New data on the morphology and systematic status of Spinitectus petrowi and Spinitectus gigi (Nematoda : Cystidicolidae) parasitic in catfishes in central China

Two little-known nematode species of the genus Spinitectus Fourment, 1883, S. petrowi Belous, 1965 (prevalence 25%, intensity 1-8) and S. gigi Fujita, 1927 (prevalence 10%, intensity 2-3), were collected from the gastrointestinal tract of the yellow catfish, Pelteobagrus fulvidraco (Richardson), from Liangzihu Lake, Hubei Province, central China, in September of 2002. The light and scanning electron microscopical examination of this material, supplemented by a few museum specimens of S. gigi collected from the catfish Clarias fuscus (Lacepede) in southern China, made it possible to study in detail the morphology of these parasite species and to redescribe them. The first species, whose correct name is S. petrowi Belous, 1965, exhibits some morphological features (e.g., unusually short vestibule, shape of pseudolabia and of the left spicule) not found in most other congeners; a unique feature is the presence of peculiar pairs of transversely oriented peg-like cuticular spines with rounded ends on the ventral surface of the female tail. Spinitectus gigi was found to have 28-31 cuticular spines in the first ring, relatively long distances between the 2nd-7th rings of spines, and anterior rings divided into 2 sectors; the excretory pore is located at the level of the 4th ring of cuticular spines; males posses 4 pairs of preanal- and 6 pairs of postanal caudal papillae and a pair of small phasmids. Spinitectus bagri Wang, Wu et Yu, 1993 and S. wulingensis Yu et Wang, 1997 are considered junior synonyms of S. petrowi, whereas S. clariasi Ky, 1971, S. ophicephali Ky, 1971 and S. yuanjiangensis Wang, Wit et Yu, 1997 are regarded to be junior synonyms of S. gigi. Spinitectus petrowi was not previously reported from China.

Characterization of development-related genes for the cestode Bothriocephalus acheilognathi

Differential gene expression of mature and immature Bothriocephalus acheilognathi cestodes was analyzed using the suppression subtractive hybridization technique. Five mature-associated cDNAs were isolated and characterized. Virtual Northern blot and RT-PCR analyses confirmed that four of the five genes were up-regulated in mature parasites. The sequence analysis revealed that one gene encoded the structural protein chorion precursor, and that three encoded functional proteins homologous to yolk ferritin, sodium/hydrogen exchanger and muscin-like protein. Another gene appeared to be specific to B. acheilognathi, encoding a putative metal-bound protein. Although results obtained in the present study are preliminary, the information about the five genes may provide clues for further investigation on the decline in parasite numbers during the maturation of B. acheilognathi.

New data on Myxobolus longisporus (Myxozoa : Myxobolidae), a gill infecting parasite of carp, Cyprinus carpio haematopterus, from Chinese lakes

The original description of Myxobolus longisporus Nie et Li, 1992, the species infecting gills of Cyprinus carpio haematopterus L., is supplemented with new data on the spore morphology and pathogenicity. Spores are elongate pyriform with pointed anterior end, 15.7 (15.5-16.5) mum long, 6.7 (6-8) mum wide and 5.5 mum thick. Sutural ridge is straight and narrow. Mucus envelope is lacking. Two equal-sized elongate pyriform polar capsules are 8.5 mum long and 2.5 mum wide with convergent long axes. Polar filament coiled perpendicularly to the long axis of the capsule makes 9 (8-10) turns. Posterior end of polar capsules exceeds mid-spore by 15-20%. Cyst-like plasmodia are localised in the gill secondary lamellae. The infection is described in adult big host specimens. Gross lesions manifested as dark red colouration of gill tissues were restricted to the ventral part of the first gill arches. Remarkable site specificity (apical part of secondary lamellae) was observed in the course of development of microscopic lesions. M. longisporus is characterised also on the molecular level using sequences of SSU rRNA gene. Phylogenetic analysis based on these sequences has allowed clearer phylogenetic relationships to be established with other species of the genus Myxobolus sequenced to date.

Scanning electron microscopy of Aspidogaster ijimai Kawamura, 1913 and A-conchicola Baer, 1827 (Aspidogastrea, Aspidogastridae) with reference to their fish definitive-host specificity

Two species of aspidogastreans, namely Aspidogaster ijimai and A. conchicola, were studied by scanning electron microscopy. In nine lakes and an old river course, the Tian'ezhou oxbow, investigated in the flood plain of the Yangtze River, A. ijimai was obtained from the common carp (Cyprinus carpio) in three lakes, and A. conchicola from the black carp Mylopharyngodon piceus in three lakes and the oxbow. In none of the localities, however, were the two species found together. It is suggested that A. ijimai may be considered as a specialist parasite for the common carp, at least in the flood-plain lakes of the Yangtze River. The two parasites were similar in many aspects of their morphology. Their bodies can both be separated into a dorsal part and a ventral disc, with the body surface of the dorsal part elevated by transverse folds, and the disc subdivided into alveoli by transverse and longitudinal septa, although the number of alveoli was different in the two species. The depression on the ventral surface of the neck region was prominent for both species, and their ventral disc was covered densely with non-ciliated bulbous papillae. The position of mouth, osmo-regulatory pore and marginal organ was also similar for A. ijimai and A. conchicola. However, microridges in the trough of the folds in the neck region and numerous small pits on the upper part of the septa were found exclusively in A. ijimai, but uniciliated sensory papillae in A. conchicola.

Genetic differentiation in populations of the cestode Bothriocephalus acheilognathi (Cestoda, Pseudophyllidea) as revealed by eight microsatellite markers

The genetic structure of populations of the fish cestode, Bothriocephalus acheilognathi collected from Bailianhe Reservoir (BLH), Changshou (CSH) and Liangzi (LZH) Lakes was investigated by using 8 microsatellite loci. A total of 108 adult worms were genotyped at each of the 8 loci. For the 3 populations, the mean number of alleles per locus ranged from 2.38 to 5.5, and the mean expected heterozygosity ranged from 0.432 to 0.559. The average polymorphic information content (PIC) was from 0.384 to 0.492. The significant F-is values indicated non-random mating within LZH and BLH populations. On the other hand, when samples were further classified into subpopulations at the level of host fish species, no or little heterozygote deficiency was detected at most loci, showing that cross-fertilization, predominantly, but not exclusively, must have occurred within the subpopulations. Microsatellite markers also revealed an unexpected high level of genetic differentiation, as measured by R-st and N-m values or by deltau(2) genetic distance among subpopulations from different hosts. Factors influencing the population genetic structure and the parasite host specificity are discussed.

Some nematodes of fishes from central China, with the redescription of Procamallanus (Spirocamallanus)fulvidraconis (Camallanidae)

The present paper comprises a systematic survey of nematodes based on helminthological examinations of 176 specimens of freshwater fishes, belonging to 22 species, from central China (mostly lakes in Hubei Province) collected during the autumn of 2001. The following six species were recorded: Procamallanus (Spirocamallanus) fulvidraconis Li, 1935, Camallanus cotti Fujita, 1927, Dentiphilometra monopteri Moravec et Wang, 2002, Pingus sinensis Hsu, 1933, Proleptinae gen. sp. larv., and Eustrongylides sp. larv. Data on their morphology, morphological variability, host range, prevalence, intensity and distribution are provided. SEM studies of P. fulvidraconis and larval Physalopterinae, used for the first time in these species, revealed some additional morphological details and made it possible to redescribe the former. In contrast to the existing description of P. fulvidraconis, this species was found to possess two spicules and a V-shaped gubernaculum with unequal arms (originally mistaken for the left spicule), as well as deirids, whose location can be considered an important taxonomic feature. Larvae of the Physalopterinae have not previously been reported from fishes in China. The finding of larval Eustrongylides in Paramisgurnus dabryanus represents a new host record. All but one nematode species from this zoogeographically interesting region are briefly described and illustrated.

Is the genus Digramma synonymous to the genus Ligula (Cestoda : Pseudophyllidea)? Evidence from ITS and 5 ' end 28S rDNA sequences

The genus Digramma (Cestoda: Pseudophyllidea) described by Cholodkovsky in 1915 differs from the genus Ligula only by the number of the reproductive organs per proglottis. However, the occurrence of transitional forms in Digramma raises much confusion concerning its generic validity. In the present study, cestodes previously designated as Digramma and Ligula were collected from lakes in the lower and middle reaches of the Yangtze River, and also from Qinghai Lake on Qingzang plateau, China. The entire internal transcribed spacer of the ribosomal DNA (ITS rDNA) and 5' end of 28S rDNA were compared between the Digramma and Ligula specimens. The low level of nucleotide variation between the two genera may imply that cestodes in the genus Digramma are paraphyletic to the Ligula genus, and Digramma is a synonym of Ligula. However, whether previously identified Digramma cestodes represent different species in the genus Ligula requires further investigation.

Myxobolus lentisuturalis sp n. (Myxozoa : Myxobolidae), a new muscle-infecting species from the Prussian carp, Carassius gibelio from China

A new highly pathogenic muscle-infecting species of the genus Myxobolus Butschli, 1882 is described from the Prussian carp, Carassius gibelio (Bloch, 1782) using spore morphology and SSU rDNA sequence data. Phylogenetic analyses elucidated relationship of the newly described Myxobolus lentisuturalis to other Myxobolus species and supported its position of an independent species.

Observations on two species of phyllodistomes (Trematoda : Gorgoderidae) from fishes of the Hubei Province, central China, with an erection of Neophyllodistomum gen. n.

Two rare trematode species so far reported in the genus Phyllodistomum Braun, 1899, P. pawlovskii (Zmeev, 1936) and P. serrispatula Chin, 1963, were found in the urinary system the yellow catfish Pelteobagrus fulvidraco (Richardson) of Bao'an Lake (prevalence 8 %) and the swamp eel Monopterus albus (Zouiev) of Liangzi Lake (prevalence 6 9/6), respectively, from the Hubei Province, central China. In contrast to the original description, P. pawlovskii showed a considerable morphological variability particularly in the shape, size and topography of testes. A unique morphological feature of P. serrispatula is the presence of many conspicuous lateral outgrowths on the hindbody and, principally based on this character, a new genus Neophyl-lodistomum is erected (type species N. serrispatula (Chin, 1963) comb. n.)) to accommodate this species. Both trematode species are briefly redescribed. P. pawlovskii and N. serrispatula are reported for the first time from the Hubei Province and the former from the Yangtze River drainage system.

Molecular variation of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda : Pseudophyllidea) in different fish host species based on ITS rDNA sequences

The molecular variation in Bothriocephalus acheilognathi Yamaguti, 1934 from 11 species of freshwater fish collected in Australia, China, the Czech Republic, England and Hawaii was investigated by determining the nucleotide sequences of the internal transcribed spacer region. The length of the first and second internal transcribed spacer sequences of multiple individuals ranged from 553 to 571 bp and 553 to 615 bp, and the G + C content from 53.1 to 53.5%. The percentage sequence divergence varied between 0 and 0.9% in the ITS1 and 0 and 6.6% in the ITS2, respectively, indicating the occurrence of intraspecific variation. It is demonstrated that the fragment length variation resulted primarily from microsatellite polymorphisms present in the ITS region, especially in the ITS2 region. Phylogenetic analyses revealed that B. acheilognathi examined in this study consisted of three closely related genotypes with certain degrees of host-specificity, and the genotype representing isolates from Cyprinus carpio L. was the most common and diverse form within the species B. acheilognathi.

Effects of Bothriocephalus acheilognathi on the polarization response of pronephric leucocytes of carp, Cyprinus carpio

An in vitro assay was used to examine the effect of Bothriocephalus acheilognathi Yamaguti, 1934 (Cestoda: Pseudophyllidea) on the polarization response of pronephric leucocytes of carp, Cyprinus carpio. Leucocytes, isolated from naive, naturally-infected fish and carp injected intraperitoneally with cestode extracts, were exposed to parasite extracts (protein concentrations 0-10.0 mu g ml(-1)), for up to 24 h in the presence or absence of carp serum. In general, polarization responses of the pronephric leucocytes, primarily neutrophils and eosinophils, increased with incubation time although there was no significant difference in the response induced by the different protein concentrations. Differences in the polarization response were, however, observed in naive, naturally infected and injected fish and the cells responded differently in the presence and absence of carp serum. In the absence of carp serum the polarization response of pronephric leucocytes in vitro was significantly reduced with cells obtained from injected and naturally infected fish compared with those obtained from naive carp. This suppression of leucocyte migration was however reduced by the addition of carp serum to the in vitro system. The role of this interaction between the possible suppression of polarization induced by the parasite and stimulation by serum is discussed.

The distribution of monogenean parasites on aquatic vertebrates inhabiting Chinese inland waters

The general distribution of monogenean parasites of aquatic vertebrates inhabiting Chinese inland waters is summarised. Five hundred and seventy-two out of a total of 581 species of monogeneans were discovered on fish, while only nine species were found on Amphibia and Reptilia. Most dactylogyrids and diplozoids parasitise cyprinids, while ancyrocephalids occur on silurids and cyprinids, and gyrodactylids are found mainly on cyprinids and cobitids. Analyses of host-specificity and host-diversity suggests that the family Ancyrocephalidae should be divided into several families parallel with the Dactylogyridae. Of 12 subfamilies of the Cyprinidae, only the Gobiobotinae was found to be free of infection with monogeneans, and the genus Dactylogyrus has more species than any other monogenean genus associated with every cyprinid subfamily.

Antibody response of carp, Cyprinus carpio to the cestode, Bothriocephalus acheilognathi

The humoral antibody response and the number of pronephric antibody-secreting cells were examined in naturally Bothriocephalus acheilognathi-infected carp. Cyprinus carpio, and in those injected intraperitoneally with an extract of the cestode. In the extract-injected fish, specific antibody was detected 3 weeks after a second injection given 2 weeks after the primary injection, and antibody levels persisted for more than 200 days. A third injection also enhanced the antibody level in the extract-injected carp. The numbers of antibody-secreting cells were significantly higher in carp injected 3 times with the extract than in the control. In naturally-infected fish, the serum antibody levels and the number of pronephric antibody-secreting cells were higher in infected fish than in uninfected individuals although this difference was not statistically significant. The relevance of these results to immune protection against infection is discussed.

Evidence of host blood feeding by the monogenean, Ancyrocephalus mogurndae (Monogenea : Ancyrocephalidae) from the gills of the mandarin fish, Siniperca chuatsi

By examining iron contents, it is demonstrated that the monogenean Ancyrocephalus mogurndae (Yamaguti, 1940) feeds on the blood of its host, the mandarin fish Siniperca chuatsi (Basilewsky). The iron content and then the quantity of blood necessary to produce this amount of iron are found different in young and fully-matured worms. Young worms contain higher levels of iron and estimated amount of blood. It is suggested that A. mogurndae may start to feed on host blood as attached on gills, and the amount of blood ingested by young worms may vary from 0.01 to 1.00 mu l before reproduction. The difference between young and fully-matured worms may be accounted for by the elimination of haematin and change of food composition in matured worms and may also be affected by reproduction. Experimental infections of the monogenean may provide supportive information for explaining the difference, and further studies should also examine the effect of immune components in host blood ol mucus on the intestines of the parasite.

Co-occurrence and microhabitat of Ancyrocephalus mogurndae (Monogenea) and Henneguya weishanensis (Myxosporea) on gills of the mandarin fish, Siniperca chuatsi

Seasonal variations in a population of the monogenean Ancyrocephalus mogurndae Gussev, 1955 were investigated on gills of cage-cultured mandarin fish, Siniperca chuatsi (Basilewsky), during the period from April 1994 to April 1995. The abundance of A. mogurndae peaked in late spring and summer. Prevalence was high (75-100 %) throughout the study period, and did not vary significantly between months. More than 50 % of all monogeneans were found on the first and second gill arches, except one occasion when the fourth gill arch had the majority in April 1995. The niche breadths were significantly correlated with the population abundance. A coexistent parasitic myxosporean, Henneguya weishanensis Hu, 1965, on the gills of the fish was found to have little influence on the gill-arch preference of the monogenean, although the monogenean abundance was higher in those fish infected with the myxosporean.