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In this report, recombinant interleukin-8 (rIL-8) was produced and its activity tested for the first time in fish. The rainbow trout rIL-8 was produced in Escherichia coli and purified using a 6xHis tag at the N-terminus. The rIL-8 induced a dose-dependent migration of head kidney leukocytes at concentrations from 0.1 to 10 ng/ml, with a peak response at 1 ng/ml. Trout rIL-8 also had a significant effect on superoxide production by head kidney cells, with maximal activity at 0.1 and 1 ng/ml. When injected intraperitoneally into trout, rIL-8 had a clear effect on total leukocyte number in the peritoneal cavity, with increasing doses (up to 5 microg) eliciting more cells. Of three leukocyte types distinguished, neutrophils were the dominant cell type, especially at higher rIL-8 concentrations. In contrast, the proportion of macrophages and lymphocytes decreased with rIL-8 administration, suggesting that they were not attracted at the same rate as neutrophils.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is a crucial component of almost the entire tumor necrosis factor receptor superfamily signaling pathway. In the present study, a TRAF2 gene has been cloned from grass carp (Ctenopharyngodon idella) by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The full-length cDNA is 3162 bp, including a 60 bp 5' untranslated region (UTR), a 1611 bp open reading frame, and a 1491 bp 3' UTR. The polyadenylation signal (AATAAA) and the mRNA instability motifs (ATTTTA, ATTTA) were followed by a poly(A) tail in the 3' UTR. No signal peptide or transmembrane region has been found in the putative amino acids of grass carp TRAF2 (gcTRAF2). Phylogenetic tree analysis clearly showed that gcTRAF2 is nearest to the TRAF2 gene of goldfish. The identity of gcTRAF2 with its homologs in other vertebrates ranges from 56% to 97%. It is characterized by one RING-type signature at the N-terminus, one zinc finger in the middle part, and one conserved TRAF domain consisting of a C-proximal (TRAF-C) subdomain and a N-proximal (TRAF-N) subdomain. The identity of TRAF-C among all TRAF2 homologs in vertebrates varies from 78% to 97%, whereas the identity of TRAF-N ranges from 56% to 100%. The recombinant gcTRAF2 has been expressed in Escherichia coli using pET-32a expression vector. The rabbit anti-gcTRAF2 polyclonal antibody was obtained. The expression of gcTRAF2 in different organs was examined by real-time quantitative polymerase chain reaction and Western blot analysis. It was widely distributed in heart, head kidney, thymus, brain, gill, liver, spleen, and trunk kidney. This is the first report of a TRAF2 homolog molecule in fish.

The complete internal transcribed spacer 1 (ITS1), 5.8S ribosomal DNA, and ITS2 region of the ribosomal DNA from 60 specimens belonging to two closely related bucephalid digeneans (Dollfustrema vaneyi and Dollfustrema hefeiensis) from different localities, hosts, and microhabitat sites were cloned to examine the level of sequence variation and the taxonomic levels to show utility in species identification and phylogeny estimation. Our data show that these molecular markers can help to discriminate the two species, which are morphologically very close and difficult to separate by classical methods. We found 21 haplotypes defined by 44 polymorphic positions in 38 individuals of D. vaneyi, and 16 haplotypes defined by 43 polymorphic positions in 22 individuals of D. hefeiensis. There is no shared haplotypes between the two species. Haplotype rather than nucleotide diversity is similar between the two species. Phylogenetic analyses reveal two robustly supported clades, one corresponding to D. vaneyi and the other corresponding to D. hefeiensis. However, the population structures between the two species seem to be incongruent and show no geographic and host-specific structure among them, further indicating that the two species may have had a more complex evolutionary history than expected.

The gene of interferon regulatory factor-2 (IRF-2) has been cloned from the mandarin fish (Siniperca chuatsi). The IRF-2 gene has 6,418 nucleotides (nt) and contains eight exons and seven introns, encoding two mRNAs. The two IRF-2 mRNAs each contained an open reading frame of 873 nt, which both translate into the same 291 amino acids but differed in their 5' untranslated region: one mRNA was transcribed initially from the exon 1 bypassing exon 2, while the other was transcribed from the exon 2. The microsatellites (CA repeats) could be found in the carboxyl terminal region of mandarin fish IRF-2, which result in the truncated form molecules. The microsatellites' polymorphism was investigated, and eight alleles were found in 16 individuals. The microsatellites were also examined in IRF-2 of several freshwater perciform fishes. The transcription of the IRF-2 in different tissues with or without poly inosine-cytidine stimulation was analyzed by real-time PCR, and the constitutive transcription of both molecules could be detected in all the tissues examined.

The first description of the male and a redescription of the female of the nematode Philometra clavaeceps Dogiel and Akhmerov, 1959, a parasite of east Asian cyprinids, are presented on the basis of specimens collected from Culter erythropterus Basilewsky and Culter dabryi Bleeker from Liangzi Lake (the Yangtze River basin), Hubei Province, central China. Gravid females from the fish abdominal cavity, penetrating often into ovaries, occurred in May-June, whereas conspecific males and young mature females on the swimbladder were recorded in January. Philometra clavaeceps seems to have a pronounced annual maturation cycle in the locality. The finding of P. clavaeceps in C. dabryi represents a new host record.

Further to the previous finding of the rainbow trout rtCATH_1 gene, this paper describes three more cathelicidin genes found in salmonids: two in Atlantic salmon, named asCATH_1 and asCATH_2, and one in rainbow trout, named rtCATH_2. All the three new salmonid cathelicidin genes share the common characteristics of mammalian cathelicidin genes, such as consisting of four exons and possessing a highly conserved preproregion and four invariant cysteines clustered in the C-terminal region of the cathelin-like domain. The asCATH_1 gene is homologous to the rainbow trout rtCATH_1 gene, in that it possesses three repeat motifs of TGGGGGTGGC in exon IV and two cysteine residues in the predicted mature peptide, while the asCATH_2 gene and rtCATH_2 gene are homologues of each other, with 96% nucleotide identity. Salmonid cathelicidins possess the same elastase-sensitive residue, threonine, as hagfish cathelicidins and the rabbit CAP18 molecule. The cleavage site of the four salmonid cathelicidins is within a conserved amino acid motif of QKIRTRR, which is at the beginning of the sequence encoded by exon IV. Two 36-residue peptides corresponding to the core part of rtCATH_1 and rtCATH_2 were chemically synthesized and shown to exhibit potent antimicrobial activity. rtCATH_2 was expressed constitutively in gill, head kidney, intestine, skin and spleen, while the expression of rtCATH_1 was inducible in gill, head kidney, and spleen after bacterial challenge. Four cathelicidin genes have now been characterized in salmonids and two were identified in hagfish, confirming that cathelicidin genes evolved early and are likely present in all vertebrates.

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 Clariasfuscus (Lacépède) 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, Wu et Yu, 1997 are regarded to be junior synonyms of S. gigi. Spinitectus petrowi was not previously reported from China.

The nematode Camallanus hypophthalmichthys Dogel and Akhmerov, 1959 is redescribed from specimens collected from the intestine of the bighead carp Aristichthys nobilis, from Liangzihu Lake (Yangtze River basin), Hubei Province, central China. The light and scanning electron microscopical examination made it possible to study in detail the morphology of this so far little-known species and to confirm its validity. The main specific features of C. hypophthalmichthys distinguishing it from the most similar Camallanus spp. is the presence of 3 small caudal processes on the male tail tip, 13-16 longitudinal ridges on the inner surface of the valve of the buccal capsule, and the arrangement of preanal and postanal genital papillae in the male. This finding represents a new host record, the first record of this parasite in the Yangtze River basin, and the first documented record of C. hypophthalmichthys from China. Camallanus hypophthalmichthys is considered a specific intestinal parasite of fishes of the cyprinid Hypophthalmichthyinae.

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) microm long, 6.7 (6-8) microm wide and 5.5 microm thick. Sutural ridge is straight and narrow. Mucus envelope is lacking. Two equal-sized elongate pyriform polar capsules are 8.5 microm long and 2.5 microm 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.

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 Hsü, 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.

The current knowledge concerning the structure of fish immunoglobulin (Ig) genes and the genetic mechanisms in generating fish antibody diversities is reviewed. The heavy (H-) and light (L-) chains of the immunoglobulin are encoded by multigenic loci on separate chromosomes. In different taxa of fish, the Ig genes are organized in different patterns. Fish antibody can recognize various foreign antigens,and the antibody diversity is generated by the following genetic mechanisms:the sequence diversity within the segments encoding the variable domain, the combinatorial diversity of V (D) J segments, the imprecision of rearrangements,gene conversion,somatic hypermutation,and the combination of H- and L-chain.

Genetic markers including allozyme,mtDNA,RAPD/RFLP and micro DNA have been used in the research of helminth population genetics. Available data on helminth genetic variability have shown that most helminth populations exhibit different levels of genetic variation resulting mainly from the pattern of life cycle,geographical distribution and parasite-host interaction,and several hypotheses have been proposed to explain the genetic variation.

A new highly pathogenic muscle-infecting species of the genus Myxobolus Bütschli, 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.