Arif Ciloglu

Detection and molecular characterization of avian Plasmodium from mosquitoes in central Turkey

Assessing vector-parasite relationship is important in understanding the emergence of vector-borne diseases and the evolution of parasite diversity. This study investigates avian Plasmodium parasites in mosquitoes collected from Kayseri province in Central Anatolian, Turkey and determines the haemosporidian parasite lineages from these mosquito species. A total of 6153 female mosquitos from 6 species were collected from 46 sites during June-August of 2008 and 2009. Each mosquitos head-thorax and abdomen were separated, categorized with respect to species and collection area and pooled for DNA extraction. A total of 1198 genomic DNA pools (599 thorax-head, 599 abdomen) were constituted of which 128 pools (59 thorax-head, 69 abdomen) were positive for avian haemosporidian parasites (Plasmodium and Haemoproteus) by Nested-PCR analysis. Culex pipens, Aedes vexans, Culex theileri and Culiseta annulata were positive with minimum infection rates (MIRs) of 16.22 and 18.15, 4.72 and 5.98, 5.18 and 10.36, 10.64 and 10.64 in their thorax-head and abdomen parts, respectively. No avian haemosporidian DNA was detected from Culex hortensis and Anopheles maculipennis. Phylogenetic analyses of the partial cytb gene of avian haemosporidian mt-DNA from 13 positive pools revealed that 11 lineages in four phylogenic groups were Plasmodium and the other two were Haemoproteus. Our results suggest that Cx. pipiens could probably be the major vector of avian Plasmodium in Central Turkey. This is the first report of molecular detection and characterization of avian Plasmodium lineages from mosquitoes in Turkey.

Molecular Investigation of Francisella-Like Endosymbiont in Ticks and Francisella tularensis in Ixodid Ticks and Mosquitoes in Turkey

This study was carried out to investigate the molecular prevalence of Francisella-like endosymbionts (FLEs) and Francisella tularensis in ticks (Acari: Ixodidae) and mosquitoes in Turkey. Genomic DNA pools were constructed from a total of 1477 adult hard ticks of Rhipicephalus (Rh.) annulatus, Rh. turanicus, Rh. sanguineus, Rh. bursa, Haemaphysalis (Hae.) parva, Hae. sulcata, Hyalomma marginatum marginatum, H. anatolicum anatolicum, H. anatolicum excavatum, H. detritum detritum, H. dromedarii, Dermacentor marginatus, and Ixodes ricinus species, which were collected from several barns, cattle, and people. Genomic DNA was also extracted from pools consisting of 6203 adult female mosquito species belonging to Aedes vexans, Culex (Cx.) pipiens, Cx. hortensis, Cx. theileri, Culiseta annulata, and Anopheles maculipennis species. Conventional PCR and TaqMan probe-based real- time PCR targeting the 16S rRNA gene for FLEs and the lpnA gene for F. tularensis, respectively, were performed on the DNA isolates obtained. FLEs and F. tularensis were not found in any genomic DNA pools constructed from ixodid ticks and mosquitos. This study represents the first investigation of F. tularensis and FLEs in potential vector ticks and mosquitoes by molecular methods in Turkey. The present study provides useful insights into the molecular epidemiology of F. tularensis and FLEs. One of the major conclusions of the study is that tularemia outbreaks may be essentially due to direct transmission from the environment (especially from water) in Turkey and not to vector-borne transmission.

Rearrangement hotspots in the sex chromosome of the Palearctic black fly Simulium bergi (Diptera, Simuliidae).

Abstract An extreme example of nonrandom rearrangements, especially inversion breaks, is described in the polytene chromosomes of the black fly Simulium bergi Rubtsov, 1956 from Armenia and Turkey. A total of 48 rearrangements was discovered, relative to the standard banding sequence for the subgenus Simulium Latreille, 1802. One rearrangement, an inversion (IIS-C) in the short arm of the second chromosome, was fixed. Six (12.5%) of the rearrangements were autosomal polymorphisms, and the remaining 41 (85.4%) were sex linked. More than 40 X- and Y-linked rearrangements, predominantly inversions, were clustered in the long arm of the second chromosome (IIL), representing about 15% of the total complement. The pattern conforms to a nonrandom model of chromosome breakage, perhaps associated with an underlying molecular mechanism.

Blood Meal Identification of the Mosquito (Diptera: Culicidae) Specimens Belong to Culex pipiens Complex that were Collected from Kayseri Province

OBJECTIVE This study aimed to determine the host preferences in blood meal of specimens belonging to Culex pipiens complex. METHODS A total of 1284 female mosquitos were morphologically examined, and genomic DNA isolations were individually performed on 376 (28.4%) specimens that were determined to be Cx. pipiens complex. PCR was performed with primers to specifically amplify the avian and mammalian mitochondrial cytochrome b (mt-cytb) gene region. Amplicons were cloned, and the obtained plasmids were sequenced to determine host species. RESULTS Of 376 specimens, 148 (39.4%) were positive for the avian and/or mammalian blood meal. Among the positive specimens, 43, 98, and seven were determined to be positive for only mammalian, avian, and both avian and mammalian blood, respectively. Avian host preference in blood meal of the specimens belonging to Cx. pipiens was found to be significant. Of 15 avian blood positive isolates, nine, three, two, and one were designated as blood meal from avian species in Passeriformes, Accipitriformes, Columbiformes, and Strigiformes orders, respectively. While six, four, three, and two out of 15 mammalian blood-positive specimens were found to be positive for human, cattle, sheep, and dog blood, respectively. CONCLUSION Molecular data regarding the host preferences of the Cx. pipiens species complex in blood meal were revealed for the first time in Turkey with this study.

Kayseri yöresinden toplanmi{dotless}ş Culex pipiens örneklerinde Wolbachia Endobakterisinin belirlenmesi ve moleküler karakterizasyonu

This study was performed to investigate Wolbachia endobacteria in Culex pipiens specimens collected from Kayseri province of Turkey. For this aim, totally 10 genomic DNA pools each including 6-15 Cx. pipiens specimens which were collected and identified within the scope of a project (No: 107O533) supported by TUBITAK, were examined by using the amplification of surface protein gene (wsp) region of the Wolbachia. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. After the genomic DNA extraction from the pools, PCR analyses were carried out with Wolbachia specific primer pair which was amplified a 590-632 bp region of the wsp gene. Out of 6 of the 10 examined genomic DNA pools were found to be positive (60.0%) by PCR analyses. The minimum infection rate of Wolbachia spp. in the totally analyzed 118 Cx. pipens specimens was determined as 5.08. One of the amplicon from the positive isolates was gel purified and sequenced in terms of wsp gene region of Wolbachia by using the same primers. Pair wise analyses of the obtained DNA sequences and multiple alignments with some other Wolbachia strains available in the GenBank were done and phylogenies were investigated. The obtained isolate (WolKys1) was deposited in GenBank International Nucleotide Sequence Database with the accession number JX474753. The phylogenetic analyses revealed that the obtained WolKys1 isolate belongs to Wolbachia Super Group B and wPIP group. According to the phylogenetic comparisons the WolKys1 showed 100.0% identity with some other Wolbachia isolates under the Group B. In conclusion, this study reports the first molecular detection and characterization of Wolbachia endobacteria in Cx. pipiens populations in Turkey.

Molecular Characterization of Myiasis-Causing Moth Flies (Diptera: Psychodidae)

OBJECTIVE The aim of this study is to examine the molecular characterization of moth flies (Diptera: Psychodidae) based on their mitochondrial DNA sequences and determine the vectorial potential and damage caused by moth flies in future researches. METHODS A total of 240 adult moth flies were collected from toilet, bathroom, and basement walls of houses from different locations of the Kayseri region between May 2016 and April 2017. The polymerase chain reaction (PCR) analyses were performed using primer pairs, specificaly targeting the mitochondrial cytochrome oxidase c subunit I (mt-COI) gene of adult flies. RESULTS In total, five isolates were gel purified and sequenced for molecular characterization and phylogenetic analyses. Two species, namely Telmatoscopus albipunctatus (ERU-Telmatos3 and ERU-Telmatos6) and Psychodidae sp. (ERU-Psycho1,4,5), were successfully identified with the sequence alignment of isolates. According to the phylogenetic analysis, it was determined that the ERU-Telmatos3 and ERU-Telmatos6 isolates are clustered in the haplogroup A, while the ERU-Psycho1,4,5 isolate was clustered within the haplogroup B. The ERU-Psycho1 isolate was characterized as a new haplotype within the haplogroup B. CONCLUSION This study represents the first molecular characterization and phylogenetic status of moth flies in Turkey. The obtained findings should be the first step in the future investigation based on detecting the transmission of bacterial pathogens by moth flies.

Occurrence and molecular characterization of Clinostomum complanatum (Trematoda: Clinostomidae) in freshwater fishes caught from Turkey

The metacercariae of Clinostomum species which known as yellow grubs have zoonotic potential by infecting humans. In the present study, a total of 403 freshwater fish specimens belonging to different genera from Central Anatolia Region of Turkey were examined for yellow grub metacercariae infections. Only three specimens belonging to Squalius cephalus were found to be infected with metacercariae with a prevalence on this host species of 2.4% and an overall prevalence of 0.7%. All the metacercariae were morphologically identified as Clinostomum complanatum. Partial fragments of mitochondrial cytochrome oxidase I (mt-COI) gene and internal transcribed spacer 2 (ITS-2) were amplified for sequence and phylogenetic analyses. The sequence analyses of ITS-2 and mt-COI revealed three and nine polymorphic sites leading to detection of four and five haplotypes within the related gene regions, respectively. Moreover, the intraspecific genetic distances for C. complanatum isolates ranged from 0.0 to 0.7% for ITS-2 and 0.0 to 1.4% for mt-COI data sets. Consequently, the present study has provided first combined morphologic and molecular data on C. complanatum infecting Turkish freshwater fishes.

Molecular characterization of the horn fly Haematobia irritans infesting horses in Central Anatolia Region in Turkey

&NA; This study reports intense horn fly infestations of horses raised in an important wetland ecosystem, Sultan Marshes in Central Anatolia, Turkey. In total, seven horses raised together were found to be harbored over 500 flies per site of each animal. Totally, 376 fly specimens were collected from the horses by using the nets and were subjected to the laboratory for species identification. All flies were morphologically identified as the adults of Haematobia irritans. Partial fragments of mitochondrial cytochrome oxidase I (mt‐COI) gene from totally 50 isolates were amplified for sequence and phylogenetic analyses. The mt‐COI sequence analyses revealed no polymorphism among the isolates and explored a unique haplotype for H. irritans. A mean haplotype and nucleotide diversities of 0.8571 and 0.00695 were determined, respectively, within the COI data set of H. irritans, and newly characterized haplotype from Turkey exhibited a mean intraspecific genetic difference of 1.0 to the all published sequences of the isolates from several countries. COI data set also revealed a mean interspecific genetic difference of 1.7 between H. irritans and Haematobia exigua.

Genetic diversity and identification of palearctic black flies in the subgenus wilhelmia (Diptera: Simuliidae)

Abstract Accurate species identifications are the essential first step in understanding the medical, economic, and ecological importance of black flies. The utility of DNA barcoding based on cytochrome c oxidase subunit 1 (COI) sequences was evaluated for identifying six common species of Palearctic black flies in the subgenus Wilhelmia, including several that are virulent pests. Chromosomally identified larvae from Turkey and Germany and COI sequences in GenBank were analyzed. Intraspecific genetic divergence was 0.7–3.5% (mean 1.6%), whereas interspecific genetic divergence was 2.7–16.9%. On the basis of COI barcodes, the six nominal species of Simulium (Wilhelmia) were clustered in three distinct clades with high levels of genetic divergence, using maximum likelihood and Bayesian analyses. All specimens of Simulium equinum (L.), Simulium pseudequinum Séguy, and Simulium paraequinum Puri were correctly identified. However, >75% of identifications were ambiguous for Simulium lineatum (Meigen) and Simulium turgaicum Rubtsov (Meigen) because of overlapping intra- and interspecific divergence of the two species and Simulium balcanicum (Enderlein), all three of which are chromosomally similar and nearly isomorphic. Phylogenetic evaluation showed that S. balcanicum, S. equinum, S. pseudequinum, and S. paraequinum were monophyletic, with high bootstrap and posterior probability values, but it also showed that S. lineatum and S. turgaicum were paraphyletic, each clustering in two distinct groups, suggesting the presence of cryptic taxa. Although DNA barcoding provided a partialmeans of identification and indications of additional biodiversity, other molecularmarkers are needed to clarify the limits of all pest species of the subgenusWilhelmia.

Investigation of avian haemosporidian parasites from raptor birds in Turkey, with molecular characterisation and microscopic confirmation.

Avian haemosporidians are common vector-borne blood parasites that have been reported in birds all over the world. Investigations of avian haemosporidian parasites are conducted mainly on passerine birds. However, studies that focus on non-passerine avian hosts are important for our understanding of the true diversity, host specificity and genetic variability among these widespread parasites. In the present study, blood samples from a total of 22 raptor birds belonging to two orders, two families and six species from the Central Anatolia Region of Turkey were investigated for three genera of avian haemosporidians (Plasmodium Marchiafava et Celli, 1885, Haemoproteus Kruse, 1890 and Leucocytozoon Sambon, 1908) using a combination of microscopic examination of blood films and nested PCR targeting the parasite mitochondrial cytochrome b gene (cyt-b). In total, six individual raptor birds identified positive for species of Plasmodium or Leucocytozoon and one individual was found co-infected with all three haemosporidian genera. We identified five parasite cyt-b haplotypes, three of which were reported for the first time. Among these, one Plasmodium haplotype is linked to a corresponding morphospecies (P-TURDUS1, Plasmodium circumflexum Kikuth, 1931). All haplotypes were clearly distinguishable in phylogenetic analyses. As one of the first studies to investigate blood parasites from non-passerine birds in the Central Anatolia Region of Turkey, this study provides important new information on the phylogenetic relationships and genetic diversity of avian haemosporidian parasites from raptor birds. We discuss these findings in the context of avian haemosporidian host-parasite relationships and we draw attention to the need for microscopy to detect parasite sexual development stages in surveys of avian haemosporidians.