Genetic identification of new alien pest species Illinoia liriodendri and its parasitoid Areopraon silvestre in Croatia

Background and Purpose: During June 2015 in Zagreb city area (Croatia) samples of tulip tree (Liriodendron tulipifera) leaves were collected with symptoms of attack by some unknown aphid. Material and methods: Aphids were collected form leaves of tulip trees on different locations in Zagreb during July 2015. Total genomic DNA was extracted from ethanol-preserved specimens. PCR analysis was carried out and PCR products were purified from 1% agarose gel for sequencing purposes. The obtained sequences were deposited in GenBank. Results: The collected aphids were genetically determined as Illinoia liriodendri (Hemiptera, Aphididae), the North American invasive pest species. Although present in several neighboring countries on tulip trees in urban environment, this research presents the first record of Illinoia liriodendri for Croatia, confirmed on the genetic level. Interestingly enough, during genetic determination of tulip tree aphid pest, another DNA, one of parasitoid Areopraon silvestre (Hymenoptera, Braconidae) was also found in the collected samples. A. silvestere is a native European specialized solitary endoparasitoid of aphids. Conclusions: The presence of alien pest species Illinoia liriodendri was successfully determined via genetic identification. Also, genetic identification of parasitoid species on Illinoia liriodendri, Areopraon silvestre, shows the quick establishment of natural regulation of new pest species in Croatia

Preliminarni rezultati istraživanja entomofaune sjemena poljskog (Fraxinus angustifolia Vahl) i pensilvanskog jasena (Fraxinus pennsylvanica Marshall) u Hrvatskoj

Native narrow-leaved ash (Fraxinus angustifolia Vahl) and introduced green ash (F. pennsylvanica Marshall) are two most common ash (Fraxinus spp.) species in Croatian lowland forests, taking a significant place in these ecosystems. In the recent past it has been observed that narrow-leaved ash seed yields have reduced, emphasising the importance of good health status of those collected, as they are used for seedling production and consecutive regeneration of forest stands or afforestation. Given that insects could be an important factor affecting ash seed health status, the aim of this research was to gain insight into the species being present and their frequency in the ash seeds. In total 2.500 narrow-leaved ash and 1.000 green ash seeds from seven different locations were screened for insect presence. Analysed seeds were categorized as undamaged, insect damaged or empty, and observed insect specimens were identified by morphological and molecular genetic analysis. Green ash seeds revealed higher insect infestation in comparison with native ash species, with weevil Lygniodes bischoffii being the most numerous pest found. Narrow-leaved ash seeds were mostly occupied by the native weevil species, L. enucleator. Besides these pests, larval, pupal, and adult stages of parasitoid wasps (Hymenoptera) and larval stage of the seed eating moth Pseudargyrotoza conwagana (Lepidoptera) were found in both ash species. However, given that pests detected in this study prefer urban areas or were present in a very small number of seeds, where they were often followed by parasitoids, it can be concluded that insects currently do not have a significant negative impact on the reproduction of narrow-leaved ash and green ash in forest stands.Autohtoni poljski jasen (Fraxinus angustifolia Vahl) i alohtoni pensilvanski jasen (F. pennsylvanica Marshall) su dvije najčešće vrste roda Fraxinus prisutne u nizinskim šumama Hrvatske, zbog čega zauzimaju važno mjesto u ovim šumskim ekosustavima. Posljednjih godina je zapaženo smanjenje uroda sjemena poljskog jasena, naglašavajući važnost dobrog zdravstvenog stanja sakupljenih količina, jer se sjeme koristi za proizvodnju sadnica koje se dalje upotrebljavaju za pomlađivanje šumskih sastojina ili pošumljavanje. Kako kukci mogu imati velik utjecaj na zdravstveno stanje sjemena, cilj ovog istraživanja je bio dobiti uvid u prisutne vrste i njihovu učestalost u sjemenu jasena. Prisutnost kukaca je analizirana u ukupno 2.500 sjemenki poljskoga i 1.000 sjemenki pensilvanskog jasena. Promatrano sjeme je kategorizirano kao neoštećeno, oštećeno i prazno (šturo), a nađene jedinke kukaca su identificirane na temelju morfoloških karakteristika i korištenjem molekularno genetičke analize. Sjeme pensilvanskog jasena pokazalo je veću oštećenost kukcima u usporedbi sa sjemenom poljskoga jasena, pri čemu je pipa Lygniodes bischoffii bila najčešći utvrđeni štetnik. Oštećeno sjeme poljskog jasena je najčešće bilo nastanjeno autohtonom vrstom pipe, L. enucleator. Osim navedenih štetnika, u sjemenu obje vrste jasena su nađene ličinke, kukuljice i imaga parazitoidnih osa (Hymenoptera) te ličinke štetnika Pseudargyrotoza conwagana (Lepidoptera). S obzirom da štetnici utvrđeni u istraživanju preferiraju urbana područja ili su nađeni na vrlo malom broju sjemena, često zajedno sa parazitoidima, može se zaključiti kako kukci trenutno nemaju značajan negativan utjecaj na zdravstveno stanje sjemena poljskog i pensilvanskog jasena u šumskim sastojinama

Comparison of molecular and morphological systematics of Carabus species (Coleoptera: Carabidae) with special emphasis on species from Dinaric karst

Background and Purpose: Despite morphological and molecular data analysed so far, phylogenetic relationships of many lineages of genus Carabus Linnaeus 1758 (Carabini, Carabinae, Carabidae) present in Europe, have not been yet fully understood and molecular data have not been fully integrated with the morphological classifications. The aim of this research was to: (i) complement the phylogenetic relationships in the systematics of carabids within the genus Carabus with endemic species from Dinaric karst not included so far in molecular systematics research, and to (ii) examine the degree of matching of the molecular data with the existing morphological classifications based on the structure of frontal shields of larvae and the structure of the endophallus in adult specimens. Materials and Methods: In this research, phylogenetic relationships between 31 species from genus Carabus were analysed. Analyses were based on the DNA sequences of mitochondrial gene for cytochrome c oxidase subunit I. For phylogenetic inference maximum likelihood and Bayesian analysis methods were used. Results: The obtained results showed a greater concordance of molecular data with the classifications based on the structure of endophallus than with the classification based on structure of frontal shields of larvae. The results mainly corresponded to the phylograms in the previous studies confirming the taxonomic status of some species. For three species, Carabus creutzeri and C. parreyssi alpine-dinaric endemic species and C. ulrichi, all included for the first time in molecular analysis, results indicated taxonomic congruence of molecular data with the classification based on the structure of endophallus. Conclusions: Systematic categories within the genus Carabus cannot be based only on the structure of the endophallus, but the results of molecular analysis should also be included. As the topology of several groups still remains uncertain, molecular phylogeny requires further investigations with a larger data set and additional molecular markers. Carabus species from the western part of Balkan Peninsula, mainly endemics, have been under-represented in hitherto molecular systematic analyses. Further studies, including their distribution and ecology as well as studies including other Balkan’s endemic species are required to explain speciation events. This study contributes to the Barcode of Life Initiative

Određivanj prisutnosti ekotoksikološki važnih Pgp i MRP-proteina u šarankama na genskoj i proteinskoj razini

One of the most intriguing defence strategies which aquatic organisms developed through evolution is multixenobiotic resistance (MXR). The key mediators of MXR activity are ATP-binding cassette (ABC) transport proteins. They provide resistance of aquatic organisms by binding xenobiotics and extruding them from cells in an energy-dependent manner. Since Cyprinid fi sh species are common target in freshwater biomonitoring programs, we have studied the presence of two main MDR/MXR effl ux transporters Pglycoprotein (Pgp, Abcb1) and MRP-like protein(s) (Abcc) in the liver of fi ve Cyprinid species: common carp, European chub, sneep, barbel, and silver prussian carp. Their presence was evaluated on the mRNA and protein level. Various pairs of primers were designed to clone homologous fragments of MXR-related genes. At the protein level, we used Western blotting with specifi c monoclonal antibodies against human Pgp (Abcb1, Ab C219), MRP1 (Abcc1; Ab MRPm6) or MRP2 (Abcc2; Ab M2I-4). Transcripts of both key types of MXR transporters were identifi ed in all species examined and here we provide the phylogenetic analysis of new partial sequences. Immunochemical determinations with mammalian antibodies failed to identify the presence of MRP(s), but Pgp expression was found in all fi ve Cyprinid species. These results support that MXR is a defence system mediated by both Pgp and MRP types of ABC transport proteins.Mehanizam multiksenobiotičke otpornosti (MXR) stanični je obrambeni sustav odgovoran za svojstvo istovremene otpornosti na različite ksenobiotike, koje se očituje smanjenjem akumulacije, odnosno povećanjem izbacivanja potencijalno toksičnih tvari iz stanica vodenih organizama. MXR-mehanizam pokazuje odlike analogne fenomenu istovremene otpornosti na različite lijekove (engl. multidrug resistance, MDR) prvi put dokazanom u tumorskim stanicama. Posredovan je istim ABC transportnim proteinima kao i MDR. Istraživanja vezana uz MXR vodenih životinja uglavnom su imala težište na određivanju prisutnosti i funkcije P-glikoproteina (Pgp). Budući da se ribe iz reda šaranki često rabe u biomonitoringu slatkovodnih voda, cilj ovog istraživanja bilo je određivanje prisutnosti dvaju glavnih MDR/MXR-tipova proteina - Pgp i MRP-tip proteina - u jetri iz pet vrsta šaranki; šarana (Cyprinus carpio), klena (Sqalius cephalus), mrene (Barbus barbus), babuške (Carassius auratus gibelio) i podusta (Chondrostoma nasus). Njihova prisutnost utvrđena je na razini mRNA te na proteinskoj razini. Različiti parovi početnica dizajnirani su kako bi se identifi cirali homologni fragmenti gena sličnih MXR-u. Detekcija na razini proteina napravljena je putem Western blot analize s pomoću specifi čnih monoklonskih protutijela proizvedenih da prepoznaju konzervirane epitope; Pgp (C219), MRP1 (MRPm6) ili MRP2 (M2I-4) u sisavaca. Transkripti obaju ključnih MXR-transportera identifi cirani su u svim jedinkama, a napravljena je i fi logenetska analiza dobivenih sekvenci. Imunokemijskom detekcijom s protutijelima sisavaca nismo uspjeli detektirati prisutnost MRP-proteina, dok je Pgp-ekspresija potvrđena u svih pet vrsta šaranki. Nove spoznaje da je za MXRmehanizam zaslužno više transportnih proteina zasigurno će pridonijeti potpunijoj karakterizaciji MXR-a kao integralnog dijela detoksikacijskog, odnosno temeljnog obrambenog sustava vodenih organizama te njegovoj znanstvenoj afi rmaciji kao vjerodostojnog pokazatelja kvalitete okoliša

Phylogeny of cave-dwelling atyid shrimp Troglocaris in the Dinaric Karst based on sequences of three mitochondrial genes

Background and Purpose: The main purposes of this study was to revise the current taxonomy of the genus Troglocaris in the Dinarids in the light of molecular phylogenetic results from three major areas of disjunct distribution (Southern France, West Dinarids, and West Caucasus), and additionally to test the subfamily relationships between Paratyinae and Typhlatyinae in the Dinarids. Materials and Methods: This study was performed on populations of the cave-dwelling shrimp Troglocaris from three disjunct areas of distribution: Southern France, Dinaric Karst and Western Caucasus using mitochondrial genes for 16S rRNA, cytochrome oxidase I and cytochrome oxidase II subunits.We combined mitochondrial data from shrimp populations to clarify the evolutionary relationship inside (within) the genus Troglocaris. Results: Our results, based on phylogenetic analysis of three mitochondrial genes from 14 populations of the closely related atyid taxa, do not support the monophyly of the genus Troglocaris. Moreover, new insights were introduced in the Atyidae subfamily status. At subfamily level, a difference in current taxonomy was observed which excluded the genus Spelaeocaris from Typhlatyinae and placed it inside Paratyinae. Additionally the closest relative to the French species Troglocaris inermis appears to be the surface-dwelling shrimp Atyaephyra desmaresti. Conclusions: The separation of the oldest Western Troglocaris lineage from the Dinaro-Caucasian lineages is estimated to have occurred in the Late Miocene. Also DNA sequence data suggest that Troglocaris hercegovinensis from South Herzegovina and Troglocaris kutaissiana complex from Western Caucasus are sister species

Wide-nosed pygmy grasshoppers (Cladonotinae: Cladonotini, Xerophyllini) of India and Sri Lanka: catalogue with an identification key and description of a new species of the genus Tettilobus

Bhaskar, Dhaneesh, Stermšek, Sara, Easa, P.S., Franjević, Damjan, Skejo, Josip (2020): Wide-nosed pygmy grasshoppers (Cladonotinae: Cladonotini, Xerophyllini) of India and Sri Lanka: catalogue with an identification key and description of a new species of the genus Tettilobus. Zootaxa 4894 (3): 474-500, DOI: https://doi.org/10.11646/zootaxa.4894.3.1

Lepocranus and Valalyllum gen. nov. (Orthoptera, Tetrigidae, Cladonotinae), endangered Malagasy dead-leaf-like grasshoppers

Only two leaf-like pygmy grasshopper species and specimens are known from Madagascar: the Leatherback Pygmy Grasshopper (Lepocranus fuscus Devriese, 1991) —which has a relatively low median carina of the pronotum; and the Malagasy Litterhopper (Valalyllum folium gen. et. sp. nov.), herein described — which has a high median carina. Lepocranus fuscus is known from the rainforests around Tampolo, Manakambahiny, and Mahavelona (Foulpointe). The new taxon, Valalyllum folium gen. et. sp. nov. is known only from the Belanono forest. Both species inhabit northeastern Madagascar. The new species could be rare or not-easy-to-spot in the rainforest leaf litter, where it most probably lives. A new tribe, Valalyllini trib. nov., is described for the two mentioned genera because its members are different from the Caribbean leaf-like Choriphyllini Cadena-Castañeda & Silva, 2019, from the African leaf-like Xerophyllini Günther, 1979, and from the Asian leaf-like Cladonotini Bolívar, 1887. A tabular key to the tribes of Cladonotinae with leaf-like representatives is provided, together with photographs of type specimens of both species belonging to the newly described tribe. The holotype of the new species belongs to the Muséum national d’Histoire naturelle Orthoptera collection, Paris