Morphology, biology and taxonomy of Dendritobilharzia loossi Skrjabin, 1924 (Trematoda: Bilharziellidae), a parasite of Pelecanus onocrotalus (Pelecanidae) and Anas plathyrinchos (Anatidae)

Life cycles of Dendritobilharzia loossi Skrjabin, 1924, a parasite of waterbirds, and its morphobiological traits are studied and described. Mollusks Anisus spirorbis, the infection rate of which in natural environments reaches 1.3-1.9%, were recorded as intermediate hosts under conditions of Uzbekistan. The development of this trematode in intermediate and definitive hosts lasts for 26 and 15 days, respectively. Diagnostic traits of the trematodes during all stages of their ontogeny are reviewed.


INTRODUCTION
T he genus Dendritobilharzia Skrjabin et Zakharov, 1920 comprises trematodes parasitizing blood vessels of waterbirds. Currently, this genus includes four species, namely Dendritobilharzia pulverulenta (Braun, 1901), D. loossi Skrjabin, 1924, D. anatinarum Cheatum, 1941, and D. asiatica Mehra, 1940. Of these, the first two species were recorded in the Commonwealth of Independent States (the former USSR) including Uzbekistan (Skrjabin, 1951;Byhovskaya-Pavlovskaya, 1962;Iskova, 1968;Ryzhikov et al., 1974;Azimov, 1975;Smogorzhevskaya, 1976;Akramova & Azimov, 2005; and others). Until 1968, no data on the biology of the species from the genus Dendritobilharzia had been available in literature. It was not until 1968 that the first publications on the biology of one of the species from this genus, D. pulverulenta (Vusse, 1979pulverulenta (Vusse, , 1980 appeared in literature. A detailed study of the morphology and biological traits of D. pulverulenta was carried out by (Khalifa, 1976). This author established the mollusks Anisus vortex (L., 1758) and Planorbis planorbis (L., 1758) as intermediate hosts of this trematode, the infection levels of which by the cercariae reached 5.6 and 1.7 %, respectively. The mature parasites were recorded in the blood vessels of three nestlings of the domestic duck Anas platyrhynchos experimentally infected by Dendritobilharzia pulverulenta. In the subsequent years, a significant interest was noted towards the study of the life cycles of trematodes from the genus Dendritobilharzia. Leite et al. (1982)

MATERIAL AND METHODS
T he material of this study is comprised of our own collection of mollusks carried out in 2000-2007 from waterbodies situated in the floodplain of the Syrdarya River (in Tashkent and Syrdarya provinces) and in the lower reaches of the Amudarya River, where farms of Khoresm province and the Republic of Karakalpakstan are situated (see map, Fig. 1). The collection of the mollusks was carried out according to a routine method described by Zhadin (1952) from the waterbodies of Gulistan, Bekabad and Chinaz districts, which are situated in the floodplain of the Original contribution Parasite, 2011, 18, 39-48 middle course of the Syrdarya River. Similar collections were carried out in the waterbodies of the lower reaches of the Amudarya River. Waterbodies situated in the floodplain were surveyed, namely, the system of Dautkul lakes, Lake Mashankul, Shegekul, Sudochie, Khodjakul and the system of Karadjar lakes. Totally, collected and studied were 5,600 freshwater mollusks, namely Lymnaea auricularia, L. stagnalis, L. truncatula, Planorbis planorbis, Anisus septemgyratus, A. spirorbis, Physa fontinalis, in spring, summer and autumn. The parasite eggs isolated from naturally infected ducks Anas platyrhynchos dom., in the farm Saikhun situated in the Syrdarya province (25 July 2000), were used as the material for the reproduction of the biological cycle of development of the trematode D. loossi. We monitored the process of D. loossi miracidia at the following temperatures: 50,40,35,30,25,20,15,10 and 5 °C. At the temperature 50 °C the eggs die quickly. Therefore, no hatching takes place. The same situation was recorded at the temperature of 40 °C. Of seven studied ducks, which were dissected using the complete helminthological method of dissection as described by Skrjabin (1928), in one female we found 13  and 11  D. loossi in the vessels of the mesentery and liver. Besides, we found the eggs typical of D. loossi in the faeces. The examination of the white pelican female (Pelecanus onocrotalus), which was shot by poachers on Lake Sudochie on 2 August 2002, revealed mature trematodes D. loossi, 5  and 3  in the vessels of the mesentery and liver. The identification of the trematode D. loossi from ducks and a pelican was carried out by using the preparation stained using known helminthological methods. We studied 18 , 14  of natural populations of adults from the ducks and the pelican and 30 , 25  mature trematodes from experimentally infected birds.
For the infection of the mollusks we used the eggs of D. loossi obtained from naturally infected ducks Anas platyrhynchos dom. In laboratory conditions, miracidia emerging from the eggs were used for the artificial infection of fresh-water mollusks. The experimental infection of the mollusks by D. loossi miracidia was carried out on both individual specimens and groups. At the individual infection, each mollusk was put in a Petri dish into which one to three active miracidia were placed. One day later, 25-30 mollusks were transferred into each of small aquariums and monitored. During the group infection, the mollusks were kept in mid-sized aquariums (ca. 75-100 individuals per aquarium). Eggs containing mature miracidia were added to the aquariums.
Morphological and biological peculiarities of miracidia and cercariae were studied by using a generally accepted technique (Ginetsinskaya & Dobrovolsky, 1963;Ginetsinskaya, 1968). For the study of the morphology of miracidia (25 individuals) and cercariae (30 individuals) we used vital stains. Morphometric parameters of cercariae were studied on anesthetized solutions of the neutral red. While studying live objects we used vital stains: the neutral red, the sulphate of Nile blue, acetic carmine, toluidine blue, etc.
For the study of the morphology of live miracidia and cercariae, we used the phase-contrast microscopy. We used the method of silvering for the revealing of the borders between epithelial plates and sensitive papillae (sensilla) of miracidia, as well as of sensilla of cercariae. Miracidia were recorded at 0.5 % or hot 1 % nitro-acid silver, after which they are placed into glycerol and studied visually. The hatching of miracidia of the considered species from eggs takes place in the water (external environment), which depends on a number of abiotic factors. Therefore, we studied the effect of various temperatures and illumination on the process of hatching of miracidia and emission of cercaria of D. loossi. We traced the emission of cercariae at the following temperatures: 45,40,35,25,20,15,10 and 5 °C during the natural alternation of the day and night. The experiments were conducted in three replications.
Material collected during 2000 to 2007 was used in the work. Experiments were repeated many times. The data given in the Tables I-IV and in the text (daily rhythms of cercariae emission) reflect average values.
The birds were infected with cercariae that had emerged from mollusks. In the experiment, we used 20 nestlings of each of domestic ducks, geese and chickens at the age of 18-20 days, all of which were grown in conditions preventing their infection with the above- The studies were carried out using modern equipment: the phasecontrast device, inverted CK2-TR (Olympus Japan), research microscope LOMO, cooling centrifuges TR7 (Dupont, USA), binocular ML-2200 (Olympus Japan).

EGGS AND MIRACIDIA
T rematodes lay eggs in the lumen of the capillaries in the intestine and other organs. Embryos develop in the eggs situated in the tissues of the definitive host (Fig. 2). Newly-shed eggs, oval with a sharp spine on one pole, are 0.066-0.074 × 0.02-0.03 mm. Mature eggs secreted with the excrements of birds are 0.10-0.12 × 0.04-0.06 mm; they are light-brown. Miracidia hatch during the contact of the egg with water. The optimal temperature providing the emergence of miracidia was 26-32 °C in our experiment. Shortly before the emergence from the egg a miracidium makes very active movements. In a bright solar light, most of miracidia emerge from the eggs in 25-45 min. At the temperature of 35 °C, miracidia hatch from 90 % of eggs, while 10 % of miracidia died inside the eggshell. The temperature of 25 °C to 32 °C is the most optimal for the hatching of miracidia (Table I). Lower temperatures (from 5 °C to 10 °C) are insufficient for the miracidia from the eggs. The hatching of active miracidia from the eggs was recorded at a gradual increase in temperatures to 25 °C and 32 °C.
It is noteworthy that illumination stimulates the process of emergence of miracidia from the eggs. A long-term maintenance of eggs with miracidia ready to hatch in  the darkness results in their weakening and a significantly lower percentage of their hatching. Our multiple observations that the hatching of miracidia takes place in a different way. It is known that the eggs of these trematodes have no cap. By the moment of the hatching of a larva, the egg as a rule splits in its anterior third, where the shell may be thinner than in the other parts and the miracidium moves out through the crack. An actively moving miracidium of Dendritobilharzia has an elongated body slightly sharpened in the anterior and tapering in the posterior part. Miracidia show a positive photo-and negative geotaxis (Azimov, 1986). The time of the active life of larvae at the temperature of 26-32 °C is 18-20 hours. The length of the miracidia reaches 0.09-0.10 mm at the maximal width 0.06 mm. The surface of the body is formed of four rows of epithelial plates, which have numerous cilia. The epithelial plates are situated by the formula 6:8:4:4=22. The part of the body free from cilia is relatively small, in which the duct of the apical gland opens up. A large apical gland is situated in the anterior part of body. A rather large cerebral ganglion of an elongated-oval shape is situated at the level of epithelial plates of the second row. Twelve sensillae lie on the border of the epithelial plates. There are two pairs of ciliary cells. The first pair lies along the sides of the cerebral ganglion; the second one in the posterior part of the miracidium. These cells are interconnected by convoluted tubules.

DEVELOPMENT IN THE ORGANISM OF AN INTERMEDIATE HOST
The mollusk Anisus spirorbis (L., 1758) was recorded as the intermediate host of D. loossi both in the wild and in the experiment (Table II). Penetrating into the body of the intermediate host, miracidia of Dendritobilharzia undergo a regressive metamorphosis and turn into a mother sporocyst, an organism, which is characterized by an extremely simple structure. They breed parthenogenetically, giving rise to morphologically more complex individuals of the next generation -the daughter sporocysts (Fig. 3). The total infection rate of mollusks A. spirorbis by the Dendritobilharzia cercariae in natural conditions reached 1.3 and 1.9 %. The cercariae were recorded in mollusks in summer (July-August) and in autumn (September). During the experimental infection, cercariae are formed in daughter sporocysts in the hepatopancreas of A. spirorbis mollusks.
As the experiments showed, the time of the development of parthenitae and formation of cercariae in intermediate hosts depend on the temperature (Table  III). So, at relatively high temperatures the formed cercariae began emerging from A. spirorbis in 26 days. The emission of cercariae continued until the death of infected mollusks.

CERCARIA
The body of a cercaria is elongated-oval in shape, rounded on the anterior side (Fig. 4). The body length 0.220-0.246 mm; the width 0.063-0.078 mm. The anterior organ is elongated-oval in shape (the length 0.070-0.082 mm; the width 0.046-0.052 mm). The ventral sucker is significantly shifted from the midpart of the body backwards. It is roundish in shape reaching 0.032-0.034 × 0.028-0.030 mm. The tail stem is 1.5 as long as the body and reaches 0.260-0.370 mm at the width of 0.026 mm. Tail furcae are shorter than the tail stem; their length is 0.126-0.146 mm. There is no natatorial membrane on the furcae (Table IV).
The digestive system consists of the oesophagus and two rudimental intestinal branches, which reach the first pair of the penetration glands. The latter are large, their number reaching five, of which two are preacetabular and three postacetabular pairs. There are two pigmented eyes. The excretory system is described in the formula 2[(1+1+1)+(3+1)]=14. The excretory bladder is small.
The sensor apparatus consists of the dorsal, lateral and ventral complexes (Fig. 5). Sensillae are situated in symmetrical rows along the body, tail stem and furca. In general, the dorsal complex is formed of 34 sensillae; lateral, 6; ventral, 38, the total number of sensillae reaching 78.  Cercariae emerging from the mollusks into the water are very active. The cercariae D. loossi has the ability of moving both the anterior end and posterior forward. This movement is the result of the contraction of the musculature of the tail stem. The furcae of the tail play the role of a rudder. Cercariae show a positive photo-and negative geotaxis (Azimov, 1986). The most intensive emission from the mollusks takes place in the morning and in the afternoon. An increase in illumination and temperature contributes to an intensive emission of cercariae from the intermediate host.
The studies have shown that the emission of cercariae from the host mollusk is stimulated or hindered by the effect of environmental factors, of which the most important are the temperature and light. Inter-relations of these two factors provide the periodicity of the emission of cercariae. As the results of the study shows, the emission of the cercariae from mollusks takes places at a certain temperature, on average from 15 to 35 °C. The death of host mollusks is recorded at high temperatures (40-45 °C). At 15 °C the emission of cercariae is significantly restricted, while at 10-5 °C the emission of the cercariae ceases. The optimal temperature for the emission of cercariae from host mollusks is 25-32 °C. The pattern of the curve of the daily rhythm of cercariae emission has two peaks. The first maximal peak is noted at 2 pm, while the second at 7 pm. Thus, the intensity of the emission of cercariae depending on the light and temperature to a certain degree connected with the pattern of their taxis. The cercariae of this species are characterized with a positive photo-and thermotaxis. The cercariae emitted from the mollusk produce quick movements. The resting posture is very characteristic. They hang motionless, attached with the sucker to the film of the water surface tension. At this point, the ventral sucker becomes elongated and the body bends dorsally, while the tail stem and furcae hang. When soaring, the furcae are significantly straightened out. The duration of the cercariae in the water is about two days.

DEVELOPMENT OF CERCARIAE IN DEFINITIVE HOSTS
A series of experiments on the experimental infection of birds with cercariae D. loossi in laboratory conditions was carried out, which was aimed at obtaining of the original material for the reproduction of the complete life cycle of Dendritobilharzia in laboratory conditions. Experiments on the infection of goslings and chickens yielded a negative result. Nineteen ducklings became infected. On days 18 and 20 mature eggs of Dendritobilharzia were recorded in their faces. After this experiment we started exploring the ontogeny of this trematode in the definitive host (Table V).
The study of birds revealed schistosomules in blood vessels of the lungs 72 hours post infection, and in the liver and kidney 5 and 10 days post infection. It was established that the differentiation of trematodes into males and females was manifested on day 12 post infection. On day 15 the trematodes reached sexual maturity, when males and females had completely formed sexual organs (Fig. 6). Mature males and females were found in the blood vessels of the mesentery, kidney, liver and intestine as a result of the study of ducks by the method of the complete helminthological autopsy.

DISCUSSION
T he female D. loossi was described by Skrjabin (1924) from blood vessels of Pelecanus onocrotalus in Kazakhstan. In 2000, we found this species in Anas platyrhynchos dom. from the waterbody of Syrdarya province in the Republic of Uzbekistan and in P. onocrotalus from Lake Sudochie in the Republic of Karakalpakstan (Akramova & Azimov, 2005). There is no additional information on the records of D. loossi. This convinces us of the restricted distribution of D. loossi, the ranges of the population of this species covering the territory of Kazakhstan and Uzbekistan. The growing interest to this group of trematodes in the last few years enabled a deeper study of the biology of Dendritobilharzia, particularly  their life cycles and morphology at all stages of development. In a relatively short period, researchers from different states have almost completely studied the life cycles of a number of species, e.g. D. pulverulenta (Khalifa, 1976;Vusse, 1979Vusse, , 1980, D. anatinarum (Leite et al., 1982), D. loossi (our data). The results of these studies have expanded our knowledge of this group of trematodes and helped to reveal the most significant specific peculiarities of the biology of each of indicated species. In this connection, of certain interest are materials on the morphology and biology of D. loossi.
The entire course of D. loossi biology passes according to the known scheme typical of the genus Dendritobilharzia. The rate of the development of the larval stages depends on the habitat of the mollusk host and factors of external environments. The most important is the factor of temperature. The studies of D. loossi showed that an increase in the temperature accelerates, while a decrease in it slows down the development of parthenitae and formation of cercariae. These conditions and interaction of taxis enables the concentration of cercariae in certain zones of a waterbody. At contact with definitive hosts, a cercaria actively penetrates into blood vessels through their cover.
The analysis of morpho-biological peculiarities of D. loossi at all phases of ontogeny shows that a number of traits in the process of development do not undergo significant changes; in males, the length and  Braun, 1901;Skrjabin, 1951;Bykhovskaya-Pavlovskaya, 1962;Ryzhikov et al., 1974;Palm, 1965;Ulmer & van de Vusse, 1970;Sulgostowska, 1972;Khalifa, 1976;Azimov, 1975;Kolarova et al., 1989Kolarova et al., , 1997Rind, 1987;Akramova & Azimov, 2005;Bayssade-Dufour et al., 2006). The life cycle of this species under conditions of Poland, as Khalifa (1976) notes, passes with the participation of mollusks Anisus vortex and Planorbis planorbis. Morpho-biological traits of cercariae and maritae D. pulverulenta are characterized with a rather distinct difference from other species. Of special attention are the results of recent studies carried out by Bayssade-Dufour et al. (2006), who established the range of the intraspecific variability of some traits in D. pulverulenta, depending on the season, hosts and geographic conditions. The morphology of recorded D. pulverulenta males and females from Cygnus olor in France significantly differed from the described specimens of this species from other geographic zones. Based on molecular studies the authors reasonably draw a conclusion about the identity of different forms and their belonging to the species D. pulverulenta. The ranges of species D. anatinarum and D. asiatica cover specific territories in America and Asia, in which a limited number of Anseriformes species are registered (Cheatum, 1941;Freitas & Costa, 1972;Chauhan et al., 1974;Azimov, 1975;Leite et al., 1982;Borgarenko, 1984). The life cycle of D. anatinarum was studied by Leite et al. (1982)  loossi show the specificity of the considered species to specific mollusk and avian species (Table VI). The specificity to hosts, in particular to mollusks, may serve as one of the arguments for the independence of the indicated species. It is noteworthy that Macko (1959) and Vusse (1980) consider D. anatinarum as a synonym of D. pulverulenta. Azimov (1975) does not share this viewpoint of Macko and Vusse and recognizes the independence of the species D. anatinarum and D. pulverulenta, which are quite easily differentiated one from another by the structure of the uterus and ovaries. Agreeing with the views of Skrjabin (1951) and Farley (1971)  Unfortunately, the authors (Macko, 1959;Vusse, 1979Vusse, , 1980 did not justify their viewpoint properly. In their conclusions they were based on insignificant differences in the traits of maritae D. anatinarum and D. pulverulenta. After the work by Khalifa, (1976) and Leite et al. (1982), who studied morpho-biological peculiarities of the indicated trematodes, the erroneous views of Macko (1959) and Vusse (1979Vusse ( , 1980 became evident. They did not pay sufficient attention to the fact that the seminal vesicle in D. anatinarum males is elongated, while in D. pulverulenta it is strombuliform. Similar differences are present in the females in the structure of the uterus, ovary and egg form (Table VII). The most significant differences are noted in a number of traits in cercariae of these species (Table VI). The conservatism of the structure of the excretory system should be noted. Thus, the formula of this system are expressed in D. anatinarum as 2[(1+1+1)+(2+1)]=12, while in D. pulverulenta as 2[(1+1+1)+(3+1)]=14 (Khalifa, 1976;Leite et al., 1982). Therefore, we cannot agree with the opinion of Macko and Vusse on the synonymy of D. anatinarum and D. pulverulenta, and consider the former species as the independent one. In general, morpho-biological traits of D. anatinarum, D. pulverulenta and D. loossi enable us to outline the main traits suitable for the identification of species of the genus Dendritobilharzia (Tables VI and VII). Note that morphometric traits turned out to be unfit for distinguishing these species. The ranges of their variability in all compared species (both males and females) are intersecting. The considered species can be differentiated by the number of testes in males and the structure of the ovary, uterus and eggs in females. The most significant groups of traits of cercariae are the formulas of the excretory system, the number and pattern of the disposition of the penetration glands.
There are also differences in the biology of cercariae, which develop as the following: D. loossi in the intermediate host mollusk A. spirorbis; D. anatinarum in B. straminea; D. pulverulenta in P. planorbis and A. spirorbis. A strict specificity of Dendritobilharzia to their mollusk hosts can be confidently stated. Certain specificity was also noted for the adults of the considered species. So, the range of definitive hosts of D. loossi is confined to two species from the family Pelecanidae and Anatidae. The definitive hosts of D. anatinarum are two species of the genera Anas and Cairina (Anatidae). The range of hosts of D. pulverulenta is wider and includes the genera Anas, Aythia, Fulica and Cygnus. Only Nettium crecca cressa (= Anas crecca) was recorded as the definitive host of D. asiatica. All the above mentioned materials suggest the peculiarity of the morpho-biological and ecological peculiarities of trematodes of the genus Dendritobilharzia at all phases of ontogeny. Summarizing the results of the conducted analysis it is possible to draw the following conclusions: 1. The taxonomical value of the entire complex of morphological traits in the adults and cercariae is not the same. By the diversity and taxonomic value of morphological traits more accent should be placed on the diagnostic criteria of cercariae. If studied in detail of their morphology, the identification of species does not pose special difficulties. To that end, it is necessary to use the single methodology of collection and processing of the material providing a high stability of morphological traits of cercariae. 2. In adults (males and females), the taxonomic value of traits is low due to a wide individual variability, which are connected with different factors.