Population Genetics of Culex tritaeniorhynchus (Diptera: Culicidae) in Türkiye.

PURPOSE: Mosquitoes are important vectors of pathogens that can affect humans and animals. Culex tritaeniorhynchus is an important vector of arboviruses such as Japanese encephalitis virus, West Nile virus among various human and animal communities. These diseases are of major public health concern and can have huge economic and health burdens in prevalent countries. Although populations of this important mosquito species have been detected in the Mediterranean and Aegean regions of Türkiye; little is known about its population structure. Our study is to examine the population genetics and genetic composition of Cx. tritaeniorhynchus mosquitoes collected from several localities using cytochrome oxidase subunit I (COI) and the NADH dehydrogenase subunit 5 genes (ND5). This is the first extensive study of Cx. tritaeniorhynchus in the mainland Türkiye with sampling spanning many of provinces. METHODS: In this study, DNA extraction, amplification of mitochondrial COI and ND5 genes and population genetic analyses were performed on ten geographic populations of Culex tritaeniorhynchus in the Aegean and Mediterranean region of Türkiye. RESULTS: Between 2019 and 2020, 96 samples were collected from 10 geographic populations in the Aegean and Mediterranean regions; they were molecularly analyzed and 139 sequences (50 sequence for COI and 89 sequence for ND5) were used to determine the population structure and genetic diversity. For ND5 gene region, the samples produced 24 haplotypes derived from 15 variable sites and for COI gene region, 43 haplotypes were derived from 17 variable sites. The haplotype for both gene regions was higher than nucleotide diversity. Haplotype phylogeny revealed two groups present in all populations. AMOVA test results show that the geographical populations were the same for all gene regions. Results suggest that Cx. tritaeniorhynchus is a native population in Türkiye, the species is progressing towards speciation and there is no genetic differentiation between provinces and regions. CONCLUSION: This study provides useful information on the molecular identifcation and genetic diversity of Cx. tritaeniorhynchus; these results are important to improve mosquito control programs.

Larvicidal Activity of Some Aromatic Thiosemicarbazone and Metal Complexes [Ni (II), Cu (II), Co (II)] Against Aedes (Stegomyia) aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1894) (Diptera: Culicidae) Larvae.

Objective: A series of aromatic thiosemicarbazone-oxime [TP1 and TP2] derivatives and their Ni(II), Cu(II), and Co(II) complexes were synthesized, and their larvicidal activity was evaluated against Aedes aegypti and Aedes albopictus larvae. The efficacy of these substances to Aedes albopictus larvae has been demonstrated for the first time. Methods: Laboratory colonized Aedes aegypti and Aedes albopictus larvae were subjected to larvicidal activity tests. Larval mortality rates at 24 and 48 hours were recorded and LC50 values were calculated. The study was carried out at Aydin Adnan Menderes University in 2021. Results: For Aedes aegypti, LC50 of TP1 and its Co(II) complex were 15.41, 9.75, µg/mL whereas for TP2 and its Co(II) complex, LC50 were 21.62, 20.50 µg/mL after 24 and 48 h respectively. For Aedes albopictus, TP1 and its Co(II) complex showed an LC50 of 12.06, 8.75 µg/mL, whereas TP2 and its Co(II) complex showed an LC50 of 32.87, 25.48 µg/mL, for 24, and 48 h respectively. Conclusion: Both TP1 and TP2 compounds and their Co(II) complexes presented high efficacy against the larvae; it can be said that C=S groups in thiosemicarbazone derivatives are effective in showing activity and for this reason, studies should be continued to make these components effective.

Distribution of Aedes (Stegomyia) cretinus in Türkiye

OBJECTIVE: Aedes cretinus, a white and black stripped Aedes species, shares morphological similarities with Aedes albopictus and Aedes aegypti which are among the most important vectors and invasive species in the world. Due to its limited distribution and low population density, information on the biology and ecology of the species has been limited so far. This study aimed to determine distribution of Ae. cretinus in Türkiye. METHODS: Field works have been carried out in the Mediterranean, Aegean and Marmara Regions of Türkiye. Larval samples were collected by larval dippers while adult mosquito catches were performed using the human landing catch method and CDC-light traps. RESULTS: A total of 44 different Ae. cretinus populations were identified in the Mediterranean, Aegean and Marmara Regions of Türkiye. The larval specimen collected from small containers, tires, tree holes and natural small habitats. A plane (Platanus orientalis), walnut (Juglans regia), Türkiye oak (Quercus cerris), chestnut (Castenea sativa) and cedar (Cedrus libani) tree holes are the main larval habitats in which the specimen collected. In some localities, larvae were found together with Anopheles plumbeus, Anopheles claviger and Aedes geniculatus larvae in tree holes. Human landing catch method captured a greater number of females than CDC-light traps. CONCLUSION: This study determined the distribution of Ae. cretinus in Türkiye for the first time. Information on respective geographic distribution of Ae. cretinus is fundamental for effective control programmes. Further studies are needed to understand the biology and ecology of these species.

Detection of L1014F knockdown resistance mutation in Culex tritaeniorhynchus populations.

Culex tritaeniorhynchus is a major Japanese encephalitis virus vector distributed in Southeast Asia and surrounding countries. The aim of the present study is to investigate insecticide resistance status among 10 Cx. tritaeniorhynchus populations of the Mediterranean region of Turkey. Bioassay results indicated that all of the populations were resistant or at least possibly resistant to 1,1'-(2,2,2-Trichloroethane-1,1-diyl) bis (4-chlorobenzene) (DDT) (4%), [(dimethoxyphosphorothioyl) sulfanyl] butanedioate, Diethyl (malathion) (5%), and 2-[(Propan-2-yl) oxy] phenyl methylcarbamate (propoxur) (0,1%). Whereas, some of the populations were still susceptible to 3-Phenoxybenzyl (1RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2-dimethyl cyclopropane carboxylate (permethrin) (0,75%) and (S)-Cyano (3-phenoxy phenyl) methyl (1R,3R)-3-(2,2-dibromoethen-1-yl)-2,2-dimethylcyclopropane-1-carboxylate (deltamethrin) (0,05%). Biochemical analysis results showed altered alpha esterase, beta esterase, para-nitrophenyl acetate (PNPA), and glutathione-s-transferase (GST) levels in some populations while all of the populations had increased oxidase levels except for the Yumurtalik population. Additionally, all of the populations had sensitive acetylcholinesterase (AChE) levels similar to the control group except for the Erzin population. Correlation analysis showed a significant correlation between mortality rates for deltamethrin and alpha esterase, beta esterase, PNPA, and GST levels while mortality rates for permethrin were significantly correlated with GST levels. An allele-specific polymerase chain reaction (AS-PCR) detected high L1014F allele frequency in the populations. Overall results indicate the urgent need for monitoring and mapping of insecticide resistance in Cx. tritaeniorhynchus populations of the study area for effective vector control management.

Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect Knockdown and Acetylcholinesterase Mutations in Anopheles superpictus

Objective: The study aims to determine the presence of L1014F, L1014S, L1014C alleles, which are responsible for knockdown resistance and Ace-1 G119S alleles, which are responsible for acetylcholinesterase insensitivity in Anopheles superpictus, the secondary vector of malaria in Turkey. Methods: In this study, 60 Anopheles superpictus adult females were collected from Aydin, Denizli, and Mugla provinces. Then, allele-specific primers for kdr L1014F, L1014S, and L1014C alleles, and the Ace-1 G119S allele were designed. The presence of these alleles was screened in three Anopheles superpictus populations by allele-specific polymerase chain reaction. Results: Although L1014S allele frequency was too low in Aydin, Mugla, and Denizli populations, neither kdr L1014F and L1014C nor Ace-1 G119S mutations were found in any population. Conclusion: In this study, kdr L1014S mutation was detected for the first time in the Aegean Anopheles superpictus populations.

Insecticide Resistance and Mechanisms of Culex pipiens Populations in the Mediterranean and Aegean Regions of Turkey During 2017-2018.

Background: Culex pipiens has a significant public health importance since it is an important vector of West Nile virus and Rift Valley fever virus. We, therefore, aimed to determine the insecticide resistance level in Cx. pipiens populations in the Aegean and Mediterranean regions of Turkey. Methods: Bioassays have been carried out against Dichlorodiphenyltrichloroethane (DDT) (4%), Malathion (5%), Fenitrothion (1%), Propoxur (0.1%), Bendiocarb (0.1%), Permethrin (0.75%) and Deltamethrin (0.05%). Biochemical analyses have been performed to detect non-specific esterase, mixed function oxidase, glutathione-s-transferase and acetylcholinesterase levels. A knockdown resistance (kdr) (L1014F) and Acetylcholinesterase (Ace-1) (G119S) mutations have been detected by using allele-specific primers and a polymerase chain reaction (PCR) amplification of specific alleles (PASA) diagnostic test was performed for detection of F290V mutation. Results: Bioassay results showed that all Cx. pipiens populations were resistant to DDT, Malathion, Fenitrothion, Bendiocarb, Propoxur and some of the populations have started to gain Permethrin and Deltamethrin resistance. Biochemical analyses results revealed that altered glutathione-s-transferases, P450 monooxygenases, esterase levels might be responsible for DDT, organophosphate, carbamate and pyrethroid resistance in Cx. pipiens populations. Results showed mild to high frequency of L1014F, low frequency of F290V but no Ace-1 G119S mutation within the populations. Additionally, acetylcholinesterase insensitivity was not significantly high within the most of these populations. Conclusion: Overall results may help to fulfil the lacking information in the literature regarding insecticide resistance status and underlying mechanism of Culex pipiens populations of the Mediterranean and Aegean region of Turkey by using all bioassays, molecular tests and biochemical assays.

The first implementation of allele-specific primers for detecting the knockdown and acetylcholinesterase target site mutations in malaria vector, Anopheles sacharovi.

Anopheles sacharovi, a primer malaria vector species of Turkey, have a significant public health importance. It is aimed to determine the insecticide resistance status in Anopheles sacharovi populations in the Aegean and Mediterranean regions of Turkey. A total of 1638 individuals were analysed from 15 populations. Bioassay results indicated all An. sacharovi populations were resistant to DDT, malathion, fenitrothion, bendiocarb, propoxur. Many populations have begun to have resistance against permethrin and deltamethrin. Biochemical analyses results revealed that glutathione-S-transferases and P450 monooxygenases might be responsible from the mechanisms of DDT resistance; esterases and acetylcholinesterase might be responsible for organophosphate and carbamate resistance; P450 monooxygenases and esterases might be responsible for pyrethroid resistance into populations sampled from the study area. Allele-specific primers detected L1014F and L1014S mutations that provide kdr resistance against pyrethroids and DDT. Increased acetylcholinesterase insensitivity was detected while Ace-1 G119S mutations were not detected in An. sacharovi populations by using allele-specific primers. Overall results indicate the presence of multiple resistance mechanisms in Turkish An. sacharovi field populations suggesting that populations might gain resistance against all possible insecticide in the future. Therefore, insecticide resistance management strategies are urgently needed for effective vector control implementation.

Current insecticide resistance status in Anopheles sacharovi and Anopheles superpictus populations in former malaria endemic areas of Turkey.

Anopheles sacharovi and Anopheles superpictus have a significant public health importance since they are primer and seconder malaria vectors of Turkey, respectively. As a result of intensive insecticide usage in historically malaria endemic regions of Turkey for long years, insecticide resistance problem has occurred inevitably. In this study, we aimed to investigate the involvement of the detoxification enzymes in insecticide resistance in Turkish An. sacharovi and An. superpictus populations in the Mediterranean and South-eastern Anatolia region where have a malaria history in the past. Bioassay results indicated that both An. sacharovi and An. superpictus populations are resistant to DDT, resistant or possible resistant to organophosphates and carbamates and finally mostly susceptible to pyrethroids. Although bioassays results indicated high DDT resistance in all mosquito populations, biochemical assays did not show significantly high GST levels in all strains. Almost all An. sacharovi and An. superpictus populations had an increased α and ß esterase activity levels while nearly half of the overall populations had an increased p-NPA esterase than the control group. Elevated levels of MFO frequency have been shown in the majority of the populations. Consequently, our results reveal that biochemical resistance mechanisms may play an important role in insecticide resistance in Turkish An. sacharovi and An. superpictus populations. These results give useful cues to monitor the insecticide resistance before it spreads throughout an entire population, enabling early intervention.

Distribution pattern and genetic structure of Aedes zammitii (Diptera: Culicidae) along the Mediterranean and Aegean coasts of Turkey.

The Mariae species complex, consisting of Aedes mariae, Aedes phoeniciae, and Aedes zammitii, has a limited distribution worldwide. All three species are found in rocky habitats on the coastal areas of Mediterranean countries. Aedes phoeniciae and Ae. zammitii are two members of the Mariae complex that exist in Turkey. The aim of this study was to determine the distribution pattern and genetic structure of Ae. zammitii along the Mediterranean and Aegean regions. For this purpose, larval and adult samples of Ae. zammitii were collected from 19 different rocky habitats along the coastal regions of Antalya, Mugla, Aydin, Izmir, Balikesir, and Çanakkale provinces. DNA isolation was performed primarily from collected samples, and mitochondrial NADH dehydrogenase 4 (ND4) gene was amplified by polymerase chain reaction. Based on ND4 sequence analyses, 21 haplotypes were detected along the distribution range of the species. Analyses of molecular variance (AMOVA) and spatial analyses of molecular variance (SAMOVA) indicated six groups, and most of the variation was among groups, demonstrating the population structuring at group level. Isolation by distance analyses (IBD) showed a correlation between geographic and genetic distances.

Comparison of transcutaneous and total serum bilirubin measurement in Turkish newborns.

Severe neonatal hyperbilirubinemia can be prevented by screening for neonatal jaundice. Transcutaneous bilirubin (TcB) measurement is a noninvasive method for screening neonates. The aim of this study was to examine the correlation between TcB measurement (using the JM-103 bilirubinometer) and total serum bilirubin (TSB) measurement. To our knowledge, this is the first study evaluating the usefulness of the JM-103 bilirubinometer in Turkish neonates. Two hundred and fifty healthy infants in our well-baby nurseries and follow-up clinic with a gestational age of ≥36 weeks who were ≤15 days old were enrolled in this study. TcB measurements were taken usinng the JM-103; almost simultaneously, TSB was checked using a spectrophotometric method. The mean±SD TSB level was 11.2±4.6 mg/dl (range, 0.9-27.0 mg/dl); 17.2% of cases had TSB>15 mg/dl. There was good correlation between transcutaneous bilirubin and total serum bilirubin measurements (Pearson's correlation coefficient 0.87 for TcB from the forehead, 0.88 for TcB from the sternum; p<0.001). The transcutaneous bilirubin measurement tended to underestimate the value with increasing discrepancy at higher TSB values. The mean difference between serum bilirubin and transcutaneous (from the sternum and forehead) bilirubin values was significantly lower in cases not requiring phototherapy than in those requiring phototherapy [2.6 mg/dl (sternum) vs 4.8 mg/dl, 2.9 mg/dl (forehead) vs. 5.2 mg/dl, respectively; p<0.001] Although the JM-103 bilirubinometer tends to underestimate serum bilirubin, especially in patients with high bilirubin levels, it is a suitable screening tool to identify jaundiced infants that require a serum bilirubin check and may reduce the need for TSB measurements.

[Investigation of the ecological characteristics of the malaria vector Anopheles claviger (Diptera: Culicidae) in Sanliurfa (Siverek)]. / Sanliurfa (Siverek)'da sitma vektörü anopheles (anopheles) claviger (Diptera: Culicidae)'in ekolojik özellikleri üzerine arastirmalar.

In this study, the seasonal larval and adult population dynamics, the seasonal gonotrophic status and feeding activity of Anopheles claviger were investigated from June 2000 to August 2002 in the Siverek district of the Sanliurfa Province. Monthly samples were collected using light, animal, and human traps as well as the mouth aspirator in the study of adult populations and the dipper methods in larval populations. At the end of the study, it was found that the adult population was reproductively active between June and September. Reproductive activity came to an end in the beginning of October and by November the adult population had totally disappeared so that the population will be carried over to the next season by larval over-wintering between November and April. Unfed, fed and gravid adults were sampled continuously from June to September in stable quadrates and Anopheles claviger was shown to display endophagic feeding and endophilic resting behavior. Results obtained from animal and human traps showed that adult feeding activity increased during sunrise and sunset hours. Anthropophilic tendency in the population was determined to be 18.78% and 14.22% in 2000 and 2001, respectively.