Ljubiša Ž. Stanisavljević

Pattern recognition methods and multivariate image analysis in HPTLC fingerprinting of propolis extracts

High‐performance thin‐layer chromatography (HPTLC) combined with image analysis and pattern recognition methods were used for fingerprinting and classification of 52 propolis samples collected from Serbia and one sample from Croatia. Modern thin‐layer chromatography equipment in combination with software for image processing and warping was applied for fingerprinting and data acquisition. The three mostly used chemometric techniques for classification, principal component analysis, cluster analysis and partial least square‐discriminant analysis, in combination with simple and fast HPTLC method for fingerprint analysis of propolis, were performed in order to favor and encourage their use in planar chromatography. HPTLC fingerprint analysis of propolis was for the first time performed on amino silica plates. All studied propolis samples have been classified in two major types, orange and blue, supporting the idea of existence of two types of European propolis. Signals at specific RF values responsible for classification of studied extracts have also been isolated and underlying compounds targeted for further investigation. Copyright

Regional tritrophic relationship patterns of five aphid parasitoid species (Hymenoptera: Braconidae: Aphidiinae) in agroecosystem-dominated landscapes of southeastern Europe.

ABSTRACT A regional survey of the complex tritrophic associations (parasitoid-aphid-plant) of aphid parasitoids (Hymenoptera: Braconidae: Aphidiinae) was carried out to determine and explore the patterns of those associations in various types of environments. Here, we present trophic relationship patterns of the five aphid parasitoid species in crop and noncrop habitats in southeastern Europe, and we contrast them in a regional (Mediterranean [MED] versus continental [CNT]) context. In total, 79 aphid host taxa were identified in this survey. Forty-two of these were recorded from noncrop plants only, 21 from crop plants only, and 18 were present on both types of plants. This means that ≈74% of all the parasitoid-aphid trophic interactions that support the persistence of the five selected parasitoids are entirely (54%) or partially (20%) associated with noncrop plants. The correspondence of parasitoid-aphid combinations among habitat/region combinations is very high and specific. Our results suggest that Mediterranean and continental regions are clearly distinguished by a contrasting pattern of trophic interactions in crop habitats, whereas the noncrop habitats contribute in lesser degree to these differences. For the crop/noncrop breakdown, the number of nonspecific interactions was larger than expected in crop habitats, whereas in noncrop habitats the abundance of partially specific and specific interactions was larger. The analysis of variance for the regional and habitat distribution of mean aphid host number per parasitoid was highly significant. When both regions were analyzed separately, the parasitoid/crop design showed significant parasitoid effects as well as interactions, whereas the habitat effect was not significant for the Mediterranean region and highly so for the continental region. This highly complex pattern suggests that the mean number of parasitized aphid species is not distributed among parasitoids, regions, and habitats in a similar manner. Even with these complexities taken into account, the overall trend is that noncrop habitats support more parasitoid-aphid combinations and more so in the continental than in Mediterranean regions, although not always statistically significant. As mentioned, large number of noncrop aphid hosts, especially for Lysiphlebus fabarum (Marshall), Praon volucre (Haliday) and Aphidius colemani Viereck, can significantly enhance the population buildup for these important parasitoids around agroecosystems. These facts can be important in biological aphid pest control in the region. Although not easily quantified, the overall positive effects of larger parasitoid diversity in noncrop habitats are undoubtedly related to the distribution and structure of noncrop habitat patches in agroecosystems at a landscape scale.

Morphological Characterization of Ephedrus persicae Biotypes (Hymenoptera: Braconidae: Aphidiinae) in the Palaearctic

ABSTRACT Ephedrus persicae Froggatt (Hymenoptera: Braconidae: Aphidiinae) represents a biologically complex parasitoid species group that parasitizes >150 aphid species worldwide, including many pests in different agroecosystems. Evidence from the host range indicated some taxonomic problems within this species-complex. Using classical and geometric morphometric approaches, we assessed the morphological differences among various E. persicae biotypes in the Palaearctic reared from 20 aphid hosts. We found that morphological variability of E. persicae biotypes was influenced by the host. Classical morphometric analysis indicated a conservative position of E. persicae biotypes in the Palaearctic. Our analysis showed that E. persicae biotypes reared from nonleaf-curling Aphis and leaf-curling Dysaphis aphid hosts have significant differences, but all other analyzed E. persicae biotypes have an intermediate position, making it impracticable to split the E. persicae group this way. However, geometric morphometrics used to analyze variation of wing shape showed better predictive ability. The high ratio of correctly assigned individuals based on the wing shape of E. persicae/Aphis viticis Ferrari, E. persicae/Brachycaudus klugkisti (Börner), and E. persicae/Tuberocephalus momonis (Matsumura) biotypes indicates that some host-specific adaptations occurred. The E. persicae/ Hyadaphis foeniculi (Passerini) biotype is clearly separated along the CV2 canonical axis, exhibiting shorter and wider wings. On the basis of this result, we describe a new taxon—Ephedrus lonicerae n. sp.

Phylogenetic relationships between the genera Aphidius and Lysaphidus (Hymenoptera: Braconidae: Aphidiinae) with description of Aphidius iranicus sp. nov

We analyzed the phylogenetic relationships between eight Aphidius Nees and six Lysaphidus Smith species on the basis of 12 morphological characters by parsimony analysis. The consensus tree does not support the generic status of Lysaphidus. Aphidius iranicus, sp. nov., associated with Titanosiphon bellicosum Nevsky on Artemisia absinthium L. from Iran, is described. The new parasitoid species is described and illustrated by line drawings, and its diagnostic characters are discussed. The taxonomic position of the subgenus Tremblayia Tizado and Núñez-Pérez is also considered. Tremblayia and Lysaphidus are newly classified as synonyms of Aphidius. The following new or revised combinations are proposed: Aphidius adelocarinus Smith, comb. rev., A. ramythirus Smith, comb. rev., A. rosaphidis Smith, comb. rev., A. viaticus (Sedlag), comb. nov., A. arvensis (Starý), comb. nov., and A. erysimi (Starý), comb. nov.

Phylogenetic relationships among Praini (Hymenoptera: Braconidae: Aphidiinae) aphid parasitoids, with redescription of two species

Phylogenetic relationships among four genera and 25 species of the tribe Praini (Braconidae: Aphidiinae) were investigated on the basis of 15 characters (13 morphological and two life history characters) using parsimony analysis. Areopraon Mackauer and Pseudopraon Starý are basal within the tribe. Both genera include species in which the external and internal types of pupation exist simultaneously [for example, Pseudopraon mindariphagum Starý, Areopraon lepelleyi (Waterston) and A. pilosum Mackauer)]. It can therefore be asserted that the external type of pupation behavior in the Praini probably originated in the polymorphic pupation behavior of ancient genera such as Pseudopraon and Areopraon. The genera Areopraon, Pseudopraon, and Dyscritulus Hincks form a monophylum separate from Praon. Areopraon is not retrieved as monophyletic. Species of the genus Praon Haliday possess conical apical spines on the top of the ovipositor sheath as an apomorphic character state. Within the genus Praon, we recognized the following closely related species groups: Parapraon + Praon dorsale-yomenae; and Praon rosaecola. Two Praini species are redescribed. Mesopraon Starý is a new synonym of Areopraon. Areopraon silvestre (Starý) and Areopraon helleni (Starý) are new combinations. A key to the recent Praini genera is given.

Morphological variation in different populations of Aceria anthocoptes (Acari: Eriophyoidea) associated with the Canada thistle, Cirsium arvense, in Serbia

The russet mite, Aceria anthocoptes (Nal.), is the only eriophyid that has been recorded on Canada thistle, Cirsium arvense (L.) Scop. It has been noted in several European countries and recently in the USA. With its apparent host specificity and because of the damage it causes to its host plant, A. anthocoptes is being studied as a potential candidate for classical biological control. The aim of the present study was to examine quantitative morphological traits in four populations of A. anthocoptes living on two infraspecific host plant taxa (C. arvense var. arvense and C. arvense var. vestitum) in two geographically separate areas of Serbia in order to test the hypothesis of absence of the possible host plant impact on mite morphology. MANOVA analysis revealed significant differences between populations from different localities in Serbia. Populations of A. anthocoptes inhabiting two thistle varieties in the vicinity of Belgrade differed significantly from mites inhabiting the same two host varieties in the vicinity of the town of Ivanjica. Canonical discriminant analysis showed that the trait which best discriminates the populations of A. anthocoptes is the number of dorsal annuli. It was not possible to ascribe morphological differences to the impact of the host plant.

Detecting population admixture in honey bees of Serbia

Summary Honey bee workers were sampled across the Serbian territory during 2009–2010 from mostly non-migratory apiaries to determine the population structure of these bees using morphometric, genetic, and spatial information. A total of 134 bees were sampled, of which 77 were analysed using classical wing morphometrics and 122 bees were successfully analysed using 24 DNA microsatellite markers. A combination of methods including multivariate statistics and assignment tests (frequency-based and Bayesian) revealed the honey bees of this region to resemble the subspecies Apis mellifera macedonica, Apis mellifera carnica or hybrids of these two subspecies. Based on Bayesian assignment (‘Structure’) and spatial PCA, honey bees within the Serbian territory were composed of 56%-58% A. m. carnica and 42%-44% A. m. macedonica. Spatial analysis showed the existence of a north-west to south-east cline in genetic differentiation. The bees in the north-west resemble A. m. carnica,while the bees in the south-east of the country are more similar to A. m. macedonica. Thus, the extent of A. m. macedonica within Serbia was greater than previously estimated. We define a line of hybridisation between A. m. carnica and A. m. macedonica within our study area. The cline of differentiation was still evident using a combination of genetic and spatial information, in spite of beekeeping activities including transhumance and breeding efforts.

Phenotypic variability in five Aceria spp. (Acari: Prostigmata: Eriophyoidea) inhabiting Cirsium species (Asteraceae) in Serbia

From about 250 Cirsium spp., only two Aceria spp. (Acari: Eriophyoidea) have been described, Aceria anthocoptes (Nal.) and Aceria cirsii Pet. B. & Shi. Host specificity, which generally characterizes eriophyoid mites, potentially leads to speciation, so we may expect more than two Aceria spp. and/or other infraspecific taxa. Furthermore, studies on host-related variability in the morphology of Aceria mites are generally lacking. The purpose of this study was to investigate quantitative morphological traits of five Aceria populations inhabiting five Cirsium spp. in Serbia. MANOVA analysis revealed significant differences in 23 commonly used morphological traits as well as four additional traits related to the prodorsal shield design. In addition, the most important qualitative traits using scanning electron microphotographs were studied in order to clarify phenotypic differences among five Aceria spp. Discriminant analysis identified eight traits that significantly differentiate five populations. UPGMA cluster analysis of the squared Mahalanobis distances indicates that A. cirsii was morphologically the most divergent, while A. anthocoptes populations from Cirsium arvense and Aceria sp. from Cirsium heterophyllum were isolated from the branch clustering Aceria spp. populations from Cirsium eriophorum and Cirsium creticum. Analysis of qualitative traits using SEM confirmed results obtained from the analysis of morphometric features.

A New Aphidius Species (Hymenoptera: Braconidae: Aphidiinae) from High-Montane Areas of Southeastern Europe

Aphidius montenegrinus sp.n. from Serbia and Montenegro is described and illustrated. It parasitizedAcyrthosiphon daphnidis Ilharco, onDaphne alpina L. The parasitoid was described and illustrated using scanning electron microscope photographs and line drawing. The new species is a member of theAcyrthosiphon aphid parasitoid guild and was collected from Zabojsko Jezero Lake in Montenegro. The parasitoid and the hyperparasitoid spectrum ofA. daphnidis are presented for the first time.

Use of honeybees (Apis mellifera L.) as bioindicators for assessment and source appointment of metal pollution

The ability of honeybees to collect particulate matter (PM) on their bodies makes them outstanding bioindicators. In this study, two cities, Pančevo (PA) and Vršac (VS), South Banat district, Vojvodina, Serbia, were covered with two sampling sites each. The aims of this study were to determine concentrations of Al, Ba, Cd, Co, Cr, Cu, Fe, Mn, Na, Ni, Sr, and Zn in the bodies of honeybees during July and September of 2013, 2014, and 2015 and to analyze their spatial and temporal variations and sources of analyzed elements, as well as to assess pollution levels in the two cities. Significant temporal differences were found for Al, Ba, Cd, Co, Cr, Cu, Fe, Mn, Na, Ni, and Zn. Trend of reduction in metal concentrations in bodies of honeybees during the years was observed. Statistically significant spatial variations were observed for Al, Ba, and Sr, with higher concentrations in VS. PCA and CA analyses were used for the first time to assess sources of metals found in honeybees. These analyses showed two sources of metals. Co, Cd, Na, Fe, Mn, Zn, and partly Cu were contributed to anthropogenic sources, while Ca, Al, Mg, Cr, Ba, Sr, and Ni were contributed to natural sources.