Molecular characterization of coi gene of ixodes ricinus (linnaeus, 1758) from Serbia

The Ixodes ricinus tick is common in the central part of the Balkan Peninsula. It is a vector of pathogenic agents causing diseases in humans and animals. Little is known about the genetic structure of I. ricinus in this region. We have investigated intraspecific variability of the COI gene among I. ricinus ticks collected from different regions of Serbia, and the correlation between the various types of habitat and genetic variability of ticks. The obtained COI gene sequences are the first barcoding sequences of I. ricinus ticks collected at localities in Serbia. Intraspecific variability of these COI gene sequences was very low, and there was no correlation between the various types of habitat and genetic variability of ticks. Samples from isolated localities (canyon/gorge) showed no genetic differentiations from the majority of samples from open areas

ON THE FIRST NON-TYPE LOCALITY FINDING OF DUVALIUS (PARADUVALIUS) STANKOVITCHI GEORGEVITCHI (JEANNEL, 1923) (COLEOPTERA: CARABIDAE: TRECHINAE)

Troglobitic trechine ground beetle, Duvalius (Paraduvalius) stankovitchi georgevitchi (Jeannel, 1923) was found outside its type locality (Lazareva Pećina Cave, village of Zlot, near Bor, Kučajske Planine Mts., eastern Serbia) for the first time. Specimens of the mentioned taxon were recently collected in Vernjikica Cave nearby. Morphological and molecular analyses confirmed that specimens from both caves belong to the same subspecies, and that there are no significant differences between them

Interference of Field Evidence, Morphology, and DNA Analyses of Three Related Lysiphlebus Aphid Parasitoids (Hymenoptera: Braconidae: Aphidiinae)

This study provides evidence on integrating the morphological, field, and laboratory data, and application of the cytochrome oxidase subunit I (COI) barcoding gene to the three asexual or sexual Lysiphlebus spp., i.e., Lysiphlebus cardui (Marshall), Lysiphlebus confusus Tremblay and Eady and Lysiphlebus fabarum (Marshall) (Hymenoptera: Braconidae: Aphidiinae). New aphid-invasive plant association, Aphis fabae Scopoli (Hemipreta: Aphididae) on Impatiens glandulifera Royle, has been used in the same model area in the Czech Republic under the same sampling and rearing method for several consecutive years and throughout the season. For molecular identification of these three species, we used DNA sequences of the barcoding region of the mitochondrial COI gene. Although our results confirmed ecological and morphological differences among L. cardui, L. confusus, and L. fabarum, genetic analysis on the basis of COI mitochondrial barcoding gene does not support species status of the mentioned Lysiphlebus taxa. The level of morphological differentiation in these Lysiphlebus Forster species is in accordance with the usual species variability within subfamily Aphidiinae. However, it should be examined how appearance of asexual lineages affects the morphological or genetical variability

First record of Aphidius ericaphidis (Hymenoptera, Braconidae) in Europe: North American hitchhiker or overlooked Holarctic citizen?

Aphidius ericaphidis, an aphid parasitoid originally described from North America, is recorded in Europe for the first time, based on morphological and molecular analyses. The species is recorded in Serbia, the Netherlands, Sweden and the United Kingdom. It was formerly recorded as Aphidius sp./Ericaphis latifrons/Vaccinium uliginosum from the Alps (France). The possible origin of A. ericaphidis, as well as its likely distribution, is discussed in relation to its host. As it is a specific parasitoid of Ericaphis aphids, especially the invasive aphid Ericaphis scammelli on blueberries, its potential as a biocontrol agent is also discussed

New strain of 'Candidatus Phytoplasma ulmi' infecting Ulmus minor and U. laevis in Serbia

Elm yellows (EY) phytoplasma (‘Candidatus Phytoplasma ulmi’) is the causal agent of a decline in American elms in North America, and in Eurasian elm species and hybrids in Europe (Lee et al., 2004). EY is known to infect different Ulmus species: U. americana, U. minor, U. rubra, U. alata, U. serotina, U. crassifolia and U. chenmoui, showing different symptoms such as stunting, witches’ broom, yellowing and general decline of the plants (Marcone et al., 1997; Griffiths et al., 1999). In September 2007 leaves with petioles from eighteen elm trees showing symptoms of discrete leaf yellowing were collected from three different sites in northeast Serbia near the villages of Srednjevo, Ljubicevo and TuvajiÇ. From each site six samples were collected. At two sites (Srednjevo and Ljubicevo) the affected plants were of European field elm (U. minor), and at the third site they were of European white elm (U. laevis). Leaves of six symptomless young elm trees (U. minor) collected near Belgrade served as the controls. Total nucleic acids were extracted from fresh leaf midribs and petioles using the CTAB method (Angelini et al., 2001). Phytoplasma identification was conducted using a nested PCR assay with P1/P7 and F2n/R2 primers on the 16S rRNA gene, followed by RFLP analysis with MseI restriction enzyme. Positive results were obtained in nine affected U. minor samples and five U. laevis samples, with RFLP profiles indicating the presence of phytoplasmas of the 16SrV group. None of the symptomless plants were positive for the presence of phytoplasma. Further characterization was performed by amplifying the ribosomal protein genes l22 and s3 using primers rp(V)F1/rpR1 followed by rp(V)F1A/rp(V)R1A, finally by digestion with MseI and Tsp509I (Lee et al., 2004). RFLP profiles with MseI enzyme showed the presence of EY phytoplasmas of 16SrV-A group, but profiles obtained with Tsp509I enzyme were different from the EY control sample and were more similar to FD-C (16Sr V-C group). Subsequently two of these products, one from U. minor and one from U. laevis, were sequenced (GenBank Acc. No. EU592500, EU592501) and showed identical nucleotide sequence to each other. blast analyses showed 99% similarity of these isolates with reference strain EY1T (AY197675). Nucleotide changes are located in two out of three unique regions of the rpl22–rps3 genes reported by Lee et al. (2004) as being species specific for ‘Candidatus Phytoplasma ulmi’. This is the first report of elm yellows phytoplasma belonging to rRNA group 16SrV-A infecting elm species in Serbia and of its association with U. laevis. It is also the first evidence of strain differences in ‘Candidatus Phytoplasma ulmi’ detectable by RFLP analysis of ribosomal protein gene PCR products

Integrative taxonomy of root aphid parasitoids from the genus Paralipsis (Hymenoptera, Braconidae, Aphidiinae) with description of new species

Species from the genus Paralipsis are obligatory endoparasitoids of root aphids in the Palaearctic. It is known that these species are broadly distributed, parasitizing various aphid hosts and showing great biological and ecological diversity. On the other hand, this group of endoparasitoids is understudied and was thought to be represented by a single species in Europe, viz., Paralipsis enervis (Nees). However, recent description of two new species indicated the possibility of cryptic speciation and recognition of additional Paralipsis species in Europe. In this research, Paralipsis specimens collected during the last 60 years from eight European countries, as well as one sample from Morocco, were subjected to molecular and morphological characterization. Newly designed genus-specific degenerative primers successfully targeted short overlapping fragments of COI of the mitochondrial DNA. Molecular analyses showed clear separation of four independent lineages, two of which are the known species P. enervis and P. tibiator, while two new species are described here, viz., P. brachycaudi Tomanovic & Stary, sp. n. and P. rugosa Tomanovic & Stary, sp. n. No clear specialization of the taxa to a strict root aphid host has been determined. The recognized mitochondrial lineages were distinct one from another, but with a substantial within-lineage divergence rate, clearly indicating the complexity of this group of parasitoids, on which further research is required in order to clarify the factors triggering their genetic differentiation. We reviewed literature data and new records of Paralipsis enervis aphid host associations and distributions. A key for the identification of all known Paralipsis species is provided and illustrated

Genetic and morphological variation in sexual and asexual parasitoids of the genus Lysiphlebus - an apparent link between wing shape and reproductive mode

Background: Morphological divergence often increases with phylogenetic distance, thus making morphology taxonomically informative. However, transitions to asexual reproduction may complicate this relationship because asexual lineages capture and freeze parts of the phenotypic variation of the sexual populations from which they derive. Parasitoid wasps belonging to the genus Lysiphlebus Foerster (Hymenoptera: Braconidae: Aphidiinae) are composed of over 20 species that exploit over a hundred species of aphid hosts, including many important agricultural pests. Within Lysiphlebus, two genetically and morphologically well- defined species groups are recognised: the "fabarum" and the "testaceipes" groups. Yet within each group, sexual as well as asexual lineages occur, and in L. fabarum different morphs of unknown origin and status have been recognised. In this study, we selected a broad sample of specimens from the genus Lysiphlebus to explore the relationship between genetic divergence, reproductive mode and morphological variation in wing size and shape (quantified by geometric morphometrics). Results: The analyses of mitochondrial and nuclear gene sequences revealed a clear separation between the "testaceipes" and "fabarum" groups of Lysiphlebus, as well as three well-defined phylogenetic lineages within the "fabarum" species group and two lineages within the "testaceipes" group. Divergence in wing shape was concordant with the deep split between the "testaceipes" and "fabarum" species groups, but within groups no clear association between genetic divergence and wing shape variation was observed. On the other hand, we found significant and consistent differences in the shape of the wing between sexual and asexual lineages, even when they were closely related. Conclusions: Mapping wing shape data onto an independently derived molecular phylogeny of Lysiphlebus revealed an association between genetic and morphological divergence only for the deepest phylogenetic split. In more recently diverged taxa, much of the variation in wing shape was explained by differences between sexual and asexual lineages, suggesting a mechanistic link between wing shape and reproductive mode in these parasitoid wasps

Phylogenetic relationships and subgeneric classification of European Ephedrus species (Hymenoptera, Braconidae, Aphidiinae)

In this study two molecular markers were used to establish taxonomic status and phylogenetic relationships of Ephedrus subgenera and species distributed in Europe. Fifteen of the nineteen currently known species have been analysed, representing three subgenera: Breviephedrus Gärdenfors, 1986, Lysephedrus Starý, 1958 and Ephedrus Haliday, 1833. The results of analysis of COI and EF1α molecular markers and morphological studies did not support this classification. Three clades separated by the highest genetic distances reported for the subfamily Aphidiinae on intrageneric level. Ephedrus brevis is separated from persicae and plagiator species groups with genetic distances of 19.6 % and 16.3 % respectively, while the distance between persicae and plagiator groups was 20.7 %. These results lead to the conclusion that the traditional subgeneric classification of Ephedrus needs revision. Species from persicae species group are raised to subgenus level as Fovephedrus Chen, 1986 and Lysephedrus syn. nov. is assigned as a junior synonym of subgenus Ephedrus. Key for identification of Ephedrus subgenera is provided. Ephedrus hyadaphidis Kocić & Tomanović sp. nov. is described and several species are confirmed as valid species for the first time. Furthermore, two species are synonymised: E. dysaphidis syn. nov. as a junior synonym of E. cerasicola and E. blattnyi syn. nov. as a junior synonym of E. plagiator

Re-visiting the Aphidius urticae s. str. group: re-description of Aphidius rubi Stary and A. silvaticus Stary (Hymenoptera: Braconidae: Aphidiinae)

Here we tested Aphidius urticae s. str. host-associated lineages from Microlophium carnosum (Buckton), Amphorophora rubi (Kaltenbach), Macrosiphum funestum (Macchiati) and Aulacorthum vaccinii Hille Ris Lambers with the barcoding region of the mitochondrial cytochrome oxidase subunit I gene used to analyse population differences and elucidate phylogenetic relationships between the separated taxa. This molecular marker has been shown to be the most informative molecular marker in resolving species complexes in aphidiine parasitoids. Analyses of the mitochondrial sequences revealed the existence of three clearly separated mitochondrial lineages of A. urticae s. str. group associated with: i) Macrosiphum funestum and Aulacorthum vaccinii aphid hosts, ii) Microlophium carnosum and iii) Amphorophora rubi. This corresponds to the initial descriptions of A. rubi, A. silvaticus and A. urticae and their aphid host associations prior to synonymization of A. rubi and A. silvaticus with A. urticae. On the other hand, significant evolutionary distances ranging from 2.3 to 9.2% between the three mitochondrial lineages were not accompanied by clear morphological differences. Therefore, re-descriptions of A. rubi and A. silvaticus are presented, together with their morphological differentiation in a key, as well as their phylogenetic relationships and genetical differentiation