Ming-Quan Xie

The radiation of Haliotrema (Monogenea: Dactylogyridae: Ancyrocephalinae): molecular evidence and explanation inferred from LSU rDNA sequences

The D1-D2 domains of LSU rDNA were used to reconstruct the phylogenetic relationships within the Ancyrocephalinae (Monogenea: Dactylogyridae) utilizing maximum-parsimony (MP), maximum-likelihood (ML), minimum evolution (ME) and neighbour-joining (NJ) methods. A total of 32 monogenean taxa were examined in the present study, including 9 Haliotrema species and 13 other species representing the Ancyrocephalinae, 4 Thaparocleidus species representing the Ancylodiscoididae, and 6 species representing the Diplectanidae which were used as multiple outgroups. All 4 analyses (i.e. MP, ML, ME and NJ) inferred the same interrelationship pattern: (Diplectanidae, (Ancylodiscoididae, Dactylogyridae)) with high bootstrap support. However, 9 Haliotrema species were dispersed to form 4 clades together with species from other genera, indicating the apparent non-monophyly of Haliotrema. Three major groups were defined based on reconstructed phylogenetic trees to explain the radiation of Haliotrema species. The morphology of the reproductive organ, particularly the male copulatory organ (MCO), was discussed to further understand the formation of each group. (1) Results of the present study indicated an intimate relationship among Metahaliotrema (2 species), Protogyrodactylus (4 species) and Haliotrema (2 of 9 species), and notably, all these species share vagina-absence. (2) Based on the present molecular analyses and the morphological characters of the MCO, we propose to transfer H. spirotubiforum and the undetermined Haliotrema sp. ZHDDb to Euryhaliotrema as new combinations. (3) We propose to erect a new genus to accommodate the Haliotrema species with horn-like shaped MCO. Taxonomic implications of the present molecular phylogenetic analyses are discussed. A wider range of taxa and more DNA markers displaying various evolutionary rates should be used to estimate phylogenetic relationships among species within the Ancyrocephalinae and Ancylodiscoididae in further studies.

Molecular and morphological evidence indicates that Pseudorhabdosynochus lantauensis (Monogenea: Diplectanidae) represents two species

Sequences of the first internal transcribed spacer (ITS-1) and the D1-D3 domains of the large subunit (LSU) of the ribosomal DNA (rDNA) were determined for multiple specimens of 4 operational taxonomic units (OTUs) of the monogenean, Pseudorhabdosynochus lantauensis. OTUs were defined based on their collecting localities, host and/or morphological characteristics. All P. lantauensis specimens of one group (OTUs 1 and 3) differed in their sequences of the ITS-1 and partial LSU rDNA when compared with specimens of a second group (OTUs 2 and 4) by 12% and 2%, respectively. Results of the phylogenetic analyses of the LSU rDNA sequence data showed total (100%) bootstrap support for the separation of P. lantauensis into 2 distinct clades. At least 11 of the 18 nucleotide differences in the LSU sequence between the two P. lantauensis clades were derived (i.e. autapomorphic) characters when the morphologically distinct species, P. epinepheli and P. coioidesis, were used as outgroups. Furthermore, there were several autapomorphic character states for each P. lantauensis clade. This provides sufficient evidence to reject the null hypothesis that P. lantauensis represents a single species. Morphological and morphometric differences between these two clades provided additional strong support for the separation of P. lantauensis into two species. These two parasite species were found to co-exist on one of the two species of serranid fish (i.e. Epinephelus coioides) examined in the South China Sea (Guangdong Province, China).

A novel protein isolated from the serum of rabbitfish (Siganus oramin) is lethal to Cryptocaryon irritans

The susceptibility of eight marine fish species cultured in South China were tested for infection by the parasitic ciliate, Cryptocaryon irritans, via a challenge examination and an immobilization assay. All species of fish (representing six different families) that we investigated were infected by C. irritans except the rabbitfish (Siganus oramin), which displayed resistance to C. irritans infection. The infection intensity of rabbitfish (0.92+/-0.97, p<0.05) was significantly lower while the immobilization titres of rabbitfish serum were significantly higher (44.51+/-22.98, p<0.05) than the other seven species of fish. Additionally, the serum of the rabbitfish presented a strong killing effect to C. irritans in vitro. Light microscopy, scanning electron microscopy and fluorescence microscopy confirmed that rabbitfish serum could induce the theront cilia fall off, rupture of the cell membrane because of the swell and rupture of the macronucleus. Rabbitfish serum could also induce the rupture of the trophont membrane and content efflux. Herein a novel antiparasitic protein (APP) was isolated and purified from the serum of rabbitfish (S. oramin) by using a series of salting-out, cation exchange chromatography and two step of reversed phase high performance liquid chromatography (RP-HPLC). Analysis of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that APP was a homogenous polymeric protein with an N-terminal amino acid sequence of SSVEKNLAACLRDND. Its monomeric molecular mass, determined by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometer (MALDI-TOF-TOF-MS), was found to be 61,739.87 Da. Results of homology analyses indicated that this protein was a newly discovered functional protein in the rabbitfish serum. Laser confocal fluorescence microscopy conformed that the action site of the APP was mainly on the cell membrane and nucleus of theront, which agreed with the results of light microscopy, fluorescence microscopy and scanning electron microscopy. These findings suggest that this protein may contribute considerably to the innate host defence mechanism to combat microbes of the rabbitfish.

The mitochondrial genome of Polylabris halichoeres (Monogenea: Microcotylidae)

This study presents the complete mitochondrial (mt) genome of Polylabris halichoeres, which is the largest mt genome sequenced of monogeneans so far and the second complete sequence after Microcotyle sebastis from the Microcotylidae. It is basically similar to that of M. sebastis, with the exception of a high level of gene rearrangement located between trnC and trnL(UUR), a translocation of trnM and trnH, as well as a highly repetitive region (HRR) in the large non-coding region (NCR). We also find a series of trnI pseudogenes (ΨI) and one unknown short open reading frame (ORF) in the large NCR. Although the ORF cannot be unambiguously regarded as an atp8 gene, we cannot rule out the possibility that it has other functional importance, but it need further study in the future.

The complete mitochondrial genome of Tetrancistrum nebulosi (Monogenea: Ancyrocephalidae)

Abstract The first complete mitochondrial (mt) genome of Ancyrocephalidae is reported herein. The mt genome of Tetrancistrum nebulosi was 13,392 bp in length containing 12 protein-coding genes (lacking atp8), 22 tRNA genes and 2 rRNA genes. The longest non-coding region was located between nad5 and trnG, and the A + T content was 72.4%. All tRNAs had the typical clover-leaf secondary structure except for trnS1(AGN), trnR, trnF and trnQ. The rrnL and rrnS subunits were separated by trnC, as documented in the monopisthocotylean groups (Benedenia and Gyrodactylus species), while they were adjacent to each other in the polyopisthocotylean species (Microcotyle sebastis, Polylabris halichoeres and Pseudochauhanea macrorchis).

PCR-based identification and delineation of members within the Pseudorhabdosynochus lantauensis complex (Monogenea: Diplectanidae)

Molecular methods using genetic markers in the nuclear ribosomal DNA (rDNA) were established to identify and distinguish between two members within the Pseudorhabdosynochus lantauensis complex and two morphologically distinct congeners, Pseudorhabdosynochus epinepheli and Pseudorhabdosynochus coioidesis, from different marine fish species and various geographical origins. Supported by selective DNA sequencing, it was demonstrated that the polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism analysis of the first internal transcribed spacer and restriction fragment length polymorphism analysis of a variable region (representing the D1–D3 domains) in the large subunit of rDNA achieved the identification and delineation of all four taxa examined. These PCR-based approaches provide useful complementary tools to traditional methods for the accurate identification of species within the genus Pseudorhabdosynochus (irrespective of developmental stage) and have major implications for studying the ecology, transmission, and population genetic structures of these and other related parasites and for the prevention and control of the diseases they cause.

The effect of turtle (Trachemys scripta elegans) shell extract in normal and cyclophosphamide-treated mice

Abstract In the present study, the immune effect of turtle shell extract on normal and cyclophosphamide (CP)-treated mice was determined. Mice treated with the turtle shell extract and CP had significantly (p<0.05) elevated levels of serum IgG, ATL, ATS and CREA compared with CP-treated mice. The turtle shell extract-treated mice had significantly elevated serum IgG (p<0.001), reduced serum CREA level (p<0.05), but the serum ATL, ATS and BUN did not change significantly compared with normal mice. Treatment with the turtle shell extract significantly improved the CD3+, CD4+CD8−, CD4−CD8+ and CD4+CD8+ cells percentages (p<0.05) in normal mice, and also significantly improved CD3+ and CD4+CD8− cells percentages in CP-treated mice (p<0.05). There was significant difference (p<0.001) in the proliferation rate of PHA-stimulated spleen lymphocyte in vitro between the turtle shell extract + CP-treated mice and CP-treated mice, and also between the turtle shell extract-treated mice and normal mice (p<0.05). These results demonstrate that the turtle shell extract can up-regulate the immune states of normal and CP-treated mice, and shows some protective effect on liver and kidney damage in CP-induced mice.