Morphostructural investigation of the female reproductive system and molecular evidence for Wolbachia in Balclutha brevis Lindberg 1954 (Hemiptera, Cicadellidae).

Balclutha brevis Lindberg 1954 (Homoptera, Cicadellidae) is an allochthonous species that is rapidly spreading in Sicily and in mainland Europe due to the wide spread of its host plant and therefore could also compete with populations of native species. Considering these ecological implications, based on the lacking ultrastructural data about the reproductive systems of the Auchenorrhyncha and since previous investigations on the male reproductive system of B. brevis have shown some interesting features, we carried out morphostructural investigations on the female reproductive system of this alien leafhopper. Moreover, given the high interest in literature on Wolbachia entomoparasite and based on our previous studies, we provided a contribution to further investigations in applied sciences. For this aim we performed a molecular analysis on males and females of B. brevis to detect the possible presence of strains of the bacterium known to alter host reproductive biology. The female reproductive system has a morphological organization comparable to the general anatomical features of most of the Auchenorrhyncha species; however, comparing our data with the literature, some considerations are discussed. As for the histological and ultrastructural investigations, our results show a secretory activity of the various examined structures. In the spermatheca of B. brevis, in particular, the secretory activity is more marked in the sac-shaped tract, where histochemical investigations showed a lipid component of the secretion; possible origin of this component is discussed. Moreover, mainly free spermatozoa are found in the sac-shaped tract of the spermatheca and in the common oviduct. As for the latter, an interesting findings is the lack of cuticular intima on the epithelial surface of the common oviduct; furthermore, the observed features and the literature in this regards led us to review the significance of the structure called as spermatheca. The molecular screening study (PCR) with three Wolbachia specific genes (16SrRNA, ftsZ,wsp) strongly supports Wolbachia infections in both males and females of the host species. This is the first study demonstrating the presence of Wolbachia in Balclutha genus, though no morphostructural alteration commonly related to the presence of the bacterium has been found in all the examined specimens.

Morphostructural analysis of the male reproductive system and DNA barcoding in Balclutha brevis Lindberg 1954 (Homoptera, Cicadellidae).

Balclutha brevis Lindberg 1954 is an allochthonous leafhopper infesting an invasive grass, Pennisetum setaceum, in Sicily and in mainland Europe; therefore, this species could compete with populations of native species, thus contributing to the loss of biodiversity. Considering the ecological implications of B. brevis, investigations on all its biological aspects represent, therefore, a premise for further studies in applied sciences. Based on the lacking ultrastructural data about the reproductive systems of the Auchenorrhyncha, we carried out morphostructural investigations on the male reproductive system of B. brevis. Further, a first report of DNA barcoding analysis (amplification and sequencing of Cytochrome Oxidase I gene) has also been performed to characterize B. brevis compared to other congeneric species. From a morphological point of view, the male reproductive system of B. brevis has an organization comparable to the general anatomical features of most of the Auchenorrhyncha species; however, comparing our data with those concerning the different groups of Cicadomorpha, some considerations are discussed. As for the histological and ultrastructural investigations, our results show a secretory activity of the various examined structures, mainly in the lateral ejaculatory ducts and in the accessory glands. The latter, in particular, show morphostructural differences comparing the distal tract to the proximal one; moreover, the histochemical techniques showed the possible presence of a lipid component in the peculiar cytoplasmic granules found in the gland cells. The significance of these findings in the accessory glands is discussed. Finally, the ultrastructural features found in the seminal vesicles are different from those of the lateral ejaculatory ducts and are indicative of the different roles played by these structures in the organization of the spermatozoa bundles.

Comparative pattern of genetic structure in two Mediterranean killifishes Aphanius fasciatus and Aphanius iberus inferred from both mitochondrial and nuclear data.

In this study, genetic variation was assessed in Aphanius fasciatus and Aphanius iberus characterized by similar ecological traits but with very different distribution ranges in the Mediterranean area. Five populations of A. iberus and five of A. fasciatus were analysed using five polymorphic microsatellite loci and partial mitochondrial control region (D-loop) sequences. Congruent results were found with both nuclear and mitochondrial molecular markers. The results showed that similar levels of genetic divergence, based on mitochondrial control region sequences, are present among populations of A. iberus and among populations of A. fasciatus despite the very different geographic distance existing among the examined populations of the two species (low geographic distance in A. iberus and high in A. fasciatus). A possible explanation could be that the populations of A. iberus were isolated for a longer time than the populations of A. fasciatus supporting the hypothesis that the split in the lineage leading to A. iberus is older than the split in the lineage leading to A. fasciatus. The possibility that the wide circum-Mediterranean distribution of A. fasciatus ensures the high connectivity of its populations, preventing, in some cases, local differentiation, however, cannot be ruled out.

Chromosomal mapping and molecular characterization of ribosomal RNA genes in Lebias fasciata (Teleostei, Cyprinodontidae).

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.