The co-evolutionary relationship between bitterling fishes and freshwater mussels: insights from interspecific comparisons

Hypothesis: In parasites that use hosts for offspring development, adults may base oviposition decisions on a range of host traits related either to host quality or the co-evolutionary relationship between parasite and host. We examined whether host quality or co-evolutionary dynamics drive the use of hosts in the bitterling-mussel relationship. Organisms: Six species of bitterling fish (Acheilognathinae) and eight species of freshwater mussels (Unionidae, Corbiculidae) that are used by bitterling for oviposition. Site of experiments: Experimental tanks in Wuhan, China, at the site of the natural distribution of the studied species. Methods: Three experiments that controlled for host accessibility and interspecific interactions were conducted to identify host preferences among bitterling fishes and their mussel hosts. We started with a broad interspecific comparison. We then tested bitterling behavioural choices, their temporal stability, and mussel host ejection behaviour of the eggs of generalist and specialist bitterling species. Finally, we measured host mussel quality based on respiration rate and used published studies on mussel gill structure to infer mussel suitability as hosts for bitterling eggs. Results: We found significant interspecific differences among bitterling species in their use of mussel hosts. Bitterling species varied in their level of host specificity and identity of preferred hosts. Host preferences were flexible even among apparently specialized species and fishes switched their preferences adaptively when the quality of individuals of preferred host species declined. Mussels varied considerably in their response to oviposition through egg ejections. Host preference by a generalist bitterling species correlated positively with host quality measured as the efficiency of the mussel gills to extract oxygen from inhaled water. Host ability to eject bitterling eggs correlated positively with their relative respiration rate, probably due to a higher velocity of water circulating in the mussel gill chamber.

Growth and energy budget of F-2 'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F-2 'all-fish' growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29.2 degrees C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I-E), and the proportion of I-E utilized for heat production (H-E) were significantly higher in the transgenics than in the controls. The proportion of I-E directed to waste products [faecal energy (F-E) and excretory energy loss (Z(E) + U-E) where Z(E) is through the gills and U-E through the kidney], and the proportion of metabolizable energy (M-E) for recovered energy (R-E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I-E = 19.3 F-E + 6.0 (Z(E) + U-E) + 45.2 H-E + 29.5 R-E or 100 M-E = 60.5 H-E + 39.5 R-E. The average energy budget equation of the controls was: 100 I-E = 25.2 F-E + 7.4 (Z(E) + U-E) + 35.5 H-E + 31.9 R-E or 100 M-E = 52.7 H-E + 47.3 R-E. These findings indicate that the high growth rate of 'all-fish' transgenic common carp relative to their non-transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.

Evidence of host specificity and congruence between phylogenies of bitterling and freshwater mussels

Evidence of host specificity and congruence between phylogenies of bitterling and freshwater mussels. Zoological Studies 45(3): 428-434. Bitterling (Cyprinidae: Acheilognathinae) are freshwater fishes with a unique spawning relationship with freshwater mussels on whose gills they lay their eggs. During the breeding season of bitterling fishes, we collected 843 mussels belonging to 16 species from Lake Qinglan, central China and examined their gill chambers for the presence of bitterling larvae. Three species of bitterling larvae were identified; Acheilognathus tonkinensis, Ach. cf. meridianus, and Ach. barbatulus, in 3 species of mussel: Unio douglasiae, Lamprotula caveata, and L. tortuosa, suggesting host specialization. Using our own and other published data, we compared the respective phylogenies of bitterling and mussels, but failed to show clear congruence. However, broad specializations are evident, with Acheilognathus and Tanakia showing preferences for mussels with a relatively simple gill structure (Ableminae), and Rhodeus spp. showing preferences for mussels of the Anodontinae and Unioninae, which have more-complex gill structures.

Population dynamics of the water mite Unionicola arcuata (Unionicolidae) in the freshwater bivalve Cristaria plicata (Unionidae) in Poyang Lake, eastern China

Population dynamics of the water mite Unionicola arcuata were investigated in the freshwater bivalve Cristaria plicata during the period from January to December 2002 in Poyang Lake, East China. A pattern of seasonal variation was observed, with prevalence and abundance peaking in early spring and autumn. The number of mites in individual hosts was significantly correlated with the size, but not with the sex, of bivalves. The change in infection level of mites on different infection sites in C. plicata was significant, with > 58% of the mites found on the outer and inner gills, indicating that U. arcuata shows site preference.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

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.

Cloning, sequencing and expression analysis of cDNA encoding a constitutive heat shock protein 70 (HSC70) in Fenneropenaeus chinensis

The cDNA encoding hsc70 of Chinese shrimp Fenneropenaeus chinensis was cloned from hepatopancreas by RT-PCR based on its EST sequence. The full length cDNA of 2090 bp contained an open reading frame of 1956 nucleotides and partial 5'- and 3'-untranslated region(5'- and 3'-UTR). PCR amplification and sequencing analysis showed the existence of introns in the region of 1-547 bp, but they did not exist in the region of 548-2090 bp of hsc70 cDNA. When the deduced 652 amino acid sequence of HSC70 was compared with the members of HSP70 family from other organisms, the results showed 85.9% similarity with HSC71 from Oncorhynchus mykiss and HSC70 from Homo sapiens. It also exhibited 85.8% similarity with HSP70 from Mus musculu and 85.4% with HSC70 from Manduca sexta. Expression analysis showed that hsc70 mRNA was espressed constitutively in hepatopancreas, muscle, eyestalks, haemocytes, heart, ovary, intestine and gills in Fenneropenaeus chinensis. No difference could be detected on hsc70 mRNA level in muscle between heat-shocked and control animals.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri. (c) 2006 Elsevier Ltd. All rights reserved.

Molecular cloning and responsive expression to injury stimulus of a defender against cell death 1 (DAD1) gene from bay scallops Argopecten irradians

Apoptosis is an active process of cell death, which is an integral part of growth and development in multicellular organisms. The defender against cell death 1 (DAD1), the regulatory protein to inhibit the apoptosis process, was first cloned from the bay scallop Argopecten irradians by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA end (RACE). The full-length cDNA of the A. irradians DAD1 was 607 bp, consist of a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 205 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 339 bp. The deduced amino acid sequence of the A. irradians DAD1 showed 75.5% identity to Araneus ventricosus, 74.5% to Drosophila melanogaster, and 73.6% to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. Excluding the Saccharomyces cerevisiae DAD1 homologue, all animal DAD1 including A. irradians DAD1 homologue formed a subgroup and all plant DAD1 proteins formed another subgroup in the phylogenetic analysis. The A. irradians DAD1 was expressed in all examined tissues including adductor muscle, mantle, gills, digestive gland, gonad and hemolymph, suggesting that A. irradians DAD1 is expressed in most body tissues. Furthermore, the mRNA expression levels of A. irradians DAD1 gene of hemolymph were particularly high after injury, suggesting that the gene is responsive to injury stimuli.

A Dicer-1 gene from white shrimp Litopenaeus vannamei: Expression pattern in the processes of immune response and larval development

Dicer is a member of the RNAase III family which catalyzes the cleavage of double-stranded RNA to small interfering RNAs and micro RNAs, and then directs sequence-specific gene silencing. In this paper, the full-length cDNA of Dicer-1 was cloned from white shrimp Litopenaeus vannamei (designated as LvDcr1). It was of 7636 bp, including a poly A tail, a 5' UTR of 136 bp, a 3' UTR of 78 bp, and an open reading frame (ORF) of 7422 bp encoding a putative protein of 2473 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other Dicer-1 homologues and showed the highest (97.7%) similarity to the Dicer-1 from tiger shrimp Penaeus mondon. Quantitative real-time PCR was employed to investigate the tissue distribution of LvDcr1 mRNA, and its expression in shrimps under virus challenge and larvae at different developmental stages. The LvDcr1 mRNA could be detected in all examined tissues with the highest expression level in hemocyte, and was up-regulated in hemocytes and gills after virus injection. These results indicated that LvDcr1 was involved in antiviral defense in adult shrimp. During the developmental stages from fertilized egg to postlarva VII, LvDcr1 was constitutively expressed at all examined development stages, but the expression level varied significantly. The highest expression level was observed in fertilized eggs and followed a decrease from fertilized egg to nauplius I stage. Then, the higher levels of expression were detected at nauplius V and postlarva stages. LvDcr1 expression regularly increased at the upper phase of nauplius, zoea and mysis stages than their prophase. The different expression of LvDcr1 in the larval stages could provide clues for understanding the early innate immunity in the process of shrimp larval development. (C) 2010 Elsevier Ltd. All rights reserved.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

Two trichodinid ectoparasites from marine molluscs in the Yellow Sea, off China, with the description of Trichodina caecellae n. sp (Protozoa : Ciliophora : Peritrichia)

The morphology and infraciliature of two ectoparasitic ciliates, Trichodina caecellae n. sp. and T. ruditapicis Xu, Song & Warren, 2000, parasitising the gills of marine molluscs from the Shandong coast of the Yellow Sea, China, were investigated following wet silver nitrate and protargol impregnation. T. caecellae was found on the small marine sand clam Caecella chinensis Deshayes and is distinguished mainly by the acute triangle-like blade, the very delicate central part and the needle-shaped ray. T. ruditapicis was studied based on four populations from three clams: two populations from Ruditapes philippinarum (Adams) and one each from Saxidomus purpuratus (Sowerby) and Solen grandis Dunker. All four populations fell within the range of morphometry and agreed closely in the overall appearance of the adhesive disc. However, variability was found in the denticle structure, especially in populations from different host clams. Our observations suggest that denticle morphology may be more or less variable between and within populations, and that such minor differences should not be overestimated. It should be emphasised that, except for the denticle morphology, the bright granules or circles in the centre of the adhesive disc represent another important feature facilitating the identification of this trichodinid species.

Trichodinid ectoparasites (Ciliophora, Peritrichia) from the tiger puffer Takifugu rubripes in the Yellow Sea, with revision of Trichodina jadranica Raabe,1958

Two ectoparasitic ciliates, Trichodina fugu Imai et al., 1997 and T. jadranica Raabe, 1958, found on the gills and skin of the maricultured tiger puffer Takifugu rubripes on the China coast of the Yellow Sea, were studied using the dry silver nitrate method. Trichodina fugu is distinguished by its almost rod-shaped denticle blades. Trichodina jadranica is usually described as a small trichodinid with a clear central circle in the adhesive disc and with a low number of denticles. However, the data available suggest that the species is highly variable in morphometry and the Chinese population represents the largest in body size and denticle dimensions found to date. Based on the revision of T. jadranica, two major morphotypes, each represented by several populations are classified, differing in the shape of the blades, viz., distinctly curved and sickle-shaped with pointed distal ends (as in the classical T. jadranica) vs. less curved and more or less rectangle-like with rounded distal ends (as in T. domerguei gobii). Trichodina domerguei gobii Raabe,.1959, which was synonymised with T. jadranica, is thus elevated to species rank. Furthermore, Trichodina jadranica noblei Lom, 1970 has straight and stout blades with broadly rounded distal ends and is raised to species rank: T noblei Lom, 1970. Trichodina jadranica sensu Xu et al., 1995 shows high similarities in denticle shape and dimensions as well as the central granule pattern with T chlamydis Xu et al., 1999. Thus, it is synonymised with the latter species.