Authoritative subspecies diagnosis tool for European honey bees based on ancestry informative SNPs.

BACKGROUND: With numerous endemic subspecies representing four of its five evolutionary lineages, Europe holds a large fraction of Apis mellifera genetic diversity. This diversity and the natural distribution range have been altered by anthropogenic factors. The conservation of this natural heritage relies on the availability of accurate tools for subspecies diagnosis. Based on pool-sequence data from 2145 worker bees representing 22 populations sampled across Europe, we employed two highly discriminative approaches (PCA and FST) to select the most informative SNPs for ancestry inference. RESULTS: Using a supervised machine learning (ML) approach and a set of 3896 genotyped individuals, we could show that the 4094 selected single nucleotide polymorphisms (SNPs) provide an accurate prediction of ancestry inference in European honey bees. The best ML model was Linear Support Vector Classifier (Linear SVC) which correctly assigned most individuals to one of the 14 subspecies or different genetic origins with a mean accuracy of 96.2% ± 0.8 SD. A total of 3.8% of test individuals were misclassified, most probably due to limited differentiation between the subspecies caused by close geographical proximity, or human interference of genetic integrity of reference subspecies, or a combination thereof. CONCLUSIONS: The diagnostic tool presented here will contribute to a sustainable conservation and support breeding activities in order to preserve the genetic heritage of European honey bees.

Synthesis of naphthalimide derivatives with potential anticancer activity, their comparative ds- and G-quadruplex-DNA binding studies and related biological activities.

Two new cytotoxic 1,8-naphthalimide derivatives have been synthesized and characterized. Their biological activities as cytotoxicity and antimicrobial activities and inhibitory activities against DNA-polymerase were evaluated. The interactions of compounds with double-stranded- and quadruple-DNA have been studied by UV-Vis, fluorescent intercalator displacement, competition dialysis, circular dichroism and the findings were compared with the parent naphthalimide and the other compounds. The results show that both compounds (1 and 2) and the parent compound NI have strong cytotoxic activities against Beas-2B, MCF-7, HepG2 and MDA-MB-231 cancer cell lines, antimicrobial activities against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and inhibitory activities towards Taq-polymerase and transcriptase. These novel cationic compounds 1 and 2 can stabilize G-quadruplexes DNA according to thermal denaturation experiments, they change the 3D structure of the DNA (see details in CD experiments) and they exhibit different binding affinities for q-DNA and ds-DNA revealed by spectrophotometric titrations and competitive dialysis studies.

The comparative study of the DNA binding and biological activities of the quaternized dicnq as a dicationic form and its platinum(II) heteroleptic cationic complex.

The square-planar heteroleptic Pt(II) coordination compound [Pt(bpy)(dicnq)](NO3)2 (1) and the quaternized dicnq ligand, namely 12,13-dicyano-5,6-dihydrodipyrazino[2,3-f:1',2',3',4'-lmn][1,10]phenanthroline-4,7-diium dibromide (2) (Fig. 1) were synthesized and fully characterized by means of FTIR, NMR, MALDI-TOF MS and the purity was confirmed by CHN analyses. The DNA binding profiles of 1 and 2 were identified in an identical condition. The biological activities of these compounds were investigated by the assays of transcription and replication inhibition, cytotoxic and antimicrobial activity. The result of this study indicates that, both compounds strongly bind to DNA via intercalation but only 1 has a strong nuclease activity. The coordination compound of dicnq (1) binds to the DNA only slightly stronger than the quaternized form of dicnq (2), and is more potent as an inhibitor of transcription and replication and therefore, 1 has more potential as an anticancer agent but the compounds did not show cytotoxic activity against MCF-7 and MDA-MB-231 breast cancer, and DLD-1 colon cancer cell lines it was found that they only had activities against HepG2 liver cancer cell line with following IC50 values; 94.75 and 159.60 µM for 1 and 2, respectively. In addition, tested bacteria are more susceptible to compound 1. These biological activities of 1 may strongly be due to its ability to digest DNA as a chemical nuclease. According to this study, the quaternization of the ligand does not make biologically more active than the coordination compound of the same ligand in this case. The compound (1) is worth further investigation for its antitumor activities.

The effects of KIR2DL4 stimulated NK-92 cells on the apoptotic pathways of HER2 + /HER-breast cancer cells.

Natural killer (NK) cells are immune cells that have attracted significant attention due to their cytotoxic properties. They are believed to be highly effective in cancer therapy. In this study, anti-KIR2DL4 (Killer cell Immunoglobulin like Receptor, 2 Ig Domains and Long cytoplasmic tail 4) was used to stimulate the NK-92 activator receptor to increase their cytotoxicity on breast cancer cell lines. Unstimulated and stimulated NK-92 cells (sNK-92) were cocultured with breast cancer (MCF-7 and SK-BR-3) and normal breast (MCF-12A) cell lines at 1:1, 1:5, and 1:10 (Target:Effector) ratios. The most effective cell cytotoxicity ratio (1:10) was used in the immunostaining and western blot assays to evaluate apoptosis pathway proteins. The sNK-92 cells showed higher cytotoxic activity on breast cancer cells than NK-92 cells. sNK-92 cells had a selective significant cytotoxicity effect on MCF-7 and SK-BR-3 cells but not MCF-12A cells. While sNK-92 cells were effective at all cell concentrations, they were most effective at a 1:10 ratio. Immunostaining and western blots showed significantly higher BAX, caspase 3, and caspase 9 protein levels in all breast cancer cell groups cocultured with sNK-92 than with NK-92 cells. NK-92 cells stimulated with KIR2DL4 showed elevated cytotoxic activity. The cytotoxic activity of sNK-92 cells on breast cancer cells is via apoptosis pathways. However, their effect on normal breast cells is limited. While the obtained data contains only basic information, additional clinical studies are needed to provide a basis for a new treatment model.

Honey bee (Apis mellifera) wing images: a tool for identification and conservation.

BACKGROUND: The honey bee (Apis mellifera) is an ecologically and economically important species that provides pollination services to natural and agricultural systems. The biodiversity of the honey bee in parts of its native range is endangered by migratory beekeeping and commercial breeding. In consequence, some honey bee populations that are well adapted to the local environment are threatened with extinction. A crucial step for the protection of honey bee biodiversity is reliable differentiation between native and nonnative bees. One of the methods that can be used for this is the geometric morphometrics of wings. This method is fast, is low cost, and does not require expensive equipment. Therefore, it can be easily used by both scientists and beekeepers. However, wing geometric morphometrics is challenging due to the lack of reference data that can be reliably used for comparisons between different geographic regions. FINDINGS: Here, we provide an unprecedented collection of 26,481 honey bee wing images representing 1,725 samples from 13 European countries. The wing images are accompanied by the coordinates of 19 landmarks and the geographic coordinates of the sampling locations. We present an R script that describes the workflow for analyzing the data and identifying an unknown sample. We compared the data with available reference samples for lineage and found general agreement with them. CONCLUSIONS: The extensive collection of wing images available on the Zenodo website can be used to identify the geographic origin of unknown samples and therefore assist in the monitoring and conservation of honey bee biodiversity in Europe.

Population structure and pattern of geographic differentiation of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) in Turkey.

BACKGROUND: The Colorado potato beetle (CPB) is the most harmful pest of potato in potato cultivation regions globally. Although it is an economically important agricultural pest, the population structure and colonization route of this species in Turkey are uncertain. We used microsatellite and mitochondrial DNA (mtDNA) markers to obtain information about the population source, structure and bio-invasion route of CPB populations in Turkey. RESULTS: The common single mtDNA haplotype in European CPB populations was obtained in all Turkish CPB populations based on mtDNA data analysis. However, microsatellites revealed a low level of genetic variation in CPB populations. The results of microsatellite analysis [factorial correspondence analysis (FCA), Bayesian analysis of genetic population structure (BAPS), unweighted pair group method with arithmetic mean (UPGMA) dendrogram, F-statistics and Nei's distances] indicated three groups for invasive CPB: Thrace-Marmara and Aegean; Black Sea, Central Anatolia and Mediterranean; Northeastern Anatolia. Region-specific alleles have been identified in regions, where commercial potato cultivation and insecticide use are intensive. CONCLUSION: The detection of a single fixed European haplotype in all Turkish populations has proved that CPB in Turkey originated from Europe as a result of a founder event occurred in European populations. Low genetic variation was due to the short time period since the spread of CPB from America to Europe. The highest number of private alleles were found in the top commercial potato cultivation region-Central Anatolia from where the CPB populations spread to other parts of Turkey. © 2021 Society of Chemical Industry.

Mitogenome Phylogeny Including Data from Additional Subspecies Provides New Insights into the Historical Biogeography of the Eurasian lynx Lynx lynx.

Previous molecular studies of the wide-ranging Eurasian lynx Lynx lynx focused mainly on its northern Palearctic populations, with the consequence that the reconstruction of this species' evolutionary history did not include genetic variation present in its southern Palearctic distribution. We sampled a previously not considered Asian subspecies (L. l. dinniki), added published data from another Asian subspecies (L. l. isabellinus), and reassessed the Eurasian lynx mtDNA phylogeny along with previously published data from northern Palearctic populations. Our mitogenome-based analyses revealed the existence of three major clades (A: Central Asia, B: SE Europe/SW Asia, C: Europe and Northern Asia) and at least five lineages, with diversification in Lynx lynx commencing at least 28kyr earlier than hitherto estimated. The subspecies L. l. isabellinus harbors the most basal matriline, consistent with the origin of Lynx lynx in this subspecies' current range. L. l. dinniki harbors the second most basal matriline, which is related to, and may be the source of, the mtDNA diversity of the critically endangered Balkan lynx L. l. balcanicus. Our results suggest that the Anatolian peninsula was a glacial refugium for Eurasian lynx, with previously unconsidered implications for the colonization of Europe by this species.

Molecular diversity of some species belonging to the genus Daphnia O. F. Müller, 1785 (Crustacea: Cladocera) in Turkey.

Daphnia is a freshwater zooplankton species with controversial taxonomy due to its high morphological variation linked to environmental factors and inter-specific hybridization and polyploidy in some groups. The aim of the present study is to examine molecular diversity of some Daphnia species in Turkey and to establish DNA barcodes of Turkish Daphnia species. Sequence analysis was performed using 540 bp region of cytochrome oxidase subunit I gene of mitochondrial DNA. A total of 34 haplotypes have been identified for Turkey. Daphnia pulex complex was divided into two clades with 16.1% sequence divergence according to molecular taxonomy based on Kimura 2-parameter. The clade which was molecularly diverged from Daphnia pulex with 16.1% sequence divergence was found to show 99% similarity with Daphnia cf. pulicaria (sensu Alonso 1996) instead of Daphnia pulicaria Forbes, 1893. Furthermore, this study has contributed to Turkish zoogeography by demonstrating the distribution of Daphnia species in Turkey.

Map position of phosphoglucomutase (Pgm) locus on autosome IV of house fly (Diptera: Muscidae).

We determined the map position of phosphoglucomutase (Pgm) locus on autosome IV of housefly, Musca domestica L. (Diptera: Muscidae), by using three and five point linkage test crosses. Test strains had visible mutant markers (car, bwb, cyw, and aabys). We analyzed 1738 offspring in total from two groups of single-pair matings by electrophoresis. Here, we report the linkage of Pgm locus to autosome IV loci curly wing (cyw) and yellow eyes (ye) with recombination frequency of 16.9 and 1.1%, respectively. We combined the distances calculated from this study and the previously published data. An updated linkage map of the M. domestica L. Autosome IV was drawn based on combined data in terms of real map units obtained from the mapping function.

Phylogenetic relations of Rhizoplaca Zopf. from Anatolia inferred from ITS sequence data.

Like many lichen-forming fungi, species of the genus Rhizoplaca have wide geographical distributions, but studies of their genetic variability are limited. The information about the ITS rDNA sequences of three species of Rhizoplaca from Anatolia was generated and aligned with other species from other countries and also with the data belonging to Lecanora species. The examined species were collected from the volcanic rocks of Mount Erciyes which is located in the middle of Anatolia (Turkey). The sequence data aligned with eight other samples of Rhizoplaca and six different species of Lecanora were obtained from GenBank. The results support the concept maintained by Arup and Grube (2000) that Rhizoplaca may not be a genus separate from Lecanora. According to the phylogenetic tree, Rhizoplaca melanopthalma from Turkey with two different samples of R. melanopthalma from Arizona (AF159929, AF159934) and a sample from Austria formed a group under the same branch. R. peltata and R. chrysoleuca samples from Anatolia located in two other branches of the tree formed sister groups with the samples of the same species from different countries. Although R. peltata remained on the same branch with other samples of the same species from other countries it was placed in a different branch within the group. When the three species from Anatolia were considered alone, it was noticed that Rhizoplaca melanopthalma and Rhizoplaca peltata are phylogenetically closer to each other than Rhizoplaca chrysoleuca; the morphological characteristics also support this result.

Genetic variations of Turkish bank vole, Myodes glareolus (Mammalia: Rodentia) inferred from mtDNA.

The bank vole, Myodes glareolus, lives in deciduous forests throughout the Palearctic region. In Turkey, this species is distributed only in northern Anatolia (the Black Sea region) where these forests exist. This study reveals genetic differentiation among bank vole populations based on two regions of mitochondrial DNA (cytochrome b and D-loop). Populations in northern Anatolia are divided into two genetic lineages (the "eastern" and "western Black Sea" lineages) by the Kizilirmak Valley. While the western Black Sea lineage is close to the Balkan lineage, in accordance with their geographical proximities, surprisingly, the Uludag lineage, also situated in Western Turkey appears related to the eastern Black Sea population. The divergence time analyses suggest a separation between the Balkan and Turkish groups around 0.26 Mya, whereas the split between the eastern and western Black sea lineages appeared a little bit later (0.20 Mya). Our results suggest that regional refuges existed for this species in Turkey and that small-scale habitat fragmentations led to genetic differentiations between Myodes populations.

Geographical distribution and frequencies of organophosphate-resistant Ace alleles and morphometric variations in olive fruit fly populations.

BACKGROUND: In the Mediterranean basin, organophosphate (OP) insecticides have been used intensively to control olive fly populations. Acetylcholinesterase (Ace) is the molecular target of OP insecticides, and three resistance-associated mutations that confer different levels of OP insensitivity have been identified. In this study, genotypes of olive fly Ace were determined in field-collected populations from broad geographical areas in Turkey. In addition, the levels of asymmetry of wing and leg characters were compared in these populations. RESULTS: Our study revealed the existence of a genetically smooth stratification pattern in OP resistance allele distribution in the olive fly populations of Turkey. In contrast to earlier findings, the frequency of Δ3Q was found to be lower in the Aegean region, where the populations have been subjected to high selection pressure. Results based on the morphological differences among the samples revealed a similar pattern for both sides and did not demonstrate a clear separation. CONCLUSION: The frequencies and geographic range of resistance alleles indicate that they were selected in the Aegean coast of Turkey and then spread westward towards Europe. One possible explanation for the absence of morphological asymmetry in olive fly samples might be the presence of modifier allele(s) that compensate for the increase in asymmetry.

Differentiation of Anatolian honey samples from different botanical origins by ATR-FTIR spectroscopy using multivariate analysis.

Botanical origin of the nectar predominantly affects the chemical composition of honey. Analytical techniques used for reliable honey authentication are mostly time consuming and expensive. Additionally, they cannot provide 100% efficiency in accurate authentication. Therefore, alternatives for the determination of floral origin of honey need to be developed. This study aims to discriminate characteristic Anatolian honey samples from different botanical origins based on the differences in their molecular content, rather than giving numerical information about the constituents of samples. Another scope of the study is to differentiate inauthentic honey samples from the natural ones precisely. All samples were tested via unsupervised pattern recognition procedures like hierarchical clustering and Principal Component Analysis (PCA). Discrimination of sample groups was achieved successfully with hierarchical clustering over the spectral range of 1800-750 cm(-1) which suggests a good predictive capability of Fourier Transform Infrared (FTIR) spectroscopy and chemometry for the determination of honey floral source.

A worldwide survey of genome sequence variation provides insight into the evolutionary history of the honeybee Apis mellifera.

The honeybee Apis mellifera has major ecological and economic importance. We analyze patterns of genetic variation at 8.3 million SNPs, identified by sequencing 140 honeybee genomes from a worldwide sample of 14 populations at a combined total depth of 634×. These data provide insight into the evolutionary history and genetic basis of local adaptation in this species. We find evidence that population sizes have fluctuated greatly, mirroring historical fluctuations in climate, although contemporary populations have high genetic diversity, indicating the absence of domestication bottlenecks. Levels of genetic variation are strongly shaped by natural selection and are highly correlated with patterns of gene expression and DNA methylation. We identify genomic signatures of local adaptation, which are enriched in genes expressed in workers and in immune system- and sperm motility-related genes that might underlie geographic variation in reproduction, dispersal and disease resistance. This study provides a framework for future investigations into responses to pathogens and climate change in honeybees.

The genetic polymorphisms and colonization process of olive fly populations in Turkey.

The olive fruit fly, Bactrocera oleae, is the most important pest of olives in olive growing regions worldwide, especially in the Mediterranean basin and North America. Despite the economic importance of the olive fly, the colonization route of this species is unclear. We used nuclear microsatellite markers and mitochondrial DNA to provide information about the population structure and invasion route of olive fly populations in Turkey, as representative of the Eastern Mediterranean region. Adult fly samples were collected from 38 sublocations covering all olive growing regions in Turkey. The simple sequence variability data revealed a significant genetic variability in olive fly populations and a certain degree of differentiation between Mediterranean and Aegean populations. Mediterranean populations harbor higher levels of microsatellite variation than Aegean populations, which points to the eastern part of the Mediterranean as the putative source of invasion. mtDNA results suggest olive flies from the western part of Turkey are closely related to Italo-Aegean flies of the Mediterranean basin and the olive fly populations have invaded the northern part of the Mediterranean basin through western Turkey. In addition, finding specific American haplotypes in high frequencies might indicate that Turkey is the possible source of American olive fly populations. In order to more precisely characterize the population structure and invasion routes of this organism, more DNA-based sequence analysis should be carried out worldwide.

Identification of Lygus hesperus by DNA barcoding reveals insignificant levels of genetic structure among distant and habitat diverse populations.

BACKGROUND: The western tarnished plant bug Lygus hesperus is an economically important pest that belongs to a complex of morphologically similar species that makes identification problematic. The present study provides evidence for the use of DNA barcodes from populations of L. hesperus from the western United States of America for accurate identification. METHODOLOGY/PRINCIPAL FINDINGS: This study reports DNA barcodes for 134 individuals of the western tarnished plant bug from alfalfa and strawberry agricultural fields in the western United States of America. Sequence divergence estimates of <3% reveal that morphologically variable individuals presumed to be L. hesperus were accurately identified. Paired estimates of F(st) and subsequent estimates of gene flow show that geographically distinct populations of L. hesperus are genetically similar. Therefore, our results support and reinforce the relatively recent (<100 years) migration of the western tarnished plant bug into agricultural habitats across the western United States. CONCLUSIONS/SIGNIFICANCE: This study reveals that despite wide host plant usage and phenotypically plastic morphological traits, the commonly recognized western tarnished plant bug belongs to a single species, Lygus hesperus. In addition, no significant genetic structure was found for the geographically diverse populations of western tarnished plant bug used in this study.

Hinf-I digestion of cytochrome oxidase I region is not a diagnostic test for A. m. lamarckii

Restriction fragment length polymorphism of whole mitochondrial DNA or PCR amplified mtDNA regions are known to be useful in discriminating among honey bee lineages and also some individual subspecies. In this study, PCR-amplified fragments of cytochrome oxidase I (CO-I) and cytochrome B (Cyt B) of honey bees sampled from different countries (Cyprus, Turkey, Ethiopia, Syria and Egypt) were digested with Hinf I and Bgl II restriction enzymes, respectively. Eastern Europe and Mediterranean honey bee subspecies were separated by the Cyt B/Bgl II analysis, although Hinf I digestion of the CO-I region yielded much finer resolution within different honey bee lineages. Here we report that CO-I/Hinf-I is a discriminative test for the mitochondrial "O" lineage, rather than a diagnostic site for A. m. lamarckii.