A review of the genus Vitrea Fitzinger, 1833 (Gastropoda, Eupulmonata, Pristilomatidae) in Serbia: diversity, distribution and the description of a new species.

In this paper, the genus Vitrea Fitzinger, 1833 in Serbia is reviewed. All previous literature data on this genus from Serbia are summarised and used to discuss its distribution in the country and create distribution maps, supplemented by new material collected by the authors. All Serbian species are figured. For each species, a brief description of the examined specimens, data on previous findings in Serbia, the material (including types) that were analysed, the distribution and habitats in Serbia they inhabit, as well as remarks on specific species are given. A new species, Vitreavirgo Gojsina & Dedov, sp. nov., is described from a pit on Mt. Devica in eastern Serbia. Vitreapygmaea (O. Boettger, 1880) is reported for the first time for the territory of Serbia. As some Vitrea species have a narrow geographical range and prefer certain habitats, they are particularly vulnerable to habitat changes, which is also discussed in the paper. An identification key for all hitherto known Serbian species is given.

Morphology of the pygidial glands and chemical composition of their secretions in two species of tiger beetles (Carabidae: Cicindelinae).

Pygidial glands are a common feature of all adephagans and their products play an important role in defense against predators. The morphology of the pygidial glands and the chemistry of their secretion were studied for the first time in two species of tiger beetles - Cicindela (Cicindela) sylvicola Dejean, 1822 and Cylindera (Cylindera) germanica (Linnaeus, 1758). The glands were examined by both bright-field microscopy and nonlinear microscopy. All morphological structures of the glands were measured and described in detail. The structures mentioned were compared with those of related taxa. The secretion extracts from the pygidial glands of the investigated taxa contained a total of 24 compounds, which were detected by gas chromatography-mass spectrometry (GC-MS). The secretion mixture of C. (C.) germanica was more complex (21 chemicals) than that of C. (C.) sylvicola (11 ones). Benzaldehyde was present in both secretion samples. Hydrocarbons were the most abundant group of secretory compounds. The purpose of the compounds, their distribution within the subfamily Cicindelinae and their effects on the ecology of the group were discussed.

Pygidial glands of the blue ground beetle Carabus intricatus: chemical composition of the secretion and its antimicrobial activity.

Pygidial gland secretions are used as repellent defensive allomones in ground beetles. We provide the first precise data on the chemical composition and antimicrobial potency of the secretion of the blue ground beetle, as well as on the morphology of its pygidial glands. The latter structures were not previously studied chemoecologically and morphologically, and we hypothesized that their secretion may have some antimicrobial action, as is the case with certain Carabus species. Gas chromatography-mass spectrometry (GC-MS) was used to identify methacrylic and angelic acids as dominant chemicals in the secretion from individuals of three populations of the blue ground beetle in Serbia. We tested its secretion against selected strains of medically important microorganisms. The secretion exibits antimicrobial action against certain bacterial species and all tested micromycetes. The most significant antifungal effect of the secretion was against Penicillium ochrochloron, which is more sensitive to the secretion than to commercial antifungal drugs ketoconazole and bifonazole. Bifonazole achieved minimum inhibitory concentrations against Trichoderma viride at more than three times higher value than did the secretion, indicating a significant antifungal effect of the secretion against this micromycete as well. Additionally, we tested commercially available standards of two dominant chemicals in the secretion to investigate their interaction and antimicrobial role in the secretion. Finally, we describe all glandular morpho-functional units of the blue ground beetle. Our results suggest that the secretion of the blue ground beetle may serve not only defensive but also antimicrobial functions, which likely aid the survival of this beetle in the microbial-rich forest litter habitat.

Pygidial glands of three ground beetle taxa (Insecta, Coleoptera, Carabidae): a study on their morphology and chemical composition of their secretions.

Morphology of the pygidial glands and chemical compositions of their secretion were analysed in the adults of three selected ground beetle taxa. Secretions of pygidial glands of Cychrus (Cychrus) semigranosus, Patrobus atrorufus and Pterostichus (Platysma) niger were chemically tested. Additionally, pygidial glands of the latter two species were investigated using bright-field microscopy and nonlinear microscopy and morphological features of the glands were described in detail. Both C. (C.) semigranosus and P. atrorufus were studied for the first time in terms of chemical ecology, while the latter species was analysed for the first time in terms of pygidial gland morphology. Altogether, eight compounds were detected in the dichloromethane extracts of the pygidial gland secretions of the three ground beetle taxa analysed. The simplest secretion mixtures were present in C. (C.) semigranosus and P. atrorufus (with two compounds each), while the extract of P. (P.) niger contained five compounds. The presence of 1-tetradecanol in the secretion of P. (P.) niger represents the first finding of this compound from the pygidial gland secretion extracts of ground beetles.

Chemistry and morphology of the pygidial glands in four Pterostichini ground beetle taxa (Coleoptera: Carabidae: Pterostichinae).

Morphology of the pygidial glands and chemical composition of their secretions in adults of four ground beetle representatives of the Pterostichini tribe (Coleoptera: Carabidae) were analysed. Molops (Stenochoromus) montenegrinus, Pterostichus (Cophosus) cylindricus, P. (Feronidius) melas and P. (Pseudomaseus) nigrita were chemically tested, while the latter three species were morphologically investigated. Pterostichus (C.) cylindricus, P. (P.) nigrita and M. (S.) montenegrinus were chemically studied for the first time. Altogether, 23 chemical compounds were isolated using gas chromatography-mass spectrometry (GC-MS), of which some are new for Pterostichini or even Carabidae. Methacrylic acid was present in all species analysed. It was predominant in the secretion extract of P. (C.) cylindricus and P. (F.) melas. Isobutyric and 2-methylbutyric acids were the major components in the secretion of M. (S.) montenegrinus. Undecane, methacrylic and tiglic acids were the main components in the secretion of P. (P.) nigrita. The simplest chemical mixture was found in P. (C.) cylindricus (two compounds), while the most complex one was detected in P. (P.) nigrita (15 compounds). No significant differences in the chemical composition of the pygidial gland secretions were evidenced in P. (C.) cylindricus sampled from the same area and in the same season in two different years. Morphology of the pygidial glands of the studied species was analysed for the first time. Morphological features of the pygidial glands were observed using bright-field microscopy and nonlinear microscopy and described in details.

Bat guano-dwelling microbes and antimicrobial properties of the pygidial gland secretion of a troglophilic ground beetle against them.

Bat guano is an important source of microbial diversity in caves and can be a source of potential pathogens. Laemostenus (Pristonychus) punctatus is a guanophilic ground beetle species, which pygidial gland secretion exhibits action against pathogenic and other microbes. The distribution and diversity of microbes in bat guano from a karstic cave were determined in this study. Additionally, antimicrobial activity of the pygidial gland secretion of L. (P.) punctatus against guano-dwelling microbes was tested; minimal inhibitory concentration (MIC) and chemical composition of the secretion were analyzed. In total, 63 different bacterial species and 16 fungal morphotypes were isolated from guano samples by the cultivation method and confirmed using and phenotypic characterization and molecular identification. There was a difference in the composition of certain microorganisms between the sampling points (cave locations) and between the guano layers. The largest number of bacterial isolates belongs to the genera Lysinibacillus and Paenibacillus, while Pseudomonas species were highly abundant at the innermost sampling point. For the guanophilic fungi, the majority are ascomycetes, with Penicillium and Aspergillus as the most dominant genera. Meyerozyma guilliermondii was the only yeast species found in the guano samples. The most sensitive isolates were Enterococcus eurekensis (MIC 0.007 mg/mL) and Escherichia fergusonii (MIC 0.028 mg/mL). The most sensitive fungal isolates were M. guilliermondii, Penicillium expansum, and Trichoderma harzianum (MIC 0.15 mg/mL). This study opens a new possibility for better understanding of ecological relations between microorganisms and troglophilic ground beetles and for detailed investigations of morpho-anatomical aspects of pygidial glands.

Sexual size and shape dimorphism in two ground beetle taxa, Carabus (Procrustes) coriaceus cerisyi and C. (Morphocarabus) kollari praecellens (Coleoptera: Carabidae) - A geometric morphometric approach.

We investigated morphometric variation in size and shape of the head, pronotum, and abdomen between the taxa and sexes of two ground beetles, Carabus coriaceus cerisyi Dejean, 1826 and C. kollari praecellens Palliardi, 1825. These two taxa differ in overall size, and both of them are characterized by significant sexual size dimorphism. In many taxa, allometry, the relationship between changes in shape and changes in size, can be a major component of intra- and interspecific variation in body shape. In the present study, we applied landmark-based geometric morphometrics to explore allometric and non-allometric components of shape variation between the taxa and more importantly between sexes within the taxa. We were able to show that the differences of shape between the taxa cannot be explained by allometric changes, as allometry explains only a small amount of total shape variation between the taxa, which was expected due to the fact that the taxa belong to separate subgenera. Between the sexes, on the other hand, allometry contributes largely to the variation, particularly in abdomen shape. However, the differences of head and pronotum shape between the sexes cannot be entirely explained in terms of allometric scaling. Our results indicate that allometry contributes largely to differences of body shape between the sexes, while differences between the taxa are influenced by other factors and processes.

A new troglobitic species of the genus Leptomeson Jeannel, 1924 (Coleoptera: Leiodidae: Cholevinae: Leptodirini) from the Island of Solta (middle Dalmatia, Croatia).

Leptomeson Jeannel, 1924, originally treated as a subgenus of Anthroherpon Reitter, 1889 (Jeannel, 1924), was erected to a distinct genus by Guéorguiev (1990). It currently includes 13 endemic taxa (nine species and four subspecies) (Perreau, 2015), of which five species are recently described (Giachino et al., 2011). All Leptomeson taxa are troglobitic, montane or insular, and are distributed in a narrow Dinaric area in the proximity to the Adriatic Sea coast belonging to Croatia and Bosnia and Herzegovina (Perreau, 2000; Giachino et al., 2011) (Fig. 1).

A new hypogean species of the genus Chaetomargoreicheia Magrini Bulirsch, 2005 (Carabidae: Scaritinae: Clivinini) from Croatia.

Chaetomargoreicheia Magrini Bulirsch, 2005 is a recently established genus of scaritine ground beetles (Bulirsch Guéorguiev, 2008) (treated by certain authors as a subgenus of the genus Reicheadella Reitter, 1913) (Magrini Bulirsch, 2005; Balkenohl, 2017) which currently contains two endogean species inhabiting the Balkan Peninsula: Chaetomargoreicheia zoufali (Reitter, 1913) and C. lakotai (Magrini Bulirsch, 2005) (Balkenohl, 2003, 2017; Magrini Bulirsch, 2005; Bulirsch Guéorguiev, 2008). The aforementioned species are montane and inhabit confined geographic areas (Jeannel, 1957; Magrini Bulirsch, 2005). C. zoufali was found only in the surroundings of the village of Ravno (collected beneath a deep layer of leaf-litter), Mt. Bjelasnica, near Trebinje, E Herzegovina (Bosnia and Herzegovina), while C. lakotai was found at the entrance of a cave nearby a road (collected under a huge stone), in Mt. Lovcen, near Kotor, S Montenegro (Reitter, 1913; Holdhaus, 1924; Jeannel, 1957; Magrini Bulirsch, 2005). Chaetomargoreicheia species represent quite rare, stenoendemic taxa, for each of the up-to-now known species only one specimen has been collected by hand so far (Reitter, 1913; Jeannel, 1957; Magrini Bulirsch, 2005). The fourth author of the current study investigated numerous underground and endogean high-altitude habitats in Dalmatia (Croatia) in the last few years. As a result of the exploration, he has recently collected a small sample of scaritine ground beetles from a cave in S Croatia. After thorough analysis of the sample, we have identified a new Chaetomargoreicheia species.

First report of aphaenopsoid trechines (Coleoptera: Carabidae: Trechini) from Serbia, with descriptions of new taxa.

The trechine ground beetle taxa, Velesaphaenops gen. n., Velesaphaenops tarensis sp. n., and Acheroniotes lethensis sp. n., are described and diagnosed. The new taxa differ clearly from their closest relatives in a number of external characters and the shape of the genitalia. They probably belong to phyletic lineages of Pliocene age (the age of the palaeokarst of Kamena Gora and Mt. Tara). The new taxa are endemic to western and southwestern Serbia. Keys to the aphaenopsoid trechine genera in Serbia and to species of the genus Acheroniotes Lohaj Lakota, 2010 are appended.

Seven new taxa of Leptodirini (Coleoptera: Leiodidae: Cholevinae) from the Balkan Peninsula.

Seven new troglobitic leiodid beetle taxa (six new species and one new subspecies) from several caves and pits in the Balkan Peninsula (Bosnia and Herzegovina and Serbia) are described and diagnosed. Three new species belong to the genus Adelopidius Apfelbeck, 1907, two-to the genus Pholeuonopsis Apfelbeck, 1901, one-to the genus Leonhardia Reitter, 1901, while a new subspecies belongs to the genus Apholeuonus Reitter, 1889. All important morphological characteristics of the new taxa have been mentioned and imaged, including the aspects of the male and female genitalia. The new leiodid taxa are properly distinguished from their relatives and are endemics of the Dinaric mountain chain. A key to Adelopidius taxa is added.

Antifungal activity of the pygidial gland secretion of Laemostenus punctatus (Coleoptera: Carabidae) against cave-dwelling micromycetes.

The antifungal potential of the pygidial gland secretion of the troglophilic ground beetle Laemostenus punctatus from a cave in Southeastern Serbia against cave-dwelling micromycetes, isolated from the same habitat, has been investigated. Eleven collected samples were analyzed and 32 isolates of cave-dwelling fungi were documented. A total of 14 fungal species were identified as members of the genera Aspergillus, Penicillium, Alternaria, Cladosporium, Rhizopus, Trichoderma, Arthrinium, Aureobasidium, Epicoccum, Talaromyces, and Fusarium. Five isolates were selected for testing the antifungal activity of the pygidial gland secretion: Talaromyces duclauxi, Aspergillus brunneouniseriatus, Penicillium sp., Rhizopus stolonifer, and Trichoderma viride. The microdilution method has been applied to detect minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs). The most sensitive isolate was Penicillium sp., while the other isolates demonstrated a high level of resistance to the tested agent. L. punctatus has developed a special mechanism of producing specific compounds that act synergistically within the secretion mixture, which are responsible for the antifungal action against pathogens from the cave. The results open opportunities for further research in the field of ground beetle defense against pathogens, which could have an important application in human medicine, in addition to the environmental impact, primarily.

The pygidial gland secretion of the forest caterpillar hunter, Calosoma (Calosoma) sycophanta: the antimicrobial properties against human pathogens.

Recently, various insect secretions have been tested as possible antimicrobial agents. In beetles, these secretions are essentially products of various exocrine glands, of which particular emphasis is on pygidial glands that are common for the suborder Adephaga. The antimicrobial potential of the pygidial gland secretion isolated from the adults of Calosoma sycophanta against human pathogens has been tested and compared with the potential of other carabid species, particularly within the tribe Carabini. The antimicrobial assay includes a microdilution method which was applied in order to determine the minimal inhibitory, minimal fungicidal and minimal bactericidal concentrations. It has been tested the effect of the secretion against eight strains of fungal and eight strains of bacterial species. The secretion sample of the tested carabid species has shown statistically significant antifungal effect against all strains of treated micromycetes, the highest in comparison with previously tested carabids (Carabus spp., Laemostenus punctatus and Abax parallelepipedus). Aspergillus versicolor proved to be the most sensitive micromycete, while the remaining seven fungal strains have shown the same level of sensibility. In comparison with commercial mycotics ketoconazole and bifonazole, applied as positive controls, the tested secretion showed much higher antifungal activity for all fungal strains. Antibacterial effect has been manifested only against one bacterial strain (Escherichia coli), contrary to other previously studied carabid species. These observations might have a significant impact on the ecological domain and possible purpose in biomedical studies and applications in the future. Additionally, morpho-histology of the pygidial glands of C. sycophanta is investigated and discussed.

Antimicrobial activity of the pygidial gland secretion of three ground beetle species (Insecta: Coleoptera: Carabidae).

The antimicrobial properties of the pygidial gland secretions released by the adults of the three ground beetle species, Carabus ullrichii, C. coriaceus, and Abax parallelepipedus, have been tested. Microdilution method was applied for detection of minimal inhibitory concentrations (MICs), minimal bactericidal concentrations (MBCs), and minimal fungicidal concentrations (MFCs). Additionally, morpho-histology of the pygidial glands is investigated. We have tested 16 laboratory and clinical strains of human pathogens--eight bacterial both gram-positive and gram-negative species and eight fungal species. The pygidial secretion samples of C. ullrichii have showed the strongest antimicrobial effect against all strains of treated bacteria and fungi. Staphylococcus aureus, Lysteria monocytogenes, and Salmonella typhimurium proved to be the most sensitive bacterial strains. Penicillium funiculosum proved to be the most sensitive micromycete, while P. ochrochloron and P. verrucosum var. cyclopium the most resistant micromycetes. The pygidial secretion of C. coriaceus has showed antibacterial potential solely against Pseudomonas aeruginosa and antifungal activity against Aspergillus fumigatus, A. versicolor, A. ochraceus, and P. ochrochloron. Antibacterial properties of pygidial gland secretion of A. parallelepipedus were achieved against P. aeruginosa, while antifungal activity was detected against five of the eight tested micromycetes (A. fumigatus, A. versicolor, A. ochraceus, Trichoderma viride, and P. verrucosum var. cyclopium). Commercial antibiotics Streptomycin and Ampicillin and mycotics Ketoconazole and Bifonazole, applied as the positive controls, showed higher antibacterial/antifungal properties for all bacterial and fungal strains. The results of this observation might have a significant impact on the environmental aspects and possible medical purpose in the future.

Two new high-altitude subspecies of Tapinopterus (Tapinopterus) cognatus (Dejean, 1831) (Coleoptera: Carabidae: Pterostichinae) from Serbia.

The genus Tapinopterus Schaum, 1858 currently comprises around 60 species belonging to eight subgenera, inhabiting mostly the Balkan Peninsula and Asia Minor (Schatzmayr 1942, 1943; Bousquet 2003; Curcic et al. 2008; Arndt et al. 2011; Lohaj et al. 2012). The taxa live in limited geographic areas and are mostly montane (Trautner & Geigenmüller 1987; Guéorguiev & Guéorguiev 1998). The territory of Serbia is inhabited by two species of Tapinopterus, T. (T.) cognatus (Dejean, 1831) (recorded in Bulgaria and Romania as well) and T. (T.) miridita (Apfelbeck, 1904) (recorded in Albania and the Republic of Macedonia as well), as well as by the three subspecies, T. (T.) cognatus cognatus (Dejean, 1831) (known from Mt. Suva Planina in Serbia and from Bulgaria), T. (T.) cognatus winkleri Mandl, 1936 (known from the Svrljiske Planine Mts. and Mt. Stara Planina in both Serbia and Bulgaria), and T. (T.) miridita miridita (Apfelbeck, 1904) (known from Mt. Sar Planina in both Serbia and the Republic of Macedonia), respectively (Mandl 1936; Schatzmayr 1942, 1943; Guéorguiev & Guéorguiev 1995; Drovenik & Peks 1999; Bousquet 2003; Curcic et al. 2007; Guéorguiev & Giachino 2008). In our opinion, T. (T.) cognatus cognatus does not live in Bulgaria (as was previously reported) (Bousquet 2003), while the older Schatzmayr's (1943) record "Klisura" should refer to the Iron Gate Gorge in SW Romania and NE Serbia, not to any Bulgarian place. The other mentioned locality, i.e., Mt. Suva Planina in Serbia, is situated far from the type locality (Iron Gate). Since nearly all Tapinopterus taxa are regional or local endemics, this southernmost record in SE Serbia for T. cognatus cognatus seems unlikely. In view of the high diversity of Tapinopterus on the Balkan Peninsula, it is expected that new taxa exist in border areas of the distribution in Serbia. During the last years, the first (SC), second (NI) and fourth author (DA) of the paper investigated a number of epigean montane habitats in SE and C Serbia in order to confirm such an assumption, by collecting a series of ground beetles at high altitude sites. After careful examination of the samples, we identified two Tapinopterus subspecies new to science.

The genus Omphreus in Bosnia and Herzegovina and Montenegro, with two new subspecies of O.morio (Coleoptera, Carabidae, Omphreini).

Two new ground beetle subspecies, Omphreus (Omphreus) moriosandelissp. n. (from Mts. Zelengora and Maglic, eastern Bosnia and Herzegovina) and Omphreus (Omphreus) moriodurmitorensisssp. n. (from Mt. Durmitor, northwestern Montenegro) are here described and diagnosed. The male and female genitalia and other taxonomically important characters are illustrated. The new taxa are distinctly different from the nearest relatives and represent both endemics and relicts inhabiting limited high-altitude Dinaric areas in Bosnia and Herzegovina and Montenegro. A key to Omphreus taxa from Montenegro and a key to subspecies of Omphreus (Omphreus) morio Dejean, 1828 are presented.

Molecular Diversity of Compounds from Pygidial Gland Secretions of Cave-Dwelling Ground Beetles: The First Evidence.

Three adult cave-dwelling ground beetle species were induced to discharge secretions of their pygidial glands into vials. Dichloromethane extraction was used to obtain the secretions. In total, 42 compounds were identified by GC/MS analysis. Pheggomisetes ninae contained 32 glandular compounds, Laemostenus (Pristonychus) punctatus 13, whereas Duvalius (Paraduvalius) milutini had nine compounds. Caproic, oleic, palmitic, and stearic acids were present in the samples of all analyzed species. Undecane was predominant in the extract of L. punctatus. Palmitic acid was the major component in the secretion of D. milutini. Finally, the most abundant compounds in P. ninae secretion were heptacosene and nonacosadienes. Herein, we present the first data on the identification of pygidial gland secretion components in both troglophilous and troglobite cave-dwelling ground beetles. Some compounds are reported for the first time in the secretions of ground beetles and other higher or lower taxa. The adaptation to underground life has not led to a reduction or changes in the chemical defense mechanism in the analyzed troglophilous and troglobitic Platyninae and Trechinae taxa.

A new troglobitic species of the genus Pholeuonopsis (Coleoptera: Leiodidae: Cholevinae: Leptodirini) from western Serbia, with a key to the species from Serbia.

A new leptodirine leiodid beetle species, Pholeuonopsis (Pholeuonopsis) sljivovicensis sp. n., from a cave in western Serbia is described and diagnosed. The views of both male and female genitalia and other taxonomically important characters are imaged. The new species is clearly distinct from the closest relatives. It probably belongs to an old phyletic lineage of Mesogeid origin, like other known Pholeuonopsis taxa from the western part of the Balkan Peninsula. The new species is both an endemic and a relict inhabiting solely western Serbia. Morphological comparisons among the Serbian Pholeuonopsis species are provided, together with a key to the species. The genus Serbopholeuonopsis B. Curcic & Boskova, 2002 is regarded as a junior synonym of the genus Pholeuonopsis Apfelbeck, 1901.