Biosystems Diversity Life cycle of Bilharziella polonica (Trematoda, Schistosomatidae) parasite of semi-aquatic birds in Uzbekistan

cycle of poloni- Schistosomatidae are an actively studied ecological group of trematodes. Their ability to cause various parasitic diseases in an- imals and humans makes them an interesting object of study for a number of research centres worldwide. One of the commonest species in this group is Bilharziella polonica (Kowalewsky, 1895), whose mature stages have been recorded in aquatic and semi- aquatic birds in Uzbekistan. Our research team established that the following birds were infected with mature trematodes B. polonica : Anas platyrhynchos (23%), A. crecca (18%), Podiceps ruficollis (11%), Ardea cinerea (14%) and one individual of Oxyura leucocephala . The highest infection rate was shown by the mallard A. platyrhynchos (23%) and common teal A. crecca (18%). The infection intensity ranged between 2 and 27 individuals. Research into various types of water bodies in Karakalpakstan identi- fied 10 mollusc species – Lymnaeidae (4 species), Planorbidae (4 species) and Physidae (2 species). Cercariae morphologically similar to larvae of B. polonica were found in two species, Planorbis planorbis and P. tangitarensis . 6 chicks of domestic ducks were experimentally infected with those cercariae to track the life cycle of B. polonica in the organism of a definitive host. Helmin-tholological dissections showed that every duck was infected with B. polonica , which became mature 23–27 days after the infec- tion. Eggs of B. polonica were recorded in the excrement of one of the birds 33–35 days after the infection. Based on field and experimental research, we identify the mollusc P. tangitarensis as a new intermediate host for B. polonica in Uzbekistan.


Introduction
Birds are one of the world's largest groups of vertebrates, with over 460 species inhabiting Uzbekistan. They play a huge role in natural life and human activities. Aquatic and semi-aquatic birds are a specific group of birds, important objects of hunting. They traditionally have been harvested in the Aral Sea area located in the middle of the main Central Asian flyway, where historically aquatic and semi-aquatic birds have formed large aggregations as they migrate from Siberia and Kazakhstan to their wintering grounds by the Caspian Sea, in India, Pakistan and Africa. The Aral Sea region was also the location of Uzbekistan's largest breeding colonies of nesting semi-aquatic birds, such as herons, cormorants and pelicans (Kreysberg-Mukhina et al., 2005). This is a breeding ground for some species (cormorants, herons, ducks, geese, waders and gulls), but most birds use the territory in winter and during migrations. As is wellknown, birds, including aquatic and semi-aquatic species, are hosts for various helminth species and groups causing grave infectious diseases in animals and humans.
The ecology of aquatic and semi-aquatic birds makes them very susceptible to infection with Schistosomatidae in many parts of the world (Horák & Kolárová, 2000;Bayssade-Dufour et al., 2006;Akramova, 2011). They include trematodes from the genus Bilharziella Looss, 1899 -parasites of aquatic and semi-aquatic birds across Europe, Asia, Africa and North America. Szidat (1929; quoted from K. I. Skrjabin, 1951) studied the life cycle of В. polonica and established that eggs laid by the female enter the vessels of the intestine and further penetrate through the walls into the intestinal lumen, from where they exit the body with excrement. A miracidium emerges from the egg in water to enter the trematode's intermediate host, the mollusc (Coretus corneus, Limnaea stagnalis and L. limosa), where it turns into a sporocyst producing furco cercariae. As they leave the mollusc's body, cercariae enter the water, stick to birds' feathers and penetrate into their organisms through the skin. Szidat (1930) established that birds may become infected through the digestive tract. Litvishko (1963) reports on intermediate hosts of В. polonica in Ukraine. During an experiment on molluscs Coretus corneus, Planorbis planorbis and Limnaea stagnalis, only C. corneus proved to be infected, while no traces of furco cercariae were detected in the other two species. The role of some mollusc species in the life cycle of В. polonica in the wild across Europe and Asia is described in the works (Kolárová et al., 1997;Horák & Kolárová, 2000;Akimova, 2010), which also inform about dermatitis in humans caused by cercariae of В. polonica.
Nevertheless, the role of cercariae of B. polonica in causing human cercarial dermatitis in Poland was not supported by later studies (Zbikowska, 2003(Zbikowska, , 2004. In addition, opinions diverge on the intermediate hosts of B. polonica. Initially, molluscs from the family Planorbidae and Lymnaeidae (Szidat, 1929;Litvishko, 1963) were identified as intermediate hosts of B. polonica. However, later research into the biology of B. polonica (Khalifa, 1972;Akramova, 2011) did not confirm the conclusions made by Szidat (1929) on the participation of molluscs Lymnaea stagnalis and Lymnaea limosa in the life cycle of B. polonica in Europe and Asia. In this article, we present the results of the field and laboratory studies of the biology and life cycle of B. polonica, with a focus on the specification of mollusc species participating in the distribution of infection in the wild.

Material and methods
The material for this paper was the results of faunistic and experimental research into the morphology and biology of B. polonica carried out in 2019-2022. The material was collected on various bodies of water in the deltas and floodplains of the Amudarya and Zeravshan frequented by aquatic and semi-aquatic birds. Birds were caught during the hunting season in the Karajar and Kyzyljar areas of the Republic of Karakalpakstan and Lakes Karakir and Dengizkul in the central portion of Uzbekistan. A total of 41 bird individuals were studied using known parasitological methods (Skrjabin, 1928;Dubinina, 1971;Kotelnikov, 1976).
A study of some aquatic and semi-aquatic birds in the Republic of Karakalpakstan and Bukhara province, Uzbekistan, in August-October 2019-2021 established that some individuals were infected with mature trematodes from the genus Bilharziella Looss, 1889: 3 individuals of Anas platyrhynchos Linnaeus, 1758, (out of 13 that were examined), 2 Anas crecca Linnaeus, 1758 (out of 11), 2 Podiceps ruficollis Pallas, 1764 (out of 5), 1 Ardea cinerea Linnaeus, 1758 (out of 3) and 1 Oxyura leucocephala Scopoli, 1769 (out of 1). Mature trematodes identified as B. polonica (Kowalewsky, 1895) were extracted from the blood vessels in the intestines, mesenteries and livers of the infected birds. Infection intensity was low, ranging between 2 and 27 individuals (Table 1). During this period, large numbers of aquatic molluscs, potential intermediate hosts, were examined for spontaneous infection with larval stages of the life cycle of B. polonica ( Table 2). The research covered most of bodies of water in the Aral Sea area and central part of Uzbekistan. The molluscs were collected using a common hydrobiological method (Jadin, 1952). A total of 2108 individuals of freshwater molluscs from the families Lymnaeidae, Planorbidae and Physiidae were collected and studied in different seasons (spring, summer and autumn). In the water bodies of the Karajar, Kyzyljar and Dautkul lake systems in Karakalpakstan and in Lakes Karakir and Dengizkul in Bukhara province, we recorded 10 species of molluscs from the families Lymnaeidae (4 species), Planorbidae (4 species) and Physidae (2 species). In two species -Planorbis planorbis (Linnaeus, 1758) and P. tangutarensis Germain, 1918 -we detected cercariae similar in morphology to larvae of B. polonica (Table 2). Cercariae of naturally infected molluscs Planorbis planorbis, P. tangitarensis were used to reconstruct the life cycle of B. polonica. The molluscs were put in individual small vessels and kept in a laboratory room. The water in the vessels was examined daily for cercariae. Healthy birds from farms and nurseries were artificially infected with cercariae that emerged from the molluscs P. planorbis and P. tangitarensis. Active cercariae of the same age -that is, those collected within 3-5 hours after they had emerged from their host mollusc -were used in the experiment. The experimental birds were 6 chicks of domestic ducks, aged 15-20 days. Each was infected with 80-120 individuals of cercariae by keeping one of its legs in the water with cercariae for 10, 25 and 30 minutes at a temperature of 28-30 °С. Starting from the 20th day after the infection, the excrement of the experimental birds was regularly examined for B. polonica eggs.
The molluscs were infected with miracidia individually and in groups. When infected individually, each mollusc was placed in a Petri dish, where 1-3 active miracidia of the same age were added (the miracidia were taken within 1-2 hours after their emergence from the eggs). 24 hours later, the molluscs were placed in small aquariums, 25-30 individuals in each, and were surveyed for some time. In the case of group infection, molluscs were put in medium-size aquariums, 75-100 individuals in each. Miracidia from the eggs of B. polonica were added to the aquariums.
Larval stages were studied by dissecting live experimental molluscs. The morphology and biology of larval stages were studied using a common method (Ginetsinskaya & Dobrovolsky, 1963;Ginetsinskaya, 1968). The morphology of trematoda miracidia (25 individuals) and cercariae (25 individuals) was studied with the help of intravital staining. The morphometric parameters of the cercariae were measured with the use of anaesthetic solutions of neutral red, following the method by Ginetsinskaya (1968). Modern equipment was used in the research: phase-contrast inverted microscope CK2-TR (Olympus, Japan), research microscope LOMO, cooling centrifuges TR7 (Dupont, USA), binocular microscope ML-2200 (Olympus, Japan). The drawings were made with the help of drawing machine РА-4.
The detected trematoda were males and females (Fig. 1). Males are generally more numerous than females. In our research the male-tofemale ratio was 10♂:2♀. The grey heron and white-headed duck had only male trematodes (13 individuals in each). The general morphology and dimensions of males and females of B. polonica detected in Anas platyrhynchos and Podiceps ruficollis are given in Table 3. Males and females of B. polonica showed almost the same morphology in different bird species. However, trematodes detected in A. platyr-hynchos had the largest dimensions. Only males were found in the whiteheaded duck and grey heron, which means these birds had been infected with unisexual cercariae. In the water bodies of the Karajar, Kyzyljar and Dautkul lake systems in Karakalpakstan and in Lakes Karakir and Dengizkul in Bukhara province, we recorded 10 species of molluscs from the families Lymnaeidae (4 species), Planorbidae (4 species) and Physidae (2 species). In two species -Planorbis planorbis and P. tangutarensis -we detected cercariae similar in morphology to larvae of B. polonica (Table 3). The natural infestation of shellfish was 1.4% and 0.9%, respectively.
Cercariae of B. polonica were not found in other studied species of molluscs. A morphological study of the cercariae from molluscs P. planorbis and P. tangitarensis made it possible to identify them as B. polonica.
Given below are the general morphology and dimensions of cercariae taken from individuals painted with acetate carmine (Fig. 2). Mature cercariae leave the body of their host (mollusc) in water. Cercariae emerge from molluscs most actively in the morning and daytime hours, less actively at dusk, between 5 and 7 p.m. For 24 hours, one mollusc can emit up to 11,000 cercariae at a temperature of 25-30 °С.
Cercariae that have just left the mollusc body are very active and assume a very characteristic and highly specific position, when at rest. Cercariae stay in the upper water level or stick to aquatic plants with their ventral sucker. In this position they wait for their definitive host. In water, cercariae stay alive for 36-48 hours. The mechanical vibration of water and plant substrate cause cercariae to move. The cercariae of the studied trematode species have an elongated oval body, 218-288 μm long and 68-98 μm wide at the level of the ventral sucker (Table 4).
The head organ is elongated, pear-shaped. The ventral sucker is round and is noticeably shifted from the centre towards the tail. The tail stem is long. The tail furcae are much shorter than the tail stem. The digestive system comprises the mouth, esophagus, which branches into short intestinal canals, at the base of which (on the two sides of the body) there are two pigmented spots clearly discernable under a microscope. The cuticle consists of small spurs. There are 5 pairs of penetration glands, 2 of which are situated before the sucker and 3 behind it. The large and twisting ducts of the penetration glands are directed forward, going inside the head organ and then outside through pores at the sides of the mouth. The excretory system consists of 7 pairs of flame cells, interconnected by excretory ducts. The excretory system can be expressed with the formula: 2[(3) + (3) + (1)] = 14.
The sensory apparatus consists of dorsal, lateral and ventral complexes, located on the body, tail stem and furcae. The dorsal complex consists of 32 sensilla, 8 of which are situated in the terminal part, 4 behind the ventral sucker and the other 20 form groups on the dorsal side of the body. The tail stem has 10 sensilla, and the furcae bear 2 sensilla. The lateral complex comprises 8 sensilla. The ventral complex consists of 53-54 sensilla grouped mainly on the head organ and the ventral sucker (Fig. 3). By their morphological characteristics, cercariae from molluscs Planorbis planorbis and P. tangitarensis proved identical. Their identity is highlighted by the almost complete coincidence with basic parameters of cercariae (Table 4) and the affinity of their intermediate hosts (P. planorbis and P. tangitarensis). The cercariae we detected (Table 4) did not show any differences in form or size from those described in well-known literary sources (Szidat, 1929;Khalifa, 1972) (Table 5).

Table 5
Morphological parameters of cercariae of Bilharziella polonica (Kowalewsky, 1895) (according to Szidat, 1929;Khalifa, 1972) Parameter According to Szidat (1929), mm According to Khalifa (1972) To confirm the species of cercariae detected in the molluscs P. planorbis and P. tangitarensis, we infected experimentally 6 young individuals of domestic ducks Anas platyrhynchos dom. It was established that the experimental ducks were highly susceptible to infection and showed a fluke survivability of 30.0-55.0% (Table 5). When studying infected birds on various dates, we detected schistosomula in lung and liver vessels after 10-11 days of their infection. By day 18, the trematodes could be differentiated by sex, and by day 23-27, those discovered within the veins of the mesentery, intestine and liver had reached sexual maturity. Single eggs of B. polonica with miracidia were detected in the excrement of bird No. 6 33-35 days after the infection.
As is seen from Table 7, the morphometric parameters of B. polonica are very similar in naturally and experimentally infected individuals. Males are larger than females. Our data on the morphological parameters of males and females of B. polonica correspond with data from known literary sources (Szidat, 1929;Khalifa, 1972;Filimonova, 1985;Bayssade-Dufour et al., 2006) and do not show any considerable differences.
Egg and miracidium. The egg has the form of a flask and is provided with a curved spike. The length of immature eggs is 362-385 μm, mature eggs are 515-545 μm long (Fig. 5).
Miracidia develop in eggs, while the latter migrate within the organism of the definitive host. Mature eggs with developed miracidia are ejected from birds with excrement. Our experiments show that eggs laid by female trematodes under the intestine's mucous membrane undergo considerable morphological and biological changes as they migrate. Generally retaining their form and structure, the eggs grow in size; miracidia inside the eggs develop, as the eggs migrate within the bird's tissues; eggs found in birds' excrement usually contain developed miracidia; mature eggs become light yellow in colour. When eggs contact water, miracidia emerge. The swimming miracidium has an elongated cylindrical body with a slightly widened head and a slightly narrowed tail. It is 150-160 μm long and 36-55 μm wide. In water, miracidia move fast in search of their victim, stretching the body strongly. The body is densely covered with cilia resting on a total of 22 epithelial laminae. The epithelial laminae form four rows that can be expressed by the formula 6 : 8 : 4 : 4 = 22. The large apical gland is located in the front part of the miracidium. In the front part of the body, on the sides of the apical gland, is a pair of glandular cells. The excretory system includes two pairs of flame cells. The twisting canals of the front and rear cell in each pair join into a single lateral duct going out on each the side of the body between row 3 and 4 of the epithelial laminae. The nervous system is represented by a large cerebral ganglion right behind the apical gland. The sensory system consists of 14 sensilla located at the edges of the epithelial laminae. The rudimentary reproductive system is represented by 7-8 propagatory cells in the rear part of the miracidium's body.
The miracidium is highly mobile, making abrupt linear movements. By the end of its lifespan, the rate of the movements drops sharply. At a temperature of 18-20 °С it stays alive for a few hours.
Our team established that molluscs P. planorbis and P. tangitarensis acted as intermediate hosts in the life cycle of B. polonica, both in the wild and in the laboratory. This is supported by data in Tables 2 and 8.
The experiments showed that molluscs G. truncatula, S. corvus, R. auricularia and L. stagnalis were resistant to miracidia of B. polonica. Similar resistance was manifested by P. fontinalis, P. acuta, A. spirоrbis and G. albus. In the meanwhile, molluscs P. planorbis and P. tangitarensis proved highly susceptible to the infection with miracidia of B. polonica. The infection rate was 100%. In the organisms of these molluscs, trematodes developed through various larval stages, from mother and daughter sporocysts to cercariae. Cercariae developed in daughter sporocysts in the hepatopancreas of molluscs P. planorbis and P. tangitarensis. At 26-32 °С, mature cercariae began to emerge from the bodies of the two mollusc species, P. planorbis and P. tangitarensis, in 23-27 days after their infection. The cercariae continued to emerge up until the mollusc's death. The high susceptibility of these molluscs to miracidia of B. polonica in the wild and at the laboratory makes it quite possible that these mollusc species act as intermediate hosts in the distribution of the infection in the biocoenoses of Uzbekistan.

Discussion
Literary sources provide diverse information on the life cycle and biology of trematodes and on the role of various species as the parasite's host (Szidat, 1929(Szidat, , 1930Litvishko, 1963;Khalifa 1972). The life cycle of B. polonica in Europe was studied 90 years ago by Szidat (1929), who identified its intermediate host to be Planorbarius corneus. Later studies of molluscs within the range of B. polonica (Vergun, 1956;Wisnewski, 1958;Zdyn, 1959;Zdraska, 1963) also showed that Planorbarius corneus was an intermediate host of this trematode. Data were also published on the infection of molluscs Planorbis planorbis with cercariae of B. polonica in Europe and Asia (Wisnewski, 1958;Butenko 1967;Arystanov, 1968). Khalifa (1972) conducted most detailed research into the life cycle of B. polonica in Poland. The researcher detected cercariae similar to those of B. polonica in molluscs Planorbarius corneus, Planorbis planorbis and Bathyomphalus contortus. Furthermore, Khalifa wrote that, according to Skrjabin (1951) and Litvishko (1963), who cited Szidat (1929), molluscs Lymnaea stagnalis and L. limosa could be experimentally infected with larvae of B. polonica. However, as she scanned the original work by Szidat (1929) "Zur Entwicklungs geschichte des Blut Trematoden der Enten Bilharziella polonica Kow. 1 Morphologie and Biologie der Cercaria von Bilharziella polonica Kow. 2 Zentll. Bakt. ParasitKde. Abt. 1. Orig., 3., 461-470", Khalifa did not find this statement, and suggested it might be a literary error. We have scrutinised the original by Szidat (1929), which was kindly provided to us by the Lenin Library in Moscow, Russia, and support the version offered by Khalifa (1972). Molluscs Lymnaea were mentioned as intermediate hosts of B. polonica by Skrjabin (1951) as a result of incorrect translation of Szidat's work (1929) into Russian. Thus, molluscs from the genera Planorbarius and Planorbis can be regarded as the main intermediate hosts for trematoda B. polonica within its range. These molluscs are usual components of diverse freshwater biocoenoses and are widely distributed across Europe and Asia. Their role in the transmission of cercariae of B. polonica and spread of infection in the wild is currently indisputable. This is also supported by the results of our research, where our team experimentally infected various mollusc species -Galba truncatula, Stagnicola corvus, Radix auricularia, Lymnaea stagnalis, Anisus spirorbis, Gyraulus albus, Planorbis planorbis and P. tangitarensis with miracidia of B. polonica. Under equal conditions, only two species proved susceptible to the infection -P. planorbis and P. tangitarensis, while Lymnaeidae and some Planorbidae (Anisus spirorbis, Gyraulus albus) were totally resistant to miracidia of B. polonica. Numerous parasitological publications also confirm the participation of Planorbarius corneus and Planorbis planorbis in the life cycle of B. polonica (Nowak & Zbikowska, 2003;Chrisanfova et al., 2009;Akimova, 2010Akimova, , 2014Khrisanfova et al., 2010;Akramova, 2011).

Conclusion
The results of our research into the morphology and biology of B. polonica and the discovery of mature forms of this trematode in a new definitive host, white-headed duck Oxyura leucocephala, complement the knowledge about the range of intermediate and definitive hosts and the circulation of infections in the wild, in the parasite-molluscs-birds system.
Thus, the group of intermediate hosts of B. polonica includes three mollusc species -Planorbarius corneus, Planorbis planorbis and P. tangitarensis, in which the parasite goes through its larval stage in the wild. The range of definitive hosts for this trematode is much broader and in-