Settlement Behaviour of Marine Invertebrate Larvae Measured by EthoVision 3.0

Submerged marine surfaces are rapidly colonized by fouling organisms. Current research is aimed at finding new, non-toxic, or at least environmentally benign, solutions to this problem. Barnacles are a major target organism for such control as they constitute a key component of the hard fouling community. A range of standard settlement assays is available for screening test compounds against barnacle cypris larvae, but they generally provide little information on mechanism(s) of action. Towards this end, a quick and reliable video-tracking protocol has been developed to study the behaviour of the cypris larvae of the barnacle, Balanus amphitrite, at settlement. EthoVision 3.0 was used to track individual cyprids in 30-mm Petri dishes. Experiments were run to determine the optimal conditions vis-à-vis acclimation time, tracking duration, number of replicates, temperature and lighting. A protocol was arrived at involving a two Petri dish system with backlighting, and tracking over a 5-min period after first acclimating the cyprids to test conditions for 2 min. A minimum of twenty replicates was required to account for individual variability in cyprid behaviour from the same batch of larvae. This methodology should be widely applicable to both fundamental and applied studies of larval settlement and with further refinements, to that of smaller fouling organisms such as microalgae and bacteria.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Stylochus tauricus,a predator of the barnacle Balanus improvisus in the Black Sea

The flatworm Stylochus tauricus Jacubova has been found associated with the barnacle Balanus improvisus Darwin, on which it feeds. The predation rate (the number of barnacles eaten by one polyclad in a month) ranges between 5–10. Inside the empty shells of B. improvisus some egg-plates of S. tauricus were observed. Pelagic Götte's larvae aged 2–3 days possess 4 lobes while those aged 7–8 days have 5 lobes. Flatworms can prey on the young of another species Balanus eburneus Gould, whereas predation on the mussels Mytilus galloprovincialis Lam. is rare. There is a direct correlation between predator abundance and prey ingested.     

DNA barcoding of blastocystis

We have developed a simple method for subtyping the intestinal protistan parasite Blastocystis using an approach equivalent to DNA barcoding in animals. Amplification of a 600 bp region of the small subunit ribosomal RNA gene followed by single primer sequencing of the PCR product provides enough data to assign isolates to specific subtypes unambiguously. We believe that this approach will prove useful in future epidemiological studies.     

Molecular characterization of larval anisakid nematodes from marine fishes off the Moroccan and Mauritanian coasts

A total of 242 larval forms of Anisakis collected from marine fishes at different sites off the Moroccan and Mauritanian coasts, recognised as belonging to Type I and Type II larvae, were identified by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms) of the ITS (Internal Transcribed Spacers) region (ITS-1, 5.8 subunit rRNA gene and ITS-2), using a previously established molecular key. The Type I larvae were found with a frequency of 98.34% and were identified as belonging to the following species: A. simplex s.str., A. pegreffii, A. simplex s.str/A. pegreffii heterozygote genotypes, A. typica, A. ziphidarum and Anisakis sp. A. The Type II larvae were found to belong to A. physeteris, with the frequency of 1.65%. The results reported in the present study provide further epizootiological and biological data on the Anisakis spp. in marine fishes off the Moroccan and Mauritanian coasts, improving the picture of the occurrence of these species in the central Atlantic coasts.     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Development of Molecular Identification Method for Genus Alexandrium (Dinophyceae) Using Whole-Cell FISH

We have developed a method to identify species in the genus Alexandrium using whole-cell fluorescent in situ hybridization with FITC-labeled oligonucleotide probes that target large subunit ribosomal rRNA molecules. The probes were designed based on the sequence of the rDNA D1-D2 region of Alexandrium species. DNA probes specific for toxic A. tamarense and A. catenella and nontoxic A. affine, A. fraterculus, A. insuetum, and A. pseudogonyaulax, respectively, were applied to vegetative cells of all above Alexandrium species to test the sensitivity of the probes. Each DNA probe hybridized specifically with vegetative cells of the corresponding Alexandrium species and showed no cross-reactivity to noncorresponding Alexandrium species. In addition, no cross-reactivity of the probes was observed in experiments using concentrated natural seawater samples. The TAMAD2 probe, which is highly specific to A. tamarense, a common toxic species in Korean coastal waters, provides a simple and reliable molecular tool for identification of toxic Alexandrium species.     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

Molecular identification of toxic Alexandrium tamiyavanichii (Dinophyceae) using two DNA probes

To improve labeling-intensity of whole-cell fluorescence in situ hybridization (FISH) in the molecular identification of toxic Alexandrium tamiyavanichii, two DNA probes (TAMID2 plus TAMIS1 designed from the LSU and SSU rDNA regions, respectively) were used to test the labeling intensity of targeted cultured A. tamiyavanichii cells. The cross-reactivity of the DNA probe to natural seawater samples and six Alexandrium species: A. affine, A. catenella, A. fraterculus, A. insuetum, A. pseudogonyaulax and A. tamarense, was also tested. The labeling intensity of the DNA probe TAMID2S1, a combination of two separate probes that target different regions of the rRNA, was 1.7–2.7 times higher than that of the single DNA probe TAMID2. With cultured A. tamiyavanichii cells in the dead growth phase at 30 days, the TAMID2S1 intensity was 1.9 times higher than that of TAMID2. During a 30-day culture, the labeling intensity of A. tamiyavanichii cells hybridized with TAMID2S1 decreased to 49.4% of the original intensity. No cross-reactivity to various microorganisms in natural seawater samples was found. The two DNA probes together, designated as TAMID2S1, readily detected A. tamiyavanichii added to natural seawater samples, even aged cultured cells.     

Utilization of a Photosystem by Drosophila melanogaster Larvae (Diptera: Drosophilidae)

Foraging-stage third-instar larvae from most wild-type (normal) Drosophila melanogaster stocks are generally repelled by light. To identify factors that affect the larval photoresponse, we elucidated the effects of age, temperature, and time on the photoresponse of larvae from a wild-type Canton-S stock. In addition, we analyzed the larvae from the LI2 isofemale line, which are unresponsive to light in a photoassay. To determine whether LI2 larvae behave abnormally on other behavioral paradigms, in comparison to Canton-S controls, we tested larvae in taste and olfactory assays and observed them to determine whether they dispersed in a food source. Like Canton-S larvae, LI2 larvae and other isofemale lines whose progenitors were collected from the same natural population are responsive to taste and olfactory stimuli. Moreover, LI2 larvae disperse in the food source, as do Canton-S larvae tested in the dark. Larvae expressing para^sbl mutations, which respond normally to light but not to chemical stimuli, do not disperse normally in the food source, suggesting that dispersal may be mediated by perception of chemical cues.     

Larvae of phoronids (Phoronida) from Terpeniya Bay (Southeastern Sakhalin)

Phoronid larvae were found in planktonic samples from the northern coast of Terpeniya Bay. In some samples, their density was up to 220 specimens/m³. Larval stages having 10, 12, 16, 20, and 22 tentacles are described. Larvae were identified as Actinotrocha branchiata and belong to the species Phoronis muelleri Selys-Longchamps, 1903. However, unlike the Ph. muelleri larvae described in the literature, the larvae we found are smaller (not more than 900 μm) before metamorphosis and have fewer tentacles (24). They lack paired vacuolated diverticula of the stomach, which are characteristic of Ph. muelleri larvae. However, judging by all other characters, namely transparency, the absence of coelomic cylinder in the preoral lobe, and the presence of adult tentacle primordia, one pair of blood cell aggregations, and a pyriform organ, these larvae are similar to the previously described larvae of Ph. muelleri. Adult forms of Ph. muelleri were previously found in Terpeniya Bay and described by Mamkaev (1962) and Emig (1984).     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

巴西橡胶树HbSUT3和HbSUT5 mRNA在嫩茎和中脉中的原位杂交分析

为研究巴西橡胶树(Hevea brasiliensis)中HbSUT3和HbSUT5基因的功能,采用地高辛标记的RNA探针与橡胶树嫩茎和中脉两种组织切片分别进行RNA原位杂交,对这2种SUT基因在组织中的表达区域与表达特点进行了分析。结果表明,在橡胶树嫩茎中,两个SUT基因主要在树皮的韧皮部和皮层细胞中表达;在中脉中,两个SUT基因在除木质部导管系统外的其它部位均有表达;HbSUT3基因在嫩茎和中脉中的表达量相近,而HbSUT5基因在嫩茎中的表达量远高于中脉。这些揭示HbSUT3和HbSUT5基因可能广泛参与韧皮部装载、蔗糖运输与库细胞供给等活动,同时两个SUT基因也存在功能分化。     

Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques

Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles. In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied. Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP). Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment.     

单分子荧光原位杂交（smFISH）技术及应用

单分子荧光原位杂交（single-molecule fluorescence in situ hybridization,smFISH）技术是一种通过用偶联荧光基团的寡核苷酸探针,对固定细胞或组织中单个mRNA分子进行成像的方法。smFISH可对RNA进行定位、定量,以此对目标转录本进行实时研究。smFISH适用于细胞、组织切片等多种类型生物样本。近年来,多种基于基础smFISH的改进技术被发明,进一步促进了该技术的实际应用。smFISH良好的RNA单分子可视化能力,使得其在发育生物学、神经生物学及肿瘤生物学等基础生物学科中得到了广泛的应用。本文综述了smFISH技术基本原理、smFISH技术的局限性、smFISH衍生技术方法、smFISH在不同生物学科中的应用进展,并对smFISH技术的发展前景做出展望。     

Molecular Analysis of Anisakis Type I Larvae in Marine Fish from Three Different Sea Areas in Korea

Anisakiasis, a human infection of Anisakis L3 larvae, is one of the common foodborne parasitic diseases in Korea. Studies on the identification of anisakid larvae have been performed in the country, but most of them have been focused on morphological identification of the larvae. In this study, we analyzed the molecular characteristics of 174 Anisakis type I larvae collected from 10 species of fish caught in 3 different sea areas in Korea. PCR-RFLP and sequence analyses of rDNA ITS and mtDNA cox1 revealed that the larvae showed interesting distribution patterns depending on fish species and geographical locations. Anisakis pegreffii was predominant in fish from the Yellow Sea and the South Sea. Meanwhile, both A. pegreffii and A. simplex sensu stricto (A. simplex s.str.) larvae were identified in fish from the East Sea, depending on fish species infected. These results suggested that A. pegreffii was primarily distributed in a diverse species of fish in 3 sea areas around Korea, but A. simplex s.str. was dominantly identified in Oncorhynchus spp. in the East Sea.