Buccal swab: A tissue sampling method for refinement of experimental procedures involving rainbow trout

Buccal swabbing is a minimally invasive method to obtain DNA and biological material from humans and animals, including fish. Reports on buccal swabbing in fish are few and only for a limited number of species. Rainbow trout (Oncorhynchus mykiss) is an important animal model and because the yield of DNA may vary among and within different species in individuals of different sizes, it was selected as useful to optimize the buccal DNA collection in this species. Different storage methods were evaluated, aimed at DNA preservation by limiting DNA degradation and bacterial growth, using commonly available and inexpensive reagents. DNA quality was also tested by amplification of a single‐copy nuclear gene and a mitochondrial gene. The results suggest that ethanol is the best storage choice for buccal swab sampling in fish genetic studies, as well as suitable for small‐bodied rainbow trout.     

The Syndrome of Sea Cucumber （Apostichopusjaponicus） Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

The Syndrome of Sea Cucumber (Apostichopus japonicus) Infected by Virus and Bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75-200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added,healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%-100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%-80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal.     

Cost-effective analysis of genotyping using oral cells in the geriatric population

We evaluated the cost-effectiveness of using buccal swab brushes in comparison with blood samples for obtaining DNA for large epidemiological studies of the elderly population. The data reported here are from the third phase of the Integral Study of Depression among the Elderly in Mexico City's Mexican Institute of Social Security, conducted in 2007. The total cost of the two procedures was determined. The measurement of effectiveness was the quality and quantity of DNA measured in ng/μL and the use of this DNA for the determination of apolipoprotein E (APO E) polymorphism by PCR. Similar rates of amplification were obtained with the two techniques. The cost of the buccal swab brushes, including sample collection and DNA extraction, was US$16.63, compared to the cost per blood sample of US$23.35. Using the buccal swab, the savings was US$6.72 per patient (P < 0.05). The effectiveness was similar. Quantity and quality of DNA obtained were similar for the oral and blood procedures, demonstrating that the swab brush technique offers a feasible alternative for large-scale epidemiological studies.     

Isolation and characterization of a sea cucumber fucoidan‐utilizing marine bacterium

Aims: To isolate a fucoidan‐utilizing strain from seawater for sea cucumber fucoidan degradation. Methods and Results: The utilization of sea cucumber fucoidan was monitored by H₂SO₄–phenol assay for neutral sugar. The bacterium CZ1127 was isolated from seawater and shown to have a relatively large maximum fucoidan‐utilizing rate of 81·5%. CZ1127 was confirmed to belong to the family Flavobacteriaceae by 16S rDNA and physiological analyses. This strain has an ability to utilize fucoidans extracted from various sea cucumbers to different degrees. Both extracellular and intracellular enzymes of CZ1127 could degrade sea cucumber fucoidan, as confirmed by high‐performance size exclusion chromatography. The M_r of sea cucumber fucoidan could be reduced from 792·6 kDa to at least 3·7 kDa by the crude intracellular enzyme of this strain. Conclusions: The marine bacterial strain CZ1127, which belongs to the family Flavobacteriaceae, was found to utilize various sea cucumber fucoidans and furthermore showed promise in sea cucumber fucoidan enzymatic degradation and oligosaccharide preparation. Significance and Impact of the Study: The finding of a novel source can be applied in sea cucumber fucoidan enzymatic degradation. Furthermore, it is the first definite report of a bacterial strain that can utilize the fucoidans from various sea cucumbers.     

The syndrome of sea cucumber (Apostichopus japonicus) infected by virus and bacteria

A outbreak of disease with symptoms of evisceration and skin ulteration led to mass mortality in sea cucumber Apostichopus japonicus cultivated in indoor ponds near the Dalian coast from December 2004 to April 2005. Spherical virus particles with a diameter of 75–200 nm were found in the cytoplasm of cells in the water-system, the alimentary canal and in the respiratory trees of the diseased and dying sea cucumber individuals by electron microscopic observation of ultrathin sections. Examination by negative stained samples revealed that all the diseased sea cucumbers were infected by the virus, while the healthly ones cultivated outside the contagious area were not. Two bacterial strains were also isolated from the diseased animals. When exposed to a medium containing the virus particles, regardless of whether the bacterial suspension was added, healthy sea cucumbers exhibited identical disease symptoms as the ones in the indoor ponds, and had a mortality of 90%–100%. However, when exposed to a medium in which there was only one of the two bacterial strains, 30%–80% of the sea cucumbers were infected and nearly 20% died. Negative staining showed that the viral particles were detected only in the bodies of the tested animals that were exposed to the viral medium. Histopathologically, the diseased sea cucumbers are characterized by karyopycnosis, and disintegration of the endoplasmic reticula and mitochondria in the epithelial cells in the water-system, the respiratory tree and the alimentary canal. Fundation items: Supported by commonweal program of State Oceanic Administration of China (200705007), Science and Technology Project of Liaoning Province (2004203001) and Science and Technology Project of Dlian (2004 BINS030).     

Pros and cons of external swabbing of amphibians for genetic analyses

Non-invasive DNA sampling is an important tool in amphibian conservation. Buccal swabs are nowadays replacing the wounding toe-clipping method. Skin and cloaca swabbing are even less invasive and easier to handle than buccal swabbing, but could result in contaminations of genetic material. Therefore, we test if external skin and cloaca swabs are as reliable as buccal swabs for genetic analysis of amphibians. We analysed eight microsatellite loci for the common frog (Rana temporaria, Linnaeus 1758) and compared genotyping results for buccal, skin and cloaca swabs regarding allelic dropouts and false alleles. Furthermore, we compared two DNA extraction methods regarding efficiency and cost. DNA quality and quantity (amplification success, genotyping error rate, in nanogram per microlitre) were comparable among DNA sources and extraction methods. However, skin and cloaca samples exhibited high degrees of contamination with foreign individuals, which was due to sample collection during mating season. Here, we established a simple low budget procedure to receive DNA of amphibians avoiding stressful buccal swabbing or harmful toe clipping. However, the possibility of contaminations of external swabs has to be considered.     

Clonal structure through space and time: High stability in the holothurian Stichopus chloronotus (Echinodermata)

Sea cucumbers are increasingly exploited for human consumption and for their curative properties, and many wild populations are now depleted or in danger of extinction. While aquaculture is seen as an alternative to fisheries and as a mean to restore wild populations, more knowledge is needed on their reproductive strategies to render this practice efficient, notably for fissiparous holothurians, which are some of the mobile animals able of asexual reproduction by transverse fission. Little information is available on their population genetic diversity and structure. Here, the clonal structure of populations of the fissiparous sea cucumber Stichopus chloronotus has been investigated using nine microsatellite loci and a random sampling, at different spatial (intra‐reef and inter‐reef) and temporal (inter‐season and inter‐year) scales. Our findings highlight the importance of asexual reproduction in maintaining these populations, and the prevalence of the “initial seedling recruitment” strategy (ISR), leading to a high stability of clonal composition over seasons and years. It also seemed that clonal propagation was limited to the reef scale (<10 km) while reefs were connected by sexual dispersal. This is the first time that clonal structure in sea cucumbers has been studied at such a fine scale, with a specific sampling strategy. It provides key findings on the genetic diversity and structure of fissiparous sea cucumbers, which will be useful for the management of wild populations and aquaculture.     

Neuronal peptides induce oocyte maturation and gamete spawning of sea cucumber, Apostichopus japonicus

Extracts prepared from tissues containing buccal ring nerve or longitudinal radial nerve of sea cucumber induce oocyte maturation and ovulation from ovarian tissues. We purified two small peptides, a pentapeptide and a heptapeptide, from the buccal tissues of Japanese common sea cucumber, Apostichopus japonicas. Both peptides induced oocyte maturation and gamete spawning. The pentapeptide was identified as NGIWYamide. This peptide induced in vitro germinal vesicle breakdown and ovulation of fully-grown oocytes at less than 1 pM and in vivo spawning at 10 nM. A synthetic derivative of the pentapeptide, NGLWYamide, was 10-100 times more potent compared to the natural NGIWYamide. The heptapeptide was less potent, inducing ovulation at 1 μM. NGIWYamide and NGLWYamide induced a characteristic spawning behavior when injected into sexually matured individuals. Mature eggs artificially spawned were fertilized, and developed normally and metamorphosed into young sea cucumbers. The details of the production and the mechanism of action of NGIWYamide are still unclear, but the high biopotency of the peptide will aid understanding of the neuronal and hormonal control of reproduction of sea cucumber.     

Evaluation of Methods to Improve the Extraction and Recovery of DNA from Cotton Swabs for Forensic Analysis

Samples for forensic DNA analysis are often collected from a wide variety of objects using cotton or nylon tipped swabs. Testing has shown that significant quantities of DNA are retained on the swab, however, and subsequently lost. When processing evidentiary samples, the recovery of the maximum amount of available DNA is critical, potentially dictating whether a usable profile can be derived from a piece of evidence or not. The QIAamp DNA Investigator extraction kit was used with its recommended protocol for swabs (one hour incubation at 56°C) as a baseline. Results indicate that over 50% of the recoverable DNA may be retained on the cotton swab tip, or otherwise lost, for both blood and buccal cell samples when using this protocol. The protocol’s incubation time and temperature were altered, as was incubating while shaking or stationary to test for increases in recovery efficiency. An additional step was then tested that included periodic re-suspension of the swab tip in the extraction buffer during incubation. Aliquots of liquid blood or a buccal cell suspension were deposited and dried on cotton swabs and compared with swab-less controls. The concentration of DNA in each extract was quantified and STR analysis was performed to assess the quality of the extracted DNA. Stationary incubations and those performed at 65°C did not result in significant gains in DNA yield. Samples incubated for 24 hours yielded less DNA. Increased yields were observed with three and 18 hour incubation periods. Increases in DNA yields were also observed using a swab re-suspension method for both cell types. The swab re-suspension method yielded an average two-fold increase in recovered DNA yield with buccal cells and an average three-fold increase with blood cells. These findings demonstrate that more of the DNA collected on swabs can be recovered with specific protocol alterations.     

Commercial rearing of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> in Peter the great bay: Methodical peculiarities and results of the work of a mariculture farm in Sukhodol Bight

Methodical approaches to commercial rearing of sea cucumber at mariculture farms in Peter the Great Bay in the Sea of Japan are discussed. This includes two basic phases, viz., intermediate rearing of underyearlings in cages and sea ranching. The survival rate of juvenile sea cucumbers during intermediate rearing in cages in Sukhodol Bight (Ussuri Bay) was shown to depend on the body size and rearing density of the juveniles and its value varied from 35 to 82%. The survival rate of two-year-old juveniles on artificial reefs reached 85%. Monitoring studies of the bottom sites showed that for the entire period of functioning of the farm, 8 years, a sea cucumber colony with the average distribution density of 1.5 inds/m² was formed on an area of about 30 ha. The harvest of the commercial production of sea cucumbers purchased at hatcheries can be calculated taking the survival rates of juveniles during the first years of their life into account. The calculation of the production of juvenile sea cucumbers picked by collectors was based on an appraisal of the current state of their aggregation (abundance, biomass, and size structure) with use of conservation protection measures (the allowable harvest rate of 6.4%).     

Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples

Next‐generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI‐based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post‐adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250‐bp, paired‐end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian‐specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.     

Non-invasive method for sampling and extraction of mouse DNA for PCR

We adapted a non-invasive, fast, reliable and inexpensive procedure for the sampling and extraction of deoxyribonucleic acid (DNA) for genetic testing of mice. The procedure is based on a simple DNA extraction procedure used in the forensic genetic testing of humans. It involves mouth swabbing of the inner cheek using a cotton stick, followed by alkaline lysis of the harvested buccal epithelial cells. This procedure allows for repeated sampling and genetic testing of the individual mouse, and it is faster, simpler and, in our hands, more reliable than the currently used routine procedures for the sampling and extraction of mouse DNA. Current procedures all involve biopsy of a piece of the tail, ear or toe, followed by lengthy procedures to release and isolate the DNA.     

Validation of a cheap and simple nondestructive method for obtaining AFLPs and DNA sequences (mitochondrial and nuclear) in amphibians

The use of nondestructive methods for obtaining DNA from amphibians (e.g. buccal swabs) allows genetic studies to be performed without affecting the survival of the studied individuals. In this study, we compared two methods of nondestructive DNA sampling, buccal swabs and interdigital membrane or toe‐clipping, in several amphibian species of different size: Rhinella spinulosa, R. atacamensis, six species of the genus Telmatobius and Pleurodema thaul. We evaluated the integrity of the DNA extracted by sequencing fragments of mitochondrial and nuclear genes and by generating amplified fragment length polymorphisms markers (AFLPs). In all cases, we obtained an adequate amount of DNA (mean range 55–298 ng/μL). We obtained identical DNA sequences from buccal swab and interdigital membrane/toe‐clip for all individuals. The differences in the coding of AFLP markers between the tissues were similar to those reported for replicas of the same type of sample in similar analyses in other species of amphibians. In conclusion, the use of buccal swabs is a trustworthy and inexpensive method to obtain DNA for mitochondrial and nuclear sequencing and AFLP analyses. Given the types of markers evaluated, buccal swabs may be used for phylogenetic, phylogeographic and population genetic studies, even in small amphibians (<33 mm).     

A Simple Method of Genomic DNA Extraction from Human Samples for PCR-RFLP Analysis

Isolation of DNA from blood and buccal swabs in adequate quantities is an integral part of forensic research and analysis. The present study was performed to determine the quality and the quantity of DNA extracted from four commonly available samples and to estimate the time duration of the ensuing PCR amplification. Here, we demonstrate that hair and urine samples can also become an alternate source for reliably obtaining a small quantity of PCR-ready DNA. We developed a rapid, cost-effective, and noninvasive method of sample collection and simple DNA extraction from buccal swabs, urine, and hair using the phenol-chloroform method. Buccal samples were subjected to DNA extraction, immediately or after refrigeration (4–6°C) for 3 days. The purity and the concentration of the extracted DNA were determined spectrophotometerically, and the adequacy of DNA extracts for the PCR-based assay was assessed by amplifying a 1030-bp region of the mitochondrial D-loop. Although DNA from all the samples was suitable for PCR, the blood and hair samples provided a good quality DNA for restriction analysis of the PCR product compared with the buccal swab and urine samples. In the present study, hair samples proved to be a good source of genomic DNA for PCR-based methods. Hence, DNA of hair samples can also be used for the genomic disorder analysis in addition to the forensic analysis as a result of the ease of sample collection in a noninvasive manner, lower sample volume requirements, and good storage capability.     

Sea cucumbers of the Arabian Peninsula and Iran – A review of historical and current research trends

Sea cucumbers are benthic marine invertebrates with immense ecological and commercial value. Processed sea cucumbers known as “Beche-de-mer” are a delicacy in southeast Asian countries with an ever-increasing demand depleting wild stocks on a global scale. Aquaculture techniques are well developed for commercially important species (e.g. Holothuria scabra) to aid in conservation and trade. In the Arabian Peninsula and Iran, where the major land mass is surrounded by marginal seas (Arabian Gulf, Gulf of Oman, Arabian Sea, Gulf of Aden, and Red Sea), studies on sea cucumbers are rather limited and its economic value is underestimated. Historical and current research trends indicate impoverished diversity (82 species) due to environmental extremes. Artisanal fisheries exist for the sea cucumbers of Iran, Oman, and Saudi Arabia, with Yemen and United Arab Emirates (UAE) playing a key role in collection and export to Asian countries. Stock assessment and data on export indicates depletion of natural stocks in Saudi Arabia and Oman. Aquaculture trials of high value species (H. scabra) were successful in Saudi Arabia, Oman and Iran with prospects for further expansion. Research on ecotoxicological properties and bioactive substances conducted in Iran demonstrates an immense research potential. Molecular phylogeny, biology, use in bioremediation, and characterisation of bioactive compounds were identified as potential gaps in research. Expanding aquaculture operations could revive exports and recuperate damaged stocks through sea ranching. Furthermore, regional cooperation, networking, training, and capacity building could help fill the gaps in sea cucumber research, which will aid in its effective conservation and management.     

Sea cucumbers of the Arabian Peninsula and Iran – A review of historical and current research trends

Sea cucumbers are benthic marine invertebrates with immense ecological and commercial value. Processed sea cucumbers known as “Beche-de-mer” are a delicacy in southeast Asian countries with an ever-increasing demand depleting wild stocks on a global scale. Aquaculture techniques are well developed for commercially important species (e.g. Holothuria scabra) to aid in conservation and trade. In the Arabian Peninsula and Iran, where the major land mass is surrounded by marginal seas (Arabian Gulf, Gulf of Oman, Arabian Sea, Gulf of Aden, and Red Sea), studies on sea cucumbers are rather limited and its economic value is underestimated. Historical and current research trends indicate impoverished diversity (82 species) due to environmental extremes. Artisanal fisheries exist for the sea cucumbers of Iran, Oman, and Saudi Arabia, with Yemen and United Arab Emirates (UAE) playing a key role in collection and export to Asian countries. Stock assessment and data on export indicates depletion of natural stocks in Saudi Arabia and Oman. Aquaculture trials of high value species (H. scabra) were successful in Saudi Arabia, Oman and Iran with prospects for further expansion. Research on ecotoxicological properties and bioactive substances conducted in Iran demonstrates an immense research potential. Molecular phylogeny, biology, use in bioremediation, and characterisation of bioactive compounds were identified as potential gaps in research. Expanding aquaculture operations could revive exports and recuperate damaged stocks through sea ranching. Furthermore, regional cooperation, networking, training, and capacity building could help fill the gaps in sea cucumber research, which will aid in its effective conservation and management.     

热带珊瑚礁区海参的生境选择与生态作用

珊瑚礁生态系统是一个高生产力、高生物多样性的特殊海洋生态系统,具有为生物提供栖息地、参与生物地球化学循环、防浪护岸、指示水体污染程度等生态功能。珊瑚礁生态系统的突出特点是其生境异质性很高,各种各样的生境斑块为种类繁多、习性各异的游泳和底栖生物提供栖息场所,这些礁栖生物通过参与各项生态过程而形成各种特定的功能群,共同完成重要的生态功能。在热带珊瑚礁生态系统中,海参是大型底栖动物区系的重要一员。种类繁多的海参具有各自不同的生境选择特征,通过摄食、运动等行为活动发挥着改良底质、促进有机物矿化和营养盐再生等生态作用。近几年来,全球热带海参受人类过度捕捞和珊瑚礁退化的影响而面临资源衰退、物种多样性丧失等问题,深入认识其生态学功能、加强热带海参资源保护迫在眉睫。综述了国内外热带珊瑚礁海参的基础生态学研究进展：海参对珊瑚礁生境斑块呈现显著的偏好选择特征以及种间差异和季节变动,不同生境斑块的食物质量、底质类型和水动力条件是影响海参生境偏好的重要因素;海参通过生物扰动可以改变珊瑚礁生境沉积物的含水量、渗透性、颗粒组成、再矿化率、无机营养物质释放速率以及孔隙水的化学梯度,并增加沉积物中的溶氧浓度、促进溶解...     

海参资源及其生物活性物质的研究

海参属棘皮动物门海参纲,是海洋中重要的食物和药物资源。全世界海参资源比较丰富,目前已知约有1000种,我国约有140多种。海参含有多种生物活性物质,如多糖、甾醇、皂苷、脂肪酸和毒素等,具有增加免疫力、抗肿瘤、抗凝血、保护神经组织、镇痛、抗真菌、降低血黏度等作用。本综述了国内外海参资源分布、海参生物活性物质组成及其药理作用。     

饲料中添加甘草酸对刺参生长、免疫及抗病力的影响

以初始体重为(6.80±0.00)g左右的刺参(Apostichopus japonicus Selenka)为研究对象,在室内循环水系统中进行8周饲喂实验,研究饲料中添加不同梯度的甘草酸对刺参生长、免疫及其抗病力的影响。以基础饲料为对照组,在基础饲料中分别添加50、100和200mg/kg的甘草酸,共配制4种实验饲料。结果表明:饲料中添加甘草酸对刺参的成活率没有影响,各处理组均为100%。饲料中添加200mg/kg甘草酸可显著提高刺参的特定生长率(SGR)(P0.05)。养殖实验结束后,通过注射刺参腐皮综合症致病菌灿烂弧菌(Vibriosplendidus)进行刺参攻毒实验,攻毒后14d内对照组与50mg/kg添加组的累计发病率(分别为38.3%和36.7%)显著高于100和200mg/kg添加组(分别为30.0%和26.7%)(P<0.05)。实验可得到以下结论:(1)饲料中添加200mg/kg甘草酸可以提高刺参养殖的产量,同时可以提高刺参的非特异性免疫力和抗病力;(2)在研究中,全周期养殖期间投喂甘草酸不会产生免疫疲劳或其他副作用。