Guohua Sun

Critical roles of sea cucumber C-type lectin in non-self recognition and bacterial clearance

C-type lectin is one important pattern recognition receptor (PRR) that plays crucial roles in multiple immune responses. A C-type lectin from sea cucumber Apostichopus japonicus (AjCTL-1) was characterized in the present study. The amino acid sequence of AjCTL-1 shared high similarities with other C-type lectins from invertebrates and vertebrates. The C-type lectin domain (CTLD) of AjCTL-1 contained a Ca(2+)-binding site 2 and four conserved cysteine residues. AjCTL-1 mRNA expression patterns in tissues and after bacterial challenge were then analysed. Quantitative PCR revealed that AjCTL-1 mRNA was widely expressed in the tested tissues of healthy sea cucumber. The highest expression level occurred in gonad followed by body wall, coelomocytes, tentacle, intestinum and longitudinal muscle, and the lowest expression level was in respiratory tree. AjCTL-1 mRNA expression in coelomocytes was significantly induced by gram-negative Listonella anguillarum and gram-positive Micrococcus luteus, with different up-regulation patterns post-challenge. Recombinant AjCTL-1 exhibited the ability to bind peptidoglycan directly, agglutinate M. luteus, Staphylococcus aureus and Escherichia coli, in a Ca(2+)-dependant manner, and enhance the phagocytosis of coelomocytes against E. coli in vitro. The results indicated that AjCTL-1 could act as a PRR in Apostichopus japonicus and had critical roles in non-self recognition and bacterial clearance against invading microbes.

The development process and seasonal changes of the gonad in Octopus ocellatus Gray off the coast of Qingdao, Northeast China

Little information exists on the gonadal development and maturity scale of Octopus ocellatus, an economically important octopus species commonly found in the west Pacific Ocean. A total of 171 O. ocellatus samples were collected monthly off the coast of northeast China (Qingdao, Shandong) from December 2011 to November 2012. Total body weight, mantle length, gonadosomatic index, and reproductive tract parameters of the samples were analyzed in males and females. The process of gonadal development in O. ocellatus was defined based on gonadal histological examination. Nine stages of oocyte development were identified during oogenesis, and a maturity scale of five stages (immature, developing, maturing, mature, and spent) was proposed for both ovary and testis. Additionally, the maturation status and dominant germ cell types of O. ocellatus in relation to monthly variation were identified. The seasonal changes showed that O. ocellatus off the coast of Qingdao is a fast-growing species with a short life span, which reproduces only once in a lifetime and then dies. Its reproductive cycle can be divided into four periods, beginning with the breeding period from April. Males become sexually mature in a smaller size than females, indicating that the mating behavior occurs early with sperm stored in the oviductal glands of the females before fertilization and spawning. This work contributes to the understanding of the maturation process in O. ocellatus, laying a foundation for further study on the mechanism of sperm storage in female octopuses and providing a reference for artificial breeding and fisheries impact assessment of this species.

Construction of a full-length cDNA library of Solen grandis Dunker and identification of defense- and immune-related genes

The basic genetic characteristics, important functional genes, and entire transcriptome of Solen grandis Dunker were investigated by constructing a full-length cDNA library with the ‘switching mechanism at the 5′-end of the RNA transcript’ (SMART) technique. Total RNA was isolated from the immune-relevant tissues, gills and hemocytes, using the Trizol reagent, and cDNA fragments were digested with Sfi I before being ligated to the pBluescript II SK* vector. The cDNA library had a titer of 1048 cfu μL−1 and a storage capacity of 1.05×106 cfu. Approximately 98% of the clones in the library were recombinants, and the fragment lengths of insert cDNA ranged from 0.8 kb to 3.0 kb. A total of 2038 expressed sequence tags were successfully sequenced and clustered into 965 unigenes. BLASTN analysis showed that 240 sequences were highly similar to the known genes (E-value < 1e−5; percent identity >80%), accounting for 25% of the total unigenes. According to the Gene Ontology, these unigenes were related to several biological processes, including cell structure, signal transport, protein synthesis, transcription, energy metabolism, and immunity. Fifteen of the identified sequences were related to defense and immunity. The full-length cDNA sequence of HSC70 was obtained. The cDNA library of S. grandis provided a useful resource for future researches of functional genomics related to stress tolerance, immunity, and other physiological activities.

Construction of a normalized full-length cDNA library of cephalopod Amphioctopus fangsiao and development of microsatellite markers

Amphioctopus fangsiao is one of the most economically important species and has been considered to be a candidate for aquaculture. In order to facilitate its fine-scale genetic analyses, we constructed a normalized full-length library successfully and developed a set of microsatellite markers in this study. The normalized full-length library had a storage capacity of 6.9×105 independent clones. The recombination efficiency was 95% and the average size of inserted fragments was longer than 1000 bp. A total of 3440 high quality ESTs were obtained, which were assembled into 1803 unigenes. Of these unigenes, 450 (25%) were assigned into 33 Gene Ontology terms, 576 (31.9%) into 153 Kyoto Encyclopedia of Genes and Genomes pathways, and 275 (15.3%) into 22 Clusters of Orthologous Groups. Seventy-six polymorphic microsatellite markers were identified. The number of alleles per locus ranged from 4 to 17, and the observed and expected heterozygosities varied between 0.167 and 0.967 and between 0.326 and 0.944, respectively. Twelve loci were significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction and no linkage disequilibrium was found between different loci. This study provided not only a useful resource for the isolation of the functional genes, but also a set of informative microsatellites for the assessment of population structure and conservation genetics of A. fangsiao.

The complete mitochondrial genome of Cynoglossus joyneri (Teleostei: Pleuronectiformes)

Abstract The complete mitochondrial genome of a commercially important sea fish Cynoglossus joyneri was sequenced and annotated. The 16,941 bp-long genome contained 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a control region. Phylogenetic analysis indicated that C. joyneri is a sister group with C. sinicus and C. bilineatus, and corroborated the proposed paraphyly of Cynoglossus genus.