W. J. Yao

Ig heavy chain genes and their locus in grass carp Ctenopharyngodon idella.

The cDNA and genomic sequences of IgD and IgZ were characterized in grass carp Ctenopharyngodon idella in the present study, and with the identification of a BAC clone covering zeta, mu, and delta genes, the IgH locus containing these Ig genes and other V, D, J genes was also illustrated in this fish. Secretory and membrane-bound IgZ were identified, with two transmembrane exons spliced within the CH4 exon, as reported in IgM of mammals and IgZ in other teleost fish. The first and second constant domains of IgZ shows more than 90% nucleotide identity with respective domains of grass carp IgM. The IgD has a structure of delta1-(delta2-delta3-delta4)(2)-delta5-delta6-delta7-TM-UTR, with the repeat of delta2-delta3-delta4; but intron was not found between the two repeat, i.e. between the first delta2-delta3-delta4 (delta2.1-delta3.1-delta4.1) and the second delta2-delta3-delta4 (delta2.2-delta3.2-delta4.2), and the intron between delta3.1 and delta4.1 was much shorter than the intron between delta3.2 and delta4.2. The genomic organization of the IgH locus has a pattern of Vn-Dn-Jn-Czeta-Dn-Jn-Cmu-Cdelta, as reported in other teleost fish. Thirteen V(H), fourteen D, and twelve J(H) genes were observed in this locus, with the similarity of three D segments and four J(H) segments being the same in the upstream of Czeta and Cmu. The transcriptional enhancer located at the mu-delta intergenic region was also analyzed and it seems possible that this enhancer is functional as verified in zebrafish and channel catfish.

Ontogeny of IgM-producing cells in the mandarin fish Siniperca chuatsi identified by in situ hybridisation

The ontogeny of IgM-producing cells was studied in juvenile mandarin fish Siniperca chuatsi, an important fish in Chinas aquaculture sector. The IgM-producing cells were localised through in situ hybridisation with a probe complementary to the Ig mu-chain in lymphoid-related tissues, including head kidney, spleen, thymus, intestine and gills. In head kidney, transcripts of Ig mu were first detected at 20days post-hatching (dph) with a few positive signals, and the number of IgM-producing cells increased obviously from 39dph onwards. At 136dph, a large amount of positive cells were observed in the entire organ with clusters of these cells located around the blood vessels. In spleen, IgM-producing cells were found from 26dph onwards, followed by an increase until 67dph; clusters of positive cells were also detected around blood vessels at 102dph. In thymus, IgM-producing cells were first observed at 39dph; thereafter, no obvious increase was detected until 78dph. The positive cells in thymus were distributed mainly in the outer zone of thymus. A few IgM-producing cells were still observed in thymus of 1-year-old mandarin fish. IgM-producing cells were not detected in the intestine until 87dph, with several discrete positively stained cells distributed in the lamina propria. IgM-producing cells, scattered mainly in primary gill filaments around blood vessels, were detected in gills from 90dph. As in other teleosts, these results indicated that the head kidney appears to be the primary organ for IgM production in mandarin fish, and IgM-producing cells exist in all organs examined in the present study, implying their lymphoid role in fish. In addition, it is suggested that vaccination after 20dph may be much more effective in mandarin fish.

Phylogeny of freshwater parasitic copepods in the Ergasilidae (Copepoda: Poecilostomatoida) based on 18S and 28S rDNA sequences

The phylogenetic relationships among the Ergasilidae genera are poorly understood. In this study, 14 species from four genera in the Ergasilidae including Sinergasilus, Ergasilus, Pseudergasilus, and Paraergasilus were collected in China, and their phylogenetic relationships were examined using neighbor-joining, maximum parsimony, maximum likelihood, and Bayesian inference methods based on partial sequences of 18S and 28S ribosomal deoxyribonucleic acid, respectively. All the analyses suggest that the Sinergasilus and Paraergasilus are both monophyletic, but the Ergasilus is polyphyletic rather than monophyletic. Considering the relationships among the four genera, the phylogenetic analyses and subsequent hypothesis tests all suggest that Pseudergasilus clustered with some Ergasilus species may have a closer relationship with Sinergasilus rather than with Paraergasilus. It is proposed that the Sinergasilus and the Pseudergasilus species might have evolved from Ergasilus species.

Phylogeny of diplozoids in five genera of the subfamily Diplozoinae Palombi, 1949 as inferred from ITS-2 rDNA sequences.

The phylogenetic relationship of 5 genera, i.e. Diplozoon Nordmann, 1832, Paradiplozoon Achmerov, 1974, Inustiatus Khotenovsky, 1978, Sindiplozoon Khotenovsky, 1981, and Eudiplozoon Khotenovsky, 1985 in the subfamily Diplozoinae Palombi, 1949 (Monogenea, Polyopisthocotylea) was inferred from rDNA ITS-2 region using neighbour-joining (NJ), maximum likelihood (ML) and Bayesian methods. The phylogenetic trees produced by using NJ, ML and Bayesian methods exhibit essentially the same topology. Surprisingly, freshwater species of Paradiplozoon from Europe clustered together with species of Diplozoon, but separated from Chinese Paradiplozoon species. The results of molecular phylogeny and lower level of divergence (4.1-15.7%) in ITS-2 rDNA among Paradiplozoon from Europe and Diplozoon and, on the other hand, high level of divergence (45.3-53.7%) among Paradiplozoon species from Europe and China might indicate the non-monophyletic origin of the genus Paradiplozoon. Also, the generic status of European Paradiplozoon needs to be revised. The species of Paradiplozoon in China is a basal group in Diplozoinae as revealed by NJ and Bayesian methods, and Sindiplozoon appears to be closely related to European Paradiplozoon and Diplozoon with their relationship to Eudiplozoon and Inustiatus being unresolved.

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.

Occurrence of Camallanus cotti in greatly diverse fish species from Danjiangkou Reservoir in central China.

Two thousand four hundred fifty-eight fish comprised of 53 species were captured in the Danjiangkou Reservoir, in the northwestern part of Hubei Province, central China during 2004, to examine Camallanus cotti infections. We found that 19 cypriniform, 3 siluriforme, and 4 perciforme fishes were infected by the nematode. Our study revealed the species, Hemiculter bleekeri bleekeri, Culter oxycephaloide, Pseudolaubuca sinensis, Acanthobrama simony, Mylopharyngodon piceus, Ctenopharyngodon idella, Gnathopogon imberbis, G. argentatus, Saurogobio dabryi, S. dumerili, Gobiobotia ichangensis, Liobagrus marginatoides, and Ctenogobius shennongensis as new hosts of the worm. The number and range of fish host species found in this survey were much greater than any of the previous investigations. The mean prevalence, prevalence, mean abundance, and intensity of infection varied in different fish species, indicating a possible host preference. Moreover, we suggest that this nematode is a native parasite of cypriniform fishes in China, perhaps initially in the reaches of the Yangtze River.

Utility of ITS1–5.8S–ITS2 sequences for species discrimination and phylogenetic inference of two closely related bucephalid digeneans (Digenea: Bucephalidae): Dollfustrema vaneyi and Dollfustrema hefeiensis

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.

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 Fis 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 delta(u)2 genetic distance among subpopulations from different hosts. Factors influencing the population genetic structure and the parasite host specificity are discussed.

Is the genus Digramma synonymous to the genus Ligula (Cestoda: Pseudophyllidea)?

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.

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.