Effects of testosterone undecanoate as a male contraceptive candidate on rat immunological features

Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

Immunoglobulin joining (J) chain and its expression in the Chinese soft-shelled turtle (Pelodiscus sinensis)

The immunoglobulin (Ig) joining (J) chain plays an important role in the formation of polymeric Igs and their transport into secretions. In the present study, the cDNA sequence of J chain has been cloned from the Chinese soft-shelled turtle (Pelodiscus sinensis) by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE). The cDNA sequence is 2347 bp in length and contains an open reading frame of 480 bp encoding 160 aa including the signal sequence. The deduced amino acid sequence has a high degree of homology with that of an already reported turtle J chain (80.7%), and of chicken (71.3%). By using real-time quantitative RT-PCR analysis, a significant up-regulation of J-chain transcripts was observed in spleen, kidney and blood of turtles injected with inactivated Aeromonas hydrophila, indicating the immune role of J chain in response to bacterial infection. (C) 2009 Elsevier B.V. All rights reserved.

Predominant expression and cellular distribution of fish Agr2 in renal collecting system

Anterior gradient 2 (Agr2) genes encode secretory proteins, and play significant roles in anterior-posterior patterning and tumor metastasis. Agr2 transcripts were shown to display quite diverse tissue distribution in different species, and little was known about the cellular localization of Agr2 proteins. In this study, we identified an Agr2 homologue from gibe[ carp (Carassius auratus gibelio), and revealed the expression patterns and cellular localization during embryogenesis and in adult tissues. The full-length cDNA of CagAgr2 is 803 nucleotides (nt) with an open reading frame of 510 nt encoding 169 amino acids. The Agr2 C-terminus matches to the class I PDZ-interacting motif, suggesting that it might be a PDZ-binding protein. During embryogenesis, CagAgr2 was found to be transcribed in the mucus-secreting hatching gland from tailbud stage and later in the pharynx region, swim bladder and pronephric duct as revealed by RT-PCR and whole mount in situ hybridization. In the adult fish, its transcription was predominantly confined to the kidney, and lower transcription levels were also found in the intestine, ovary and gills. To further localize the Agr2 protein, the anti-CagAgr2 polyclonal antibody was produced and used for immunofluorescence observation. In agreement with mRNA expression data, the Agr2 protein was localized in the pronephric duct of 3dph larvae. In adult fish, Agr2 protein expression is confined to the renal collecting system with asymmetric distribution along the apical-basolateral axis. The data provided suggestive evidence that fish Agr2 might be involved in differentiation and secretory functions of kidney epithelium. (C) 2009 Elsevier Inc. All rights reserved.

Fridericia dianchiensis sp nov (Enchytraeidae, oligochaeta) from Yunnan Province, China

Fridericia dianchiensis, a new enchytraeid species collected from Yunnan Province, is described here. It is characterized by a combination of the following characters: 1) lateral bundles containing maximum 3 chaetae; 2) esophageal appendages with 3-4 simple, terminal branches; 3) dorsal vessel originating in XX-XXIII; 4) sub-neural glands absent; 5) seminal vesicle large, occupying two segments; 6)clitellum girdle-shaped or gland cells absent between bursal slits and pre-middle ventrally; 7) coelomocytes without refractile vesicles, 8) spermatheca without diverticula and both ampullae broadly united; and 9) long spermathecal ectal duct without gland at the orifice.

Bryodrilus fuscistriatus, a new enchytraeid species (Oligochaeta : Enchytraeidae) from northeastern China

Bryodrilus fuscistriatus, a new enchytraeid species from Mt. Changbaishan, Jilin Province, north-eastern China, is described. It is characterized by brown epidermal glands, 7 pairs of preclitellar nephridia, poorly-developed clitellar glands, spermatheca with 2 sessile globular diverticula, and a long sperm funnel with a very broad collar. It is similar to the Alaskan B. tunicatus Dozsa-Farkas & Christensen, 2002 in possessing two diverticula in the spermathecal ampulla and the origin of the dorsal vessel, and the Chinese B. longifistulatus and B. macrotheca Xie et al., 2000c in body size, long sperm funnel and undeveloped clitellar glands, but it differs from B. tunicatus by the presence of brown-striped epidermal gland cells in III-V, a poorly-elevated clitellum, the absence of copulatory glands in XIII-XIV, the regular outline of coelomocytes, and 7 pairs of preclitellar nephridia; from B. longifistulatus and B. macrotheca by the shape of spermatheca, the color of epidermal gland cells, the position of the first pair nephridia, and the origin of dorsal vessel.

Four new terrestrial species of Marionina (Clitellata, Enchytraeidae) from China and re-examination of M. hoVbaueri Moller

Enchytraeid surveys were made in China, mainly along the Changjiang (Yangtze) River Basin, during the period 1991-1999. Among the findings, four terrestrial species of Marionina are new to science and well illustrate the taxonomic complexity of the genus as currently defined. Marionina sinica sp. n. is characterized by a specific chaetal distribution, the marionine pattern of the dorsal blood vessel, and elongate, fusiform, spermathecal ectal ducts. Marionina sacculata sp. n. is distinguished by the possession of a pair of pouch-like oesophageal appendages in IV, the lack of lateral chaetae in VII-XI, a marionine pattern of the dorsal blood vessel, and short spermathecal ectal ducts gradually expanding into spherical ampullae. Both M. sinica and M. sacculata have minute bodies (2-3 mm long in vivo) and lack spermathecal accessory glands. The former species shows its closest aYnities with the European M. brendae Rota, 1995, whereas the latter is closest to the German M. hoVbaueri Moller, 1971, for which an amended diagnosis is provided. Marionina seminuda sp. n. has only ventral chaetal bundles, distributed from III onwards and bisetose. It is similar to the Holarctic M. subterranea (Knollner, 1935) in lacking entirely the lateral chaetae and in having the brain incised posteriorly, the dorsal vessel bifurcating behind the pharynx, and coelomocytes containing opaque granules, but diVers from it in having the longest chaetae in preclitellar segments and gland cells distributed all over the spermathecal ectal ducts. Marionina righiana sp. n. is diagnosed by the location of the head pore on the prostomium, the absence of lateral chaetae from VIII ( VII or IX) onwards, the possession of free spermathecae extending backwards through one to four segments, the brain deeply incised posteriorly, the lumbricilline pattern of the dorsal blood vessel, and the opacity of coelomocytes in vivo. Prior to this study, members of the genus so atypical as M. righiana with respect to the position of the head pore and the structure of the spermathecae were known only from South American soils. Until the taxonomy of Marionina has been more thoroughly assessed and revised, the assignment of the four species to this large assemblage should be regarded as tentative.

Fridericia nanningensis, a new terrestrial enchytraeid species (Oligochaeta) from southwestern China

Fridericia nanningensis, a new species from wetland soil of Nanhu Park, Nanning city, the capital of Guangxi Zhuang Autonomous Region in southwest China, is described. It is characterized by 2-4 chaetae per bundle, poorly-developed clitellar glands, slender, unbranched peptonephridia, and spermathecae with 2 ampullar diverticula, a deep constriction in the middle of the ampulla and one large ectal gland. It is closely related to the European species, F. alata Nielsen & Christensen, 1959 and the East European species, F. tubulosa Dozsa-Farkas, 1972 by the shape of peptonephridia and the undeveloped clitellar glands. It differs from F. alata by its shorter body length and fewer chaetae per bundle, its type of coelomocytes (type "c"), its deep constriction in the middle of the spermathecae ampulla and a larger ectal gland, and it differs from F. tubulosa by its pale epidermal glands, its more anterior origin of the dorsal vessel, a deep constriction in the middle of the spermathecae ampulla, shorter ectal duct, and only one ectal gland at the spermathecal orifice.

The freeliving nematode genus Parodontophora Timm 1963 (Nematoda : Axonolaimidae) is not exclusively marine: Parodontophora limnophila sp nov from freshwater in China

Parodontophora limnophila sp. nov. is described from Poyang Lake, the largest freshwater lake of China. It is characterized by having an amphid with its posterior end close to the base of the stoma, relatively short cephalic setae, opisthocephalic setae arranged as two subdorsal groups of three longitudinally arranged setae and two single subventral setae, excretory pore at the level of the anterior part of the stoma and renette gland 34-47% of the oesophageal length. To date, the new species is the only Parodontophora species found in freshwater habitats.

九寨沟景区旅游活动扰动形成的退化地段植被恢复试验初报

日益增强的旅游活动干扰已成为九寨沟世界自然遗产的有效保护与持续管理不可回避的问题，也已成为当前区域生态保护与经济发展的焦点问题之一。评估乡土植物种的基本特征、质量、种子休眠程度以及萌发能力；筛选适宜的植被恢复乡土物种并选择有效植被恢复技术措施是开展植被恢复试验的重要前提和保障。本文以九寨沟世界自然遗产为例,通过对景区乡土植物种子基本特征的测定以及采用乡土物种作为植被恢复技术试验与恢复技术筛选的试验物种，在景区旅游干扰区的三种生境类型：原始针叶林、混交林和边坡，根据不同的影响因素设计试验，从而探索适宜的恢复措施和植被恢复物种。初步结论如下： 1． 本文研究的28种乡土植物中，17种测定的物种活力较高(>72%)，均具有作为恢复物种的潜力。但28种种子中75%的发芽率为0%。大部分种子存在一定程度的休眠，包括甘肃山楂、称花藤、疏花槭、陇东海棠、球花荚迷、巴东小檗、蔷薇、宝兴栒子、光泽栒子、光枝柳叶忍冬、甘青铁线莲、毛果铁线莲、宽叶旌节花、糙叶五加和独活。因而种子存在深度休眠是乡土物种自然更新和植被恢复的主要障碍，需要根据种子的形态生理特征，寻求打破乡土植物种子休眠的有效方法，提高种子发芽率。 2．低温层积只显著提高了蔷薇、毛果铁线莲、宽叶旌节花和假升麻3种种子的发芽率，也相对较早地提高了陇东海棠、鲜黄小檗和独活的萌发。对于其它物种的种子，低温层积没能有效打破休眠提高发芽率。综合分析结果表明九寨沟28种林下或林缘乡土植物中窄叶鲜卑花、毛果铁线莲、宽叶旌节花、假升麻、陇东海棠、鲜黄小檗和独活等12种可直播或通过低温层积处理后直播用于旅游干扰退化地段的植被恢复中，而其它的种类尚需进一步寻求打破休眠的措施以提高种子发芽能力。 3．在原始林生境下，七筋菇、巴东小檗、耳叶风毛菊、长叶毛花忍冬及独活5种乡土植物种子的出苗能力不高，因此目前急需提出有关提高种子出苗的方法措施。在相同的处理条件下，不同物种出苗数存在着较大的差异，相比而言，巴东小檗、耳叶风毛菊和长叶毛花忍冬种子出苗与幼苗存活较高，对于其环境适应能力较强，因此它们更适宜采用播种的方式应用于九寨沟原始林践踏地段地表的植被恢复实践；而七筋菇和独活在室内和自然气候条件下萌发都很困难，在植被恢复过程中，可考虑采用其它播种方式。而在边坡和混交林生境试验结果表明，供试物种无萌发和出苗现象，这可能跟外界环境条件、干扰和种子命运有关。 4． 本研究中腐殖质、松土和苔藓覆盖处理措施均未能提高物种出苗，且对样方内植被结构以及物种丰富度也无显著影响，其中的影响因素也是多方面的，因此还需进一步研究探索。但腐殖质对幼苗存活能力方面有一定的积极作用。 The increasing tourism disturbance is an unavoidable issue to effective conservation and sustaind tourist management of Jiuzhaigou Nature Reserve. It has become one of the focal problems of regional ecological protect and economic development. Assessing the traits, quality, seed dormancy and germination of native seed and choosing the suitable species and ways for vegetation restoration were the important precondition and guarantee before vegetation restoration. In this paper, as an example to Jiuzhaigou world nature heritage, through the estimation to the basic characteristics of native seeds and the use of native species for the test of vegetation restoration and the choosing of vegetation restoration techniques, in three different habitats being disturbed, including the primitive forest, mixed forest and side slope. According to different factors, designing experiments in order to explore the appropriate measures and species of vegetation restoration . Our results are as follows: 1． There are 77% seeds of the 28 species with high viability(>72%), which had the potential possibility of being restoration species. The germination of 75% of the seed was 0%.Most of the seed was dormant, So dormancy of seed was the barrier for natural regeneration and vegetation restoration, Further research is needed on investigating the other more effective methods to release seed dormancy of these native species in the future. 2． Cold stratification did not release seed dormancy of most species. In our study, there are only 3 species including Clematis peterae var. trichocarpa, Stachyurus chinensis and Aruncus sylvester (P<0.05) with germination percentage increased by cold stratification. By comprehensive analysis, the result shows: In 28 undergrowth native seeds, Sibiraea angustata, Clematis peterae var. trichocarpa, Stachyurus chinensis, Aruncus Sylvester, Malus kansuensis, Berberis diaphana,and Heracleum hemsleyanum could be used to vegetation restoration by sowing directly or sowing after cold stratification in degenerate gland being disturbed by turism, and othes seed of 28 were needed to search other methods to release seed dormancy for advancing the ability of germination. 3．In the habitat of primitive forest, the ability of emergence of the native species Clintonia udensis, Berberis henryana, Saussurea neofranchetii, Lonicera trichosantha var.xerocalyx and Heracleum hemsleyanum was low, so it is urgently needed to search about the methods of advancing emergence. In the sametreatment, the number of emergence of different species was very different. Relatively speaking, the emergence and seedling survival of the species Berberis henryana, Saussurea neofranchetii and Lonicera trichosantha var.xerocalyx was higher, and the adaptability of the species was better, so they were more suitable for vegetation restoration on trampling-induced degraded patches of primitive forest in Jiuzhaigou; But the germination of Clintonia udensis and Heracleum hemsleyanum was both difficult in laboratory and natural conditions, so it could be considered to apply other sowing methods. However, the result showed that: applied species didn’t germinate or emergent in the habitat of side slope and mixed forest, which could be related to external environment conditions, interference and the fate of the seed. 4．In this study, humus, scatification and bryophyte cover measures didn’t advance the emergence of the species or the structure of vegetation and richness in plot significantly, the impact factors of which were many, therefore further research is needed . However, humus had a certain positive effect on the ability of seedling survival.

Enhanced biological effect induced by a radioactive C-9-ion beam at the depths around its Bragg peak

To explore the potential of double irradiation source, radioactive C-9-ion beam, in tumor therapy, a comparative study oil the surviving effect of human salivary gland cells at different penetration depths between C-9 and C-12-ion beams has been carried out. The 9C-ion C beam, especially at the distal side of the beam came out more efficient in cell killing at the depths around its Bragg peak than the 12 Bragg peak. Compared to the C-12 beam, an increase in RBE by a factor of up to 2.13 has been observed at the depths distal to the Bragg peak of the 9C beam. The 9C beam showed an enhanced biological effect at the penetration depths around its Bragg peak, corresponding to the stopping region of the incident C-9-ions and where the delayed low-energy particles were emitted. Further analysis revealed that cell lethality by the emitted particles from the stopping C-9-ions is responsible for the excessive biological effect at the penetration depths around the Bragg peak of the C-9 beam.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. 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.

对虾性别分化、性腺发育中若干关键问题的研究

利用扫描电镜和组织学研究相结合的方法研究了中国对虾早期的性别分化。扫描电镜观察的结果表明，中国对虾外部性征的性别分化是在变为仔是后第35天开始的，而内部性征则是在变为仔虾后的第76天观察到生殖腺原基向精巢和卵巢的分化。促雄腺(Androgenic gland)是控制甲壳动物性别分化的一个重要器官。到目前为止，已在软甲类的多数目中报道了促雄腺的存在。不同种类促雄腺的分布位置不同，外部形态也有很大差别。中国对虾的促雄腺位于第五对步足基部的肌肉间，于片状覆盖在精荚囊与射精管连接处。当用苏木精染色时，由于其嗜碱性比较强，整个腺体被染成深蓝色。扫描电镜观察可见，中国对虾的促雄腺具有葡萄串状的外形，它是由许多葡萄形的亚单位组成的，这些亚单位的大小、形态各异组织学观察的结果表明，中国对虾的促雄腺由两种类型的细胞组成。第一类细胞，数量少，核的嗜碱性很强，被苏木精染成深蓝色，核仁不易分辨；第二类细胞，数量大，核比较膨大，嗜碱性较弱，核质疏松，具有明显可见的核仁。不同大小的虾中，腺细胞的排列方式不同。随着对虾的发育成熟，腺细胞逐渐由索状排列变为成团排列，说明促雄腺的形成存在一个逐渐发育的过程。利用手术破坏促雄腺或用促雄腺匀浆液浸泡仔虾的方法研究了中国对虾促雄腺的功能，中国对虾促雄腺的存在与否对于雄性外部性征的发育和维持起着重要作用。破坏促雄腺的雄虾的交接器的发育明显受到抑制。但是，未观察到促雄腺对对虾内部性征发育的影响，这可能与进行手术时对虾所处的发育期有关。利用促雄腺的匀浆液浸泡仔虾，对雄虾交接器的发育有一定促进作用。高温处理对中国对虾受精卵孵化率的影响随处理温度及处理时间的改变而发生变化。用热处理方法成功地诱导了中国对虾三倍体，最高诱导率达75%，并对三倍体群体的生物学特性进行了研究。在中国对虾三倍体的群体中，雌体的巢存在两种类型：一种类型卵巢，生殖细胞停留在卵原细胞阶段，没有进一步发育；另一种类型的卵巢，生殖细胞由卵原细胞发育为初级卵母细胞，发育情况与对虾的类似。对卵巢处于发育的第II或第III期的中国对虾以及卵巢发育处在第I、II、III期的几纳对虾的血液和卵巢中法呢烯酸甲酯（MF）含量进行了研究，结果表明，中国对虾和凡纳对虾中均存在MF。凡纳对虾血液和卵巢发育具有明显的刺激作用，这种刺激作用是通过血液中法呢烯酸甲酯含量的提高来实现的。这一研究结果进一步说明了法呢烯酸甲酯与甲壳动物繁殖之间的关系。

Studies on mass summer mortality of cultured zhikong scallops (Chlamys farreri Jones et Preston) in China

Mass mortalities of cultured zhikong scallops (Chlamys farreri) have occurred each summer in most culture areas of northern China since 1996. Among the hypothesized causes are high culture density, infectious disease and genetic inbreeding. To investigate these potential agents, C. farreri were deployed at three densities (low, medium and high) at three sites (Jiaonan, Penglai and Yantai) in the summer of 2000. Scallops were sampled for survival, growth and histopathology before, during and after a mortality episode. Most of the mortality occurred in July and August, during and toward the later part of the spawning season, when water temperature reached 23-26 degrees C. Final cumulative mortalities reached 85% to 90% at all three sites. Scallops in the medium and high densities had higher initial death rates than did those at the low density. High densities also inhibited growth. Ciliates from the genus Trichodina, larvae of various organisms and anomalous secretions were observed in sections of the gill cavity, with highest prevalence during and at the end of the mortality period. Prokaryotic inclusion bodies were found in the soft tissues, but their prevalence was low and apparently without correlation with mortalities. Genetic analysis with random amplified polymorphic DNA markers showed slightly lower heterozygosity in the cultured stocks (0.301) than in the wild stocks (0.331). It is possible that the mortalities are caused by a combination of several factors such as stress associated with reproduction, high temperature, overcrowding and poor circulation in the growout cages, opportunistic invaders or pathogens, and possibly inbreeding. (c) 2005 Elsevier B.V. All rights reserved.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2) mouse unit (MU) Per gram Of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus. (C) 2008 Elsevier Ltd. All rights reserved.