Aleksandra Penezić

Cryptic biodiversity presents an ongoing challenge to the accuracy and effectiveness of biodiversity assessment and conservation. To address this, we developed a non-lethal genetic monitoring framework for the subterranean rodent, European lesser blind mole rat (BMR), Nannospalax leucodon species complex, which encompasses multiple chromosomally diversified and reproductively isolated cryptic-species and subspecies. Our workflow combines species and sex detection through: karyotyping, Inter-Simple Sequence Repeat (ISSR) PCR profiling, and Sry-based sex determination of 33 individual samples from five BMR cryptic-species collected at 25 localities in Serbia. As conventional karyotyping protocols for BMR required animal sacrifice, we developed the first non-lethal fibroblast culture-based karyotyping approach from BMR skin biopsy, producing high-quality metaphase chromosomes across five cryptic-species. Among twelve tested ISSR primers, three yielded reproducible, species-specific DNA profiles that resolved four of the five cryptic-species. The Sry assay accurately determined the sex of all examined individuals. Our findings demonstrate that integrating fibroblast culture-based karyotyping, with fast, cost-effective ISSR-PCR species identification, and Sry-based sex determination, provides a reliable approach for identifying and monitoring cryptic BMR species without sacrificing individuals. This framework has potential applications in conservation programmes and contributes to an integrative taxonomy approach essential for the study and protection of other cryptic taxa.

Among the many pathways through which human activity can shape biodiversity patterns, modification of biotic interactions tends to be overlooked. Here we show how interacting anthropogenic, biotic and abiotic factors drive the continental-scale expansion of a highly adaptive mesocarnivore, the golden jackal. Using a dataset of playback surveys conducted across Europe, we find that the presence of an apex predator, the grey wolf, is the primary factor limiting jackal occurrence. However, a human presence acts as a shield that considerably reduces the strength of wolf suppression and provides jackals refuge near people. Our fitted distribution model predicts that jackals have the potential to expand until they occupy 75% of the continent, almost six times larger than their current range. The ongoing recovery of wolves in Europe could reduce suitable areas for expanding jackals by up to 18% in the near future, but a persistent human-shield effect, coupled with climate warming and landscape modification, is expected to sustain continued jackal expansion across the continent. Our results illustrate how cascading anthropogenic impacts can hamper the ecological benefits provided by apex predator recovery.

In Serbia, human echinococcosis cases are predominantly diagnosed as the cystic form, caused by Echinococcus granulosus sensu lato (s. l.) complex tapeworms. Alveolar echinococcosis, a more severe disease caused by Echinococcus multilocularis, emerged recently. Domestic dogs have been implicated in the transmission of E. granulosus sensu stricto G1/3 to humans. In an ongoing screening of definitive hosts for the presence of Echinococcus spp. to discover reservoirs of E. multilocularis, fecal samples of hunting (n = 29) and stray dogs (n = 66) were examined. Taeniid eggs were collected using flotation and mesh filtration. The presence of Cyclophyllidea, E. granulosus s. l. and E. multilocularis DNA was detected by specific PCR assays. In total, 60% of the fecal samples contained cyclophyllidean DNA, while Echinococcus spp. DNA was detected in 7.4% of the samples. E. canadensis G6–8,10 DNA was present in 4.2% and E. multilocularis in 3.2% of the samples. E. multilocularis DNA was detected only in stray dog feces, suggesting that strays are a reservoir and possibly an important link in the transmission chain. To the best of our knowledge, this study provides the first molecular identification of Echinococcus spp. in dogs in Serbia.

Purpose The knowledge of heartworm disease in free ranging wild canid populations is limited. As it is very difficult to monitor this disease in live animals, sporadic findings are mostly obtained by examining culled individuals of game species. Methods As a part of a broader study on jackal (Canis aureus) ecology in Serbia, the necropsy of legally hunted animals was performed in 2020. Results Two cases of heartworm infection with unusually located adult heartworms were diagnosed. The first case is an adult female jackal harvested in the vicinity of the capital city of Belgrade. Three adult specimens of Dirofilaria immitis were found in the right atrium of the heart, and nine adult specimens were located at an atypical site, in the vena cava caudalis. Very similar, the second case was in an adult female jackal harvested in the vicinity of Bački Monoštor. In this case, six adult specimens of D. immitis were found in the right atrium of the heart and six more adult specimens in the vena cava caudalis. Conclusion Although this nematode generally resides in the pulmonary arteries and the right ventricle of the heart, rare findings in other large blood vessels can occur. To the best of our knowledge, these are the first and only two confirmed cases of adult D. immitis found in vena cava caudalis in jackals in Europe.

The body condition of three autochthonous canid species in Serbia (wolf- Canis lupus, jackal-Canis aureus, and fox-Vulpes vulpes) was assessed based on the value of Kidney Fat Index (KFI). A total of 1232 specimens (147 wolves, 711 jackals, and 375 foxes) were collected during a ten years period. Data were analyzed according to multiple factors such as sex, age, seasons, species, and genus. The results indicate that the adult members of the Canis genus have a significantly better body conditional status than the yearlings as well as the representatives of the Vulpes genus (F (2, 900) = 71.465, p = 0.000). Significant differences in KFI across seasons are recorded in jackal (F (3, 542) = 6.912, p < 0.001) and fox (F (3, 371) = 2.675, p < 0.05) populations. Jackals have the highest KFI during winter when females are in significantly better body condition than males. None of these three canid species has shown a significant difference in body condition between sexes, although females had slightly higher KFI. Among adult female jackals, a difference in KFI between nursing ones and other adult and pregnant females is observed (H (2, n = 220) = 8.339, p = 0.016). Food deprivation which occurs naturally during the winter months does not affect the body conditional status of these predators which are well prepared for the upcoming breeding season. Considered as a whole, in spite of the differences observed at the genus level, the body condition status of these autochthonous canids measured through KFI index indicates decent average fitness status in wolves, jackals, and foxes in Serbia.

Dirofilaria immitis causes a severe and life-threatening cardio-pulmonary disease in Europe, Asia, Africa, and the Americas; also, zoonotic infections have been reported. This parasitic vector-borne disease is mostly common in domestic and wild canids and felids. Here, we present a rare finding of this roundworm in the heart of a Eurasian otter (Lutra lutra). Four mature specimens of D. immitis were found in the right ventricle of the heart. Necropsy was performed after the animal was found road-killed in Eastern Serbia, close to the Danube River. Two heartworms were females, one was male and sex for one remained unidentified. Microfilariae were present in the uterus of one of the female heartworms, indicating that otters can act as reservoirs of dirofilariasis in the wild. This is the first report of a patent infection in a free-ranging otter.

Golden jackal populations in the Balkans are increasing and spreading its range. Nowadays vagrant specimens are reaching the northern parts of Europe. As one of the prerequisites for actual population increase is a solid food base, we examined the feeding habits of adult and juvenile jackals in Serbia in the period of cub dependency stage. Stomach content of 49 adults and 28 cubs from period July-October were analyzed. Juvenile jackals had less diverse diet than the adults, represented with eight food categories. During this part of the year, jackals of both age categories were mostly consuming small mammals. Moreover, they were the main prey category in the diet of juveniles (49.82 %B and 32.08 %O). No game species were recorded in the diet of juvenile jackals. During their first summer and autumn, jackal cubs already manifest the opportunistic feeding behavior. They take advantage of easily accessible food sources such as fruit and slaughter leftovers from domestic ungulates and poultry.

Heartworm (Dirofilaria immitis) is a parasitic roundworm that causes a zoonotic disease known as dirofilariosis. Little is known about the role of wild carnivores serving as reservoirs in nature. Therefore, we examined 738 hearts and lungs of free ranging wild carnivores from Serbia to determine the presence of adult heartworms. During the period 2009–2013, the prevalence in golden jackals (Canis aureus) was 7.32 %, in red foxes (Vulpes vulpes) 1.55 %, in wolves (Canis lupus) 1.43 %, and in wild cats (Felis silvestris) 7.69 %. No adult heartworm specimens were found in beech martens (Martes foina), stone martens (Martes martes), European polecats (Mustela putorius), badgers (Meles meles) or otter (Lutra lutra). The highest recorded prevalence was in 2013 (7.30 %) and the lowest in 2012 (1.6 %). In jackals, the prevalence was higher in males (10 %) than in females (4.06 %), while in foxes the prevalence was 1.75 % in males and 1.26 % in females. The most infected host was a wolf in which 37 adult specimens were found. Because of the potentially significant role in the life cycle of D. immitis, populations of wild carnivores in Europe should be further examined and tested for heartworm infections.