorption onto thin rock sections (i.e., shale and granite) of lead (Pb) and uranium (U) was evaluated using a synthetic PbCl2 solution and uranium-containing natural groundwater as fluids. Effluent composition and element distribution on the thin rock sections before and after microfluidic testing were analyzed. Most Pb removal (9.8mg/cm2) occurred within 3.5h (140 PVF), which was 74% of the total Pb removal (13.2mg/cm2) at the end of testing (14.5h, 560 PVF). Element composition on the thin shale sections determined by µ-XRF analysis indicated that Pb removal was related primarily to Fe-containing minerals (e.g., pyrite). Two thin granite sections (biotite rich, Bt-R and biotite poor, Bt-P) exhibited no marked difference in uranium removal capacity, but a slightly higher amount of uranium was removed onto the thin Bt-R section (266µg/cm2) than the thin Bt-P section (240µg/cm2) within 120h (4800 PVF). However, uranium could not be detected by micro X-ray fluorescence (µ-XRF) analysis, likely due to the detection limit. These results suggest that microfluidic testing on thin rock sections enables quantitative evaluation of rock (mineral)-water interactions at the micro-fracture or pore scale.

Effect of Cr(VI) concentration on gas and particle production during iron oxidation in aqueous solutions containing Cl- ions.

Zero-valent iron (ZVI) is commonly used as a medium in permeable reactive barriers (PRBs) because of its high reducing ability. The generation of H2 gas in PRBs, however, can decrease the permeability of PRBs and reduce the contact area between the PRB and contaminated groundwater. This study investigated the effect of the initial Cr(VI) concentration ([Cr(VI)init]) in aqueous solutions containing Cl- ions on the generation of H2 gas. ZVI chips were reacted in reactors with 0.5-M NaCl solutions with [Cr(VI)init] ranging between 51 and 303 mg/L. The initial pH was set at 3. The oxidation of ZVI chips by Cr(VI) in aqueous solutions containing Cl- ions produced H2 gas and particles (Fe(III)-Cr(III)(oxy)hydroxides). The Cr(VI) removal from aqueous solutions increased as the [Cr(VI)init] increased, as did H2 gas generation. The positive effect of [Cr(VI)init] on H2 gas generation might be due to an increase in the redox potential gradient as [Cr(VI)init] increases. This increased gradient would enhance H+ ion penetration through the passive film (Fe(III)-Cr(III)(oxy)hydroxides), which formed on the ZVI surface, by diffusion from the solution to pits beneath the passive film.

Characterization of redox processes in shallow groundwater of Owens Dry Lake, California.

Redox status of shallow groundwaters (1-3 m depths) at Owens Dry Lake was studied to help guide mitigation efforts for attenuating dust generation from the dry lakebed. Redox conditions were characterized by Eh, oxidative capacity (OXC), and terminal electron accepting processes (TEAPs) as well as examining the energetics of TEAPs. Groundwater chemistry related to redox status was determined by major solute concentrations, dissolved gases (O2, H2, CH4), aqueous redox species (NO3, Mn2+, Fe2+/ Fe3+, SO4(2-)/HS-, DOC), and major redox sensitive components in the solid phase (extractable Fe/Mn). All of these measures of redox status indicate that sulfate reduction is the major process regulating redox conditions in most shallow groundwaters of Owens Dry Lake. Dissolved sulfate concentrations were regulated primarily by evaporation resulting in increasing concentrations as water migrates from the shoreline (<1 mM) to the center (up to 417 mM) of the dry lakebed. Eh values were generally in the range of -240 to -170 mV. The oxidative capacity demonstrates the dominant contribution of sulfate to OXC. The dominance of sulfate restricts further redox development, such as methanogenesis. Dissolved H2 concentrations ranged from 0.5 to 7.8 nM. According to the empirically defined H2 ranges, sulfate reduction was the most predominant TEAP. Moreover, thermodynamic calculations of TEAPs for H2 utilization support favorable energetics for both sulfate reduction and methanogenesis. The calculated available energy yield for sulfate reduction in the shallow groundwater of Owens Dry Lake was higher than other systems due to the high sulfate concentration.