Mitochondrial variation of the "eyed" turtles (Sacalia) based on known-locality and trade specimens

Bryan L. Stuart is thanked for his hard work to collect wild specimens, as well as providing insightful and useful comments on the data. We thank Abigail Wolf of the Field Museum for providing photographs of specimens. Robert Murphy of the Royal Ontario M

Halichondria sulfonic acid, a new HIV-1 inhibitory guanidino-sulflonic acid, and halistanol sulfate isolated from the marine sponge Halichondria rugosa Ridley & Dendy

A new sulfur-containing guanidino derivative, halichondria sulfonic acid (1) showing anti-HIV-1 activity, and halistanol trisulfate (2) with anti-tumor activity have been isolated from the marine sponge Halichondria rugosa Ridley & Dendy collected in the

Optimizing the formation of in vitro sponge primmorphs from the Chinese sponge Stylotella agminata (Ridley)

The establishment and optimization of in vitro primmorph formation from a Chinese sponge, Stylotella agminata (Ridley), collected from the South China Sea, were investigated. Our aims were to identify the key factors affecting primmorph formation in this species and to optimize the technique for developing an in vitro primmorph culture system. The size of dissociated cells from S. agminata is relatively small, in the range between 5 and 10 mum. Round-shaped primmorphs of less than 100 gm were formed 3 days after transferring the dissociated cells into seawater containing Ca2+ and Mg2+. The effect of various cell dissociation conditions, inoculum. cell density, concentration of antibiotics, pH, and temperature was further investigated upon the formation of primmorphs. The time required for primmorph formation, primmorph size distribution, and the proliferating capability were microscopically documented. Healthy sponge S. agminata, inoculum. cell density and culture temperature play a critical role for the successful formation of primmorphs and that the microbial contamination will have to be controlled. (C) 2002 Elsevier Science B.V. All rights reserved.

Expression pattern of dmrt4 from olive flounder (Paralichthys olivaceus) in adult gonads and during embryogenesis

The dmrt (doublesex and mab-3 related transcription factor) gene family comprises several transcription factors that share a conserved DM domain. Dmrt1 is considered to be involved in sexual development, but the precise function of other family members is unclear. In this study, we isolated genomic DNA and cDNA sequences of dmrt4, a member of the dmrt gene family, from olive flounder, Paralichthys olivaceus, through genome walking and real-time reverse transcriptase (RT)-PCR. Sequence analysis indicated that its genomic DNA contains two exons and one intron. A transcriptional factor binding sites prediction program identified a sexual development-related protein, Sox9 (Sry-like HMG box containing 9) in its 5' promoter. Protein alignment and phylogenetic analysis suggested that flounder Dmrt4 is closely related to tilapia Dmo (DM domain gene in ovary). The expression of dmrt4 in adult flounder was sexually dimorphic, as shown by real-time RT-PCR analysis, with strong expression in the testis but very weak expression in the ovary. Its expression was also strong in the brain and gill, but there was only weak or no expression at all in some of the other tissues tested of both sexes. During embryogenesis, its expression was detected in most developmental stages, although the level of expression was distinctive of the various stages. Whole mount in situ hybridization revealed that the dmrt4 was expressed in the otic placodes, forebrain, telencephalon and olfactory placodes of embryos at different developmental stages. These results will improve our understanding of the possible role of flounder dmrt4 in the development of the gonads, nervous system and sense organs.

Hybrids between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus: karyotype, allozyme and RAPD analyses

The hybrid between olive flounder Paralichthys olivaceus and stone flounder Kareius bicoloratus was produced by artificial insemination of olive flounder eggs with stone flounder sperm. Sinistral and dextral are two types of hybrid progeny after metamorphosis. Karyotypes of both hybrid flounders are the same as those of the two parental species. Of the 22 loci examined from 12 allozymes, 12 confirmed hybridization of the paternal and maternal loci in hybrids and no difference was found in allozyme patterns of sinistral and dextral hybrid fishes. RAPD patterns of these specimens were also studied with 38 primers selected from 104 tested. Among them, the PCR products of 30 primers showed hybridization of the paternal and maternal bands. Genetic variation between hybrids and their parental stocks was analyzed by RAPD using 10 of the above 38 primers. The average heterozygosity and genetic distance were calculated. The results suggested that the filial generation could inherit a little more genetic materials from paternal fish than that from maternal fish.

Effects of hydrostatic pressure on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Indirect immunofluorescence staining was used to detect cytological changes of isolated blastodisks during mitosis of flounder haploid eggs treated with hydrostatic pressure. Changes in microtubule structure and expected cleavage suppression were observed from blastodisk formation to the third cell cycle, with obvious differences between treated and control eggs. In most eggs, microtubules were disassembled and the nucleation capacity of the centrosome was temporarily inhibited after pressure treatment. Within 15-20 min after treatment, the nucleation capacity of the centrosome began to gradually recover, with slow regeneration of microtubules; approximately 25 min after treatment, the nucleation capacity of the centrosome recovered completely, regenerated distinct bipolar spindles, and the first mitosis ensued. During the second cell cycle, approximately 61% of the embryos were at the two-cell stage, with a monopolar spindle in each blastomere; that treatment was effective was based on second cleavage blockage. Approximately 15% of the eggs still remained at the one-cell stage and had a monopolar spindle (treatment was effective, according to the general model of first cleavage blockage). However, treatment was ineffective in approximately 15% of the embryos (bipolar spindle in each blastomeres) and in another 8% (bipolar spindle in one of the two blastomeres and a monopolar spindle in the other; both mechanisms operating in different parts of the embryo). This is the first report elucidating mitotic gynogenetic diploid induction by hydrostatic pressure in marine fishes and provides a cytological basis for developing an efficient method of inducing mitotic gynogenesis in olive flounder. (C) 2007 Elsevier Inc. All rights reserved.

Effects of cold shock on microtubule organization and cell cycle in gynogenetically activated eggs of olive flounder (Paralichthys olivaceus)

Cytological changes and subsequent mitotic processes were studied in gynogenetically activated eggs of olive flounder subjected to cold-shock treatment using indirect immunofluorescence staining of isolated blastodisks. Obvious differences between controls and treated eggs were detected during early cell division. The developmental process of haploid control was similar to that of the diploid control except several minutes delayed. Spindles disassembled by the cold-shock treatment regenerated soon after treatment, resulting in the occurrence of the first mitosis. The immature daughter centriole was easily depolymerized by cold-shock treatment, leading to the formation of the bipolar spindle in the first cell cycle and the formation of the monopolar spindle in the second cell cycle, resulting in chromosome set doubling. Some two-cell stage eggs had a monopolar spindle in one blastomere and a bipolar spindle in another during the second mitosis. These eggs had a high potency developing into haploid-diploid mosaics. To the best of our knowledge, this study is the first to clarify the mechanism of chromosome set doubling in marine fishes and provides a preliminary cytological basis for developing a reliable and efficient protocol for mitotic gynogenesis induction by cold-shock treatment in olive flounder.

Interference Competition and High Temperatures Reduce the Virulence of Fig Wasps and Stabilize a Fig-Wasp Mutualism

Fig trees are pollinated by fig wasps, which also oviposit in female flowers. The wasp larvae gall and eat developing seeds. Although fig trees benefit from allowing wasps to oviposit, because the wasp offspring disperse pollen, figs must prevent wasps fr

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.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed I week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Molecular characterization and expression profiles in response to bacterial infection of Chinese soft-shelled turtle interleukin-8 (IL-8), the first reptilian chemokine gene

In this study, an IL-8 homologue has been cloned and identified from a reptile, Chinese soft-shelled turtle for the first time. The full-length cDNA of turtle IL-8 was 1188 bp and contained a 312 bp open reading frame (ORF) coding for a protein of 104 amino acids. The chemokine CXC domain, which contained Glu-Leu-Arg (ELR) motif and four cysteine residues, was well conserved in turtle IL-8. The 4924 bp genomic DNA of turtle IL-8 contained four exons and three introns. Phylogenetic analysis showed that the amino acid sequence of turtle IL-8 clustered together with birds. RT-PCR analysis showed that turtle IL-8 mRNA was constitutively expressed liver, spleen, kidney, heart, blood and intestine tissues of control turtles. Real-time quantitative PCR analysis further indicated that the turtle IL-8 mRNA expression was apparent in various tissues at 8 h and up-regulated significantly during 8 h-7 d after Aeromonas hydrophila infection. The present studies will help us to understand the evolution of IL-8 molecule and the inflammatory response mechanism in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

Simultaneous determination of microcystin contaminations in various vertebrates (fish, turtle, duck and water bird) from a large eutrophic Chinese lake, Lake Taihu, with toxic Microcystis blooms

This is the first to conduct Simultaneous determination of microcystin (MC) contaminations in multi-groups of vertebrates (fish, turtle, duck and water bird) from Lake Taihu with Microcystis blooms. MCs (-RR, -YR, -LR) in Microcystis scum was 328 mu g g(-1) DW. MCs reached 235 mu g g(-1) DW in intestinal contents of phytoplanktivorous silver carp, but never exceeded 0.1 mu g g(-1) DW in intestinal contents of other animals. The highest MC content in liver of fish was in Carassius auratus (150 ng g(-1) DW), followed by silver carp and Culter ilishaeformis, whereas the lowest was in common carp (3 ng g(-1) DW). In livers of turtle, duck and water bird, MC content ranged from 18 to 30 ng g(-1) DW. High MC level was found in the gonad, egg yolk and egg white of Nycticorax nycticorax and Anas platyrhynchos, suggesting the potential effect of MCs on water bird and duck embryos. High MC contents were identified for the first time in the spleens of N. nycticorax and A. platyrhynchos (6.850 and 9.462 ng g(-1) DW, respectively), indicating a different organotropism of MCs in birds. Lakes with deaths of turtles or water birds in the literatures had a considerably higher MC content in both cyanobacteria and wildlife than Lake Taihu, indicating that toxicity of cyanobacteria may determine accumulation level of MCs and consequently fates of aquatic wildlife. (C) 2009 Elsevier B.V. All rights reserved.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.

Effects of animal-plant protein ratio in extruded and expanded diets on nitrogen and energy budgets of juvenile Chinese soft-shelled turtle (Pelodiscus sinensis Wiegmann)

In this study, we investigated the effects of animal-plant protein ratio in extruded and expanded diets on nutrient digestibility, nitrogen and energy budgets of juvenile soft-shelled turtle (Pelodiscus sinensis). Four extruded and expanded feeds (diets 1-4) were formulated with different animal-plant protein ratios (diet 1, 1.50:1; diet 2, 2.95:1; diet 3, 4.92:1; diet 4, 7.29:1). The apparent digestibility coefficients (ADCs) of dry matter and crude lipid for diet 1 were significantly lower than those for diets 2-4. There was no significant difference in crude protein digestibility among diets 1-4. The ADC of carbohydrate was significantly increased with the increase in animal-plant protein. Although nitrogen intake rate, faecal nitrogen loss rate and excretory nitrogen loss rate of turtles fed diet 1 were significantly higher than those fed diets 2-4, nitrogen retention rate, net protein utilization and biological value of protein in these turtles were significantly lower than those fed diets 2-4. In addition, energy intake rate, excretory energy loss rate and heat production rate of turtles fed diet 1 were also significantly higher than those fed diets 2-4. Faecal energy loss was significantly reduced with the increase in the animal-plant protein ratio. The ADC of energy and assimilation efficiency of energy significantly increased with a higher animal-plant protein ratio. The growth efficiency of energy in the group fed diet 1 was significantly lower than those in the groups fed diets 2-4. Together, our results suggest that the optimum animal-plant protein ratio in extruded and expanded diets is around 3:1.

中国非海产龟鳖类的研究

无