Fluctuating asymmetry (FA) is used in assessing the effect of environmental stress on the development stability of individuals by measuring small random deviations from perfect bilateral symmetry. Here, we checked for FA on two predatory carabid beetles, Pterostichus melas and Poecilus koyi, in order to evaluate species response to agricultural practices within Mediterranean agroecosystems, as well as FA as a method. The samples were collected in vineyards and olive groves, both under integrated pest management (IPM) and ecological pest management (EPM), and in pristine habitats in the Mediterranean region of Croatia. Geometric morphometrics (GMMs) were used to analyze the pronotum and abdomen shape variations and left–right asymmetries of each population. In respect to the FA measurements, analyzed species responded differently, with P. koyi displaying a lower intensity of FA than P. melas. On the other hand, P. melas beetles from vineyards showed a higher intensity of FA compared with populations from pristine habitats and olive groves. Accordingly, FA pointed out olive groves as potentially less adverse habitats to predatory carabids, keeping in mind the different levels of asymmetry between the two species. Our study singled out P.melas as a more suitable species for further research, in the effect that different agricultural practices can have their impact on non-target invertebrates analyzed by measuring the FA.

The Mediterranean region has a high but unevenly studied level of invertebrate diversity. Genetic-based methods, such as DNA barcoding and metabarcoding, are proposed for biodiversity assessment; however, their application is not always straightforward. The current state of data available in genetic databases limits species identification, especially in the case of certain invertebrate groups. The aim of the study was (1) to assess the diversity of seven invertebrate groups, which are potential prey for predatory arthropods, in order to facilitate the analysis of the metabarcoding of trophic interactions and, thus, expand our knowledge on biocontrol potential, and (2) to estimate the representation of local species in BOLD and NCBI GenBank databases. The DNA barcoding results consisting of sequences for 269 specimens, collected in Zadar County, within Mediterranean part of Croatia, were used for species identification and species richness assessment through the comparison of our data with the data available in BOLD and NCBI databases, and by applying species delimitation methods. Previous barcode records enabled Lepidopteran, Hemipteran, Dipteran, and Hymenopteran species identification, while Collembolan and Oligochaetes species numbers were assessed using species delimitation. Our results showed that a high number of species were unique to the study area, especially in the case of Collembolans and Oligochaetes. We confirmed that the studied area is under-researched, which was particularly evident in taxonomically demanding groups, such as Collembolans and Oligochaetes, which are rich in rare endemic species.

Spiders act as one of the major predators among arthropods in agroecosystems and are crucial to the control of pest densities. As they are almost exclusively predators, they have developed a number of hunting strategies, which have consequently impacted their complex trophic interactions. The development of molecular biology methods for environmental DNA analysis has made it easier to analyze such complex trophic networks. In order to ascertain the trophic interactions of spiders, a sampling of spider species was carried out in two olive groves in Zadar County, Croatia, under integrated and ecological pest management. To construct the trophic networks of spiders, we performed DNA metabarcoding. The combined general and spider-excluding primers were able to recognize prey from 12 distinct orders in the guts of the 57 spiders. According to our results, spiders have a complex trophic network that exhibits seasonal and site-specific variations. The results obtained from both sites having different pest management also confirmed that spiders consumed phytophagous insects in the highest ratio, including some important pests, in comparison to other prey and that management and guild type had an impact on the predation of phytophagous insects.

Orthohantaviruses are mainly carried and transmitted by wild rodents, although during the last decade, they have also been identified in multiple species of shrews and moles. Orthohantavirus, Orthohantavirus seewisense (Seewis virus, SWSV), first detected in Switzerland in a single Sorex araneus (Eurasian common shrew) specimen, has been further described in several European countries, including Croatia’s neighboring Slovenia and Hungary. Croatia is a well-known endemic region for several zoonotic agents including three different orthohantaviruses: Orthohantavirus puumalaense (PUUV), Orthohantavirus dobravaense (DOBV), and Orthohantavirus tulaense (TULV). In this study, nine shrews were tested and SWSV RNA was detected in liver, lung, and kidney belonging to two shrews (22.22%), one collected on Medvednica mountain in Zagreb County, and the other in the Stara Gradiška area in lowland Croatia. The phylogenetic analysis of the complete S segment’s open reading frame (ORF) and partial L-segment revealed that the Croatian sequences, when compared to sequences from the adjacent geographic regions, form a specific genetic lineage. Two SWSV-positive shrew species—Sorex araneus and Neomys milleri (Mediterranean water shrew)—were identified using barcode-based sequence analysis. Therefore, the SWSV detection in N. milleri throughout the course of this study is seen as a rare find in this shrew species. To our knowledge, this is the first molecular and phylogenetic analysis of SWSV in Croatia.

Branchiobdellids are small ectosymbiotic annelids living primarily on freshwater crayfish. Because humans frequently translocate crayfish, these symbionts can be co-translocated, with implications for biosecurity and conservation. Despite over a century of study, relationships between European branchiobdellids and their crayfish hosts (basibionts) remain poorly resolved. Therefore, we analysed 215 mitochondrial COI sequences (67 new and 148 from GenBank and BOLD databases) spanning most documented ectosymbiont–basibiont associations. Phylogenetic reconstruction and species delimitation identified seven recognized species and revealed deep intraspecific divergence in five, suggesting the presence of cryptic lineages. Branchiobdella hexadonta and Branchiobdella parasita each contained multiple genetically distinct groups linked to different crayfish species, whereas Branchiobdella astaci and Branchiobdella balcanica showed strong host-specific monophyly. Branchiobdella italica and Branchiobdella pentadonta showed lower divergence, consistent with a younger origin inferred by molecular clock analyses. Divergence times broadly matched crayfish basibiont history, indicating likely co-speciation. Results support an origin of Branchiobdella inhabiting crayfish gill chambers and show that crayfish translocations may blur natural epibiont–basibiont boundaries, thus complicating monitoring of symbiont lineages. This work advances our understanding of co-evolution in freshwater ecosystems and underscores the conservation importance of preserving both native crayfish and their associated symbionts, whose extinction risks may be underestimated due to unresolved taxonomy.