First finding of spirurid larva (Chromadorea, Spirurida) in the common European viper Vipera berus (Linnaeus, 1758) of the Russian fauna

The National park “Smolny” (Republic of Mordovia, Russia) is located in the Basin of Alatyr River, a second-order tributary of the Volga River. Favorable conditions are created for the development of helminths and infecting reptiles with them in floodplain biotopes. In 2018–2020 in this Protected Area we examined 25 individuals of the common viper Vipera berus (Linnaeus, 1758) by the method of complete helminthological dissection. We studied only road-killed vipers and reptiles killed by local residents. In total, eight species of helminths were registered in vipers: Leptophallus nigrovenosus, Telorchis assula, Alaria alata, larvae, Rhabdias fuscovenosa, Oswaldocruzia filiformis, Physocephalus sexalatus, larvae, Physaloptera clausa, larvae and Agamospirura minuta, larvae. Larva of spirurid nematodes Ph. sexalatus, Ph. clausa and A. minuta were recorded in the common viper for the first time in Russia. We found these nematode juveniles in walls and lining of stomach and intestine of snakes. We gave original drawings and morphological descriptions of these nematodes. Vipera berus was noted as a new host for Physaloptera clausa. Currently the helminth fauna of Vipera berus of the Russian fauna includes 15 species. Helminth infection of Vipera berus strongly depends on habitat and, especially, on the diet features. Snakes that live in dry habitats and feed on mouse-like rodents, as a rule, are weakly infested by helminths. Conversely, helminth infection is higher in vipers living in near-water habitats and feeding tailless amphibians. Participation of paratenic hosts in the helminth life cycles plays an important role in the distribution and preservation of parasites in the wild, and increases the invasion probability of the final hosts.


Introduction
The National Park "Smolny" (Republic of Mordovia, Russia) is located in the Alatyr River Basin, a second-order tributary of the Volga River. The Alatyr River traverses through Nizhny Novgorod Oblast, the Republic of Mordovia, the Republic of Chuvashia and flows into the Sura River [1]. There are many small streams, rivers, ponds and lakes in the Alatyr basin. The complex structure of floodplain biotopes contributes to the development of species richness and abundance of invertebrates intermediate hosts of helminths with a complex life cycle. On the other hand, favorable conditions are created in moist near-water habitats for the development of helminths with direct life cycle.
The purpose of this study is to describe the larva of spirurid nematodes, found in Vipera berus of the Russian fauna for the first time.

Materials and Methods
We examined 25 specimens of Vipera berus by the method of complete helminthological dissection (Skryabin, 1928). In this work, we studied snakes that died on the roads passing through the National park "Smolny" and reptiles killed by local residents in 2018-2020. The helminth fauna of Vipera berus we studied in six sites of the National park (figure 1). Snake dissection, fixation and handling of helminths were conducted according to standard techniques [17][18][19]. Collected nematodes were killed by heating in water, clarified in lactic acid and mounted in Glycerin-Jelly. Nematode identification was made according to Sharpilo [11]. Parasite taxonomy is provided according to the Fauna Europaea (http://www.fauna-eu.org/). Morphological descriptions and original drawings of nematode juveniles were given. Drawings of viper parasites were made using MBI-9 light microscope with drawing tube RA-7. Nematode and viper specimens were deposited in the helminth and reptile collections of Institute of Ecology of Volga River Basin of RAS (Togliatti).   divided into two parts: the muscular one, 0.083-0.102 mm long, and the glandular one, 0.496-0.567 mm. Nerve ring surrounds oesophagus approximately in the middle of the muscular part (slightly displaced to pharynx) at a distance of 0.114-0.126 mm from the anterior end of the body. Excretory pore is located at a distance of 0.126-0.154 mm from the anterior body end. Tail is 0.047-0.059 mm long, tapering, with a slight thickening at the end, seated with small spines. Larvae enclosed in connective tissue capsules 0.339-0.377 mm in diameter. Cysts localized in the walls of stomach and intestine. One cyst can contain two nematode larvae. The final hosts of Physocephalus sexalatus are mammals of family Suidae. The nematode parasitizes in the stomach lining of wild boars and domestic pigs. The parasite also was noted in camels, horses, donkeys, cattle and lagomorphs. Intermediate hosts are coprophagous beetles of the family Scarabaeidae. Final hosts become infected when eaten coprophagous beetles [20]. The parasite has a wide range of paratenic hosts, which include many species of vertebrates of different classes, including reptiles. Physocephalus sexalatus parasitizes in the stomach and intestine walls in reptilian hosts.

Results and Discussion
In the Middle Volga region, larva of Physocephalus sexalatus was noted in 17 species of vertebrates of different classes [21][22][23][24][25][26][27][28][29][30][31]. Parasite broadly distributed among reptiles of Eastern Europe and Central Asia [10,11]. Previously Physocephalus sexalatus, juv. recorded in Vipera berus in Ukraine and Belarus [10,11].  Physaloptera clausa parasitizes in the stomach of hedgehogs. At the larval stage, it is a common and widespread parasite of reptiles, more often found in lizards. Reptiles are paratenic hosts. In the Middle Volga region, we found parasite in the final hostthe Northern white-breasted hedgehog Erinaceus roumanicus Barrett-Hamilton, 1900, in paratenic hostsinsectivores (Sorex araneus Linnaeus, 1758) and other species of reptiles [12,15,16,23,25]. The nematode juveniles were recorded in many species of reptiles in the former USSR [10,11]. On the territory of Russia, it was found in Lacerta agilis from the Crimea [32]. Vipera berus represents a new host record for Physaloptera clausa, juv. Sharpilo, 1963, juv. (13 specimens) ( figure  4). Body 0.630-0.841 mm long with maximum width 0.037-0.047 mm. The body is covered with a slightly striated cuticle. Two cuticular crests protrude above the body surface and running along the larva body laterally. The lips are indistinguishable. Oesophagus length 0.280-0.356 mm, consists of two poorly differentiated parts. The walls of the oesophagus anterior part are sclerotized. The oesophagus is greatly widened at the base; it exceeds the intestine in width. The nerve ring surrounding the muscular part of the oesophagus approximately in its middle part, at a distance of 0.035-0.043 mm from the anterior end of the body. The excretory pore is located at a distance of 0.091-0.106 mm from the anterior end of the body. The genital primordium is oval in shape, located approximately at the mid-length of the intestine. The tail is conical, 0.037-0.049 mm long, sharpened at the end. The nematode juveniles enclosed in connective tissue capsules 0.235-0.322 mm in diameter. Cysts localized in the intestinal wall.

Morphological description of Agamospirura minuta
Agamospirura minuta, juv. is a specific parasite of reptiles, usually parasitizing the slowworm Anguis fragilis Linnaeus, 1758 and less common in other lizards and snakes [11]. Final host are unknown. Lewin [33] found similar nematode larva in reptiles of Poland and identified them as