Determination of paralytic shellfish poisoning (PSP) toxins in UK shellfish

A study was conducted to aid the interpretation of data generated by parallel testing of the qualitative Jellett Rapid Test (JRT) and the mouse bioassay (MBA) for detection of paralytic shellfish poisoning (PSP) toxins within the UK statutory shellfish biotoxin monitoring programme. A selection of stored sample extracts subjected to testing by MBA and/or JRT were further analysed by liquid chromatography with fluorescence detection (LC–FLD) to provide additional information on the concentrations of PSP toxins and toxin profiles.Results, from this study, demonstrate the potential of the JRT to effectively screen out PSP toxin negative shellfish samples and samples containing low concentrations of toxins from UK monitoring programmes. Additionally, data generated using LC–FLD highlights the potential of introducing alternative analytical techniques to completely replace the requirement for the MBA.     

Profiles of Alexandrium catenella cysts in Puget Sound sediments and the relationship to paralytic shellfish poisoning events

The magnitude of paralytic shellfish poisoning (PSP) toxins in shellfish and the geographical scope of shellfish closures in Puget Sound have increased in recent decades. PSP, monitored by the Washington Department of Health, has spread from Sequim Bay in the 1950s into central Puget Sound in the 1970s and throughout Puget Sound by the 1990s. Alexandrium catenella, the species responsible for PSP toxins, produces a benthic resting cyst that, upon germinating, can seed blooms. This study examined whether there is a relationship between profiles of cysts in the sediment and temporal variation in PSP in shellfish and if the history of the toxin's southward expansion through Puget Sound can be seen in the cyst record. To address this question, sediment cores were collected from three Puget Sound basins, Sequim Bay, Penn Cove, and Carr Inlet, and cyst profiles were determined. Activities of ²¹⁰Pb were fitted to a depth-dependent diagenetic model to date the sediment cores and determine mixing and sediment-accumulation rates. In order to compare historical variation in PSP with cyst profiles that have been altered by bioturbation, a depth and time-dependent diagenetic model was then used to predict vertical profiles of cysts that would occur under the assumption that cyst deposition rates are proportional to PSP concentration in shellfish measured over several decades at each site. The cyst profiles predicted by the model were compared to measured cyst profiles. These comparisons suggested that Alexandrium blooms and resulting PSP concentration in shellfish are more closely linked to cyst germination and deposition at some stations than at others. Sequim Bay had relatively large numbers of cysts and it is likely that the persistent toxicity here is the result of recurrent seeding from the cyst bed. Penn Cove and Carr Inlet had few cysts despite occasional blooms, suggesting that blooms are transported into those areas, perhaps from other sites of cyst germination. Sequim Bay and Penn Cove had cysts from top to bottom of the cores so it was not possible to determine the date when cysts were first introduced into these bays, but it is likely that A. catenella has been in Penn Cove since at least 1955 or for about two decades before the WDOH PSP toxicity data would indicate. The cyst profile in Carr Inlet suggested a first appearance date of 1985 that is consistent with the first appearance of PSP in shellfish in 1988.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium minutum (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium minutum (Dinophyceae) are a worldwide concern from both the economic and human health points of view. For population genetic studies of A. minutum distribution and dispersal, highly polymorphic genetic markers are of great value. We isolated 12 polymorphic microsatellites from this cosmopolitan, toxic dinoflagellate species. These loci provide one class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the estimate of gene diversity was from 0.560 to 0.862 across the 12 microsatellites; these loci have the potential to reveal genetic structure and gene flow among A. minutum populations.     

Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Mussels (Mytilus galloprovincialis) were experimentally contaminated with paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense, and changes in toxin content and specific composition during the decontamination period were analyzed by high-performance liquid chromatography (HPLC). Toxins excreted by the mussels into the seawater were also recovered using an activated charcoal column and analyzed by HPLC. The predominant toxins in A. tamarense, mussels, and seawater were the N-sulfocarbamoyl-11-hydrosulfate toxins (C1,2) and carbamate gonyautoxins-1,4 (GTX1,4). There were no remarkable differences in the relative proportions of the predominant toxins within A. tamarense, mussels and seawater. Because the relative proportion of the various toxin analogues excreted by the mussels was similar to that within their tissues during detoxification, it appeared that the selective release of particular toxins by the mussels was unlikely. The total amount of toxin lost from mussels was nearly equal to that which was found dissolved in the seawater, suggesting that, at least the early stages of mussel detoxification, most losses can be accounted for by excretion.     

Effect of storage on amnesic shellfish poisoning (ASP) toxins in king scallops (Pecten maximus)

Since 1998, king scallops (Pecten maximus) obtained from Scottish offshore sites have been monitored for domoic acid (DA) and epi-domoic acid (epi-DA), the principal toxic compounds associated with amnesic shellfish poisoning (ASP). The presence of these toxins in king scallops harvested from Scottish waters at concentrations exceeding the current regulatory limit (20 μg g⁻¹ shellfish flesh) is a recurrent event. However, little information was available to determine the effects that different storage conditions experienced during sample transportation to the monitoring laboratory may have on the toxin concentrations, which are subsequently detected. Furthermore, the stability of DA and epi-DA in the solvents (methanol:water (1:1, v/v) and citric acid buffer (0.5 M, pH 3.2)) routinely used for their extraction from shellfish has not previously been assessed. Results from this study demonstrate that when king scallop samples were stored for 2–3 days at 12 °C, a significantly higher toxin concentration was detected in the gonad than when samples were stored at 4 °C and analysed within 48 h. This implies that monitoring programmes must consider transport and storage conditions between harvest and analysis. Stability studies showed rapid decomposition of DA and epi-DA in aqueous methanol extracts while DA and epi-DA seem acceptably stable when stored refrigerated in citrate buffer.     

Variation in MHC genotypes in two populations of house sparrow (Passer domesticus) with different population histories

Small populations are likely to have a low genetic ability for disease resistance due to loss of genetic variation through inbreeding and genetic drift. In vertebrates, the highest genetic diversity of the immune system is located at genes within the major histocompatibility complex (MHC). Interestingly, parasite‐mediated selection is thought to potentially maintain variation at MHC loci even in populations that are monomorphic at other loci. Therefore, general loss of genetic variation in the genome may not necessarily be associated with low variation at MHC loci. We evaluated inter‐ and intrapopulation variation in MHC genotypes between an inbred (Aldra) and a relatively outbred population (Hestmannøy) of house sparrows (Passer domesticus) in a metapopulation at Helgeland, Norway. Genomic (gDNA) and transcribed (cDNA) alleles of functional MHC class I and IIB loci, along with neutral noncoding microsatellite markers, were analyzed to obtain relevant estimates of genetic variation. We found lower allelic richness in microsatellites in the inbred population, but high genetic variation in MHC class I and IIB loci in both populations. This suggests that also the inbred population could be under balancing selection to maintain genetic variation for pathogen resistance.     

Root responses to cereal cyst nematode (Heterodera avenae) in hosts with different resistance genes

The development of cereal cyst nematode (CCN; Heterodera avenae ) induced syncytia in the host roots of infected resistant bread wheat ( Triticum aestivum cv. AUS10894), diploid wheat ( Aegilops tauschii ), barley ( Hordeum vulgare cv. Chebec and cv. Galleon) and in the susceptible wheat cv. Meering and barley cv. Clipper were studied over a period of 13 d. The resistance to CCN in these cereal plants is conferred by the resistance genes Cre1 in the wheat cv. AUS10894, Cre3 in A. tauschii , Ha2 in barley cv. Chebec and Ha4 in barley cv. Galleon. Anatomical observations were made on the development of the syncytia in CCN-infected wheat and barley roots, which carry each of these four sources of resistance genes. Accelerated development of the syncytia in resistant plants, especially in the barley cultivars, was observed. The sites of syncytia development in susceptible wheat and barley were also closely associated with the vascular tissues in the stele, but less so in the resistant plants. The syncytia in the infected susceptible wheat and barley were also metabolically active at day 13. By contrast, the syncytia of resistant wheat plants carrying the Cre1 or Cre3 genes remained extensively vacuolated and less metabolically active. In barley plants with the Ha2 or Ha4 genes, the syncytia appeared non-functional and in early stages of degeneration by day 13 after inoculation.     

Body size, prey handling efficiency and choice of diet in three coniferous forest tits (Paridae)

The main purpose of this study was to link morphological differences between great tit ( Parus major ), willow tit ( P. montanus ) and coal tit ( P. ater ) and their rate of energy acquisition and choice of diet in order to explore the potential for competitive relations between them more directly. Handling times were measured in the laboratory by presenting mealworms of different sizes to the birds. Great tits were more efficient in handling large prey than were the smaller-bodied willow- and coal tits; for small prey sizes the coal tit was the least efficient species. Using the ratio of prey mass to the handling time value, a utility function for each species was constructed. These results suggests a potential for a segregation of the species on the food axis. However, results from the prey choice experiment show that despite considerable differences in functional morphology between the three species they do not differ significantly in the range of prey size exploited. My results suggest that the alleged importance of prey size partitioning is not likely to play the major role for the coexistence of these coniferous forests tits.     

褐飞虱种群的广义逻辑斯谛曲线模型的研究

用广义逻辑斯谛曲线模型及预测饱和增长趋势,解释了该模型包含的实际生物学意义,并给出了相应的参数估计算法,对褐飞虱Nilaparvatalugens（Stal）种群资料进行具体的分析,结果与其它模型相比,拟合程度高,用广义逻辑斯谛曲线模型作为预报,可以提高预测的效果。     

The release of glutamate and aspartate from rat brain synaptosomes in response to domoic acid (amnesic shellfish toxin) and kainic acid

Kainic acid is known to stimulate the release of glutamate (GLU) and aspartate (ASP) from presynaptic neurons. It has been suggested that the enhanced release of these endogenous EAA's plays a significant role in the excitotoxic effects of KA. Domoic acid (DOM), a shellfish toxin, is structurally similar to KA, and has been shown to be 3–8 times more toxic than KA. In this study, effects of KA and DOM on the release of GLU and ASP from rat brain synaptosomes were investigated. Amino acid analysis was performed by the reversed phase HPLC, following derivatization with 9-fluorenylmethyl chloroformate (FMOC). Potassium chloride (40 mM) was used as a positive control, and stimulated GLU release from rat brain synaptosomes in presence or absence of Ca²⁺. DOM enhanced the release of GLU, whereas KA stimulated the release of both GLU and ASP from synaptosomes in the presence of Ca²⁺. However, their potency to stimulate GLU and ASP release was enhanced in absence of Ca²⁺. These results indicate that diferent mechanisms may be involved in the release of GLU and ASP in response to DOM and KA, and that neurotransmitter release appeared to be highly specific for these agonists. It would appear that DOM and KA may interact with different receptors on the presynaptic nerve terminal, and/or activate different subtypes of voltage-dependent Ca²⁺ channels to promote influx of Ca²⁺ which is targeted for different pools of neurotransmitters.Abbreviations ANOVA analysis of variance - ASP aspartate - DOM domoic acid - DHKA dihydrokainic acid - EAA excitatory amino acid - FMOC 9-fluorenylmethyl chloroformate - GLU glutamate - KA kainic acid     

Application of whole genome re-sequencing data in the development of diagnostic DNA markers tightly linked to a disease-resistance locus for marker-assisted selection in lupin (Lupinus angustifolius)

Background

Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding.

Results

Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding.

Conclusions

We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1878-5) contains supplementary material, which is available to authorized users.     

Sex difference in the neurotensin-immunoreactive cell populations of the preoptic area in quail (Coturnix japonica)

The distribution of neurotensin-immunoreactive cells and fibers was analyzed by immunocytochemistry in the forebrain of male and female Japanese quail (Coturnix japonica) by using an antibody directed against the C-terminal part of the molecule. Immunoreactive perikarya were located almost exclusively in the medial preoptic area with small populations also being present in the nucleus paraventricularis and in the tuberal region. Immunoreactive fibers were observed not only throughout the preoptic area-hypothalamus, but also in the septal region, nucleus intercollicularis, substantia grisea centralis and the classical catecholaminergic areas of the mesencephalon, such as the area ventralis of Tsai and the nucleus tegmenti pedunculo-pontinus, pars compacta. The preoptic neurotensin-immunoreactive cells were exclusively located within the boundaries of the sexually dimorphic medial preoptic nucleus. They were significantly more numerous in females than in males. In females, the number of neurotensin cells varied during the ovulatory cycle: fewer cells were observed in birds that were about to lay an egg (they had a calcified egg in the oviduct) than in those that had already laid or were not going to lay on that day. These data indicate major variations in the expression of neurotensin in response to neurochemical or neuroendocrine changes associated with ovulation.     

Multitrait selection system using populations with a small number of interploid (4x-2x) hybrid seedlings in potato: Degree of high-parent heterosis for yield and frequency of clones combining quantitative agronomic traits

One of the most attractive features of the 4x-2x breeding scheme via unilateral sexual polyploidization (USP) is the possibility of obtaining heterotic clones with a combination of desirable traits based upon selection in a small number of hybrid seedlings. A set of experiments was carried out to verify this characteristic of the USP scheme using 42 families (with 20 plants each) derived from 2x Phureja-haploid Tuberosum or haploid Tuberosum-Solanum chacoense hybrids as male parents. These clones were 2n-pol-len-producers by either first-division restitution with crossing over (FDR-CO) or without crossing over (FDR-NCO). The 4x parents were eight cultivars of USA and European origin. A total of 168 out 840 clones was initially selected (four clones/family) based on general tuber appearance (GTA). An additional round of selection (not taking into account the parentage of the clone) for total tuber yield (TTY) reduced the sample to 96 clones. These selected clones and the original 4x parents were evaluated in two locations at Wisconsin (Hancock – E?1, and Rhinelander – E?2). The average high-parent heterosis values for TTY ranged from 27.8% (E?2) to 48% (E?1). The population of experimental clones also had a TTY range wider than that of the 4x parents. The best clone gave a yield of 101.6% and 63.2% over the best 4x parent at E?1 and E?2, respectively. For TTY, a significant genotype×environment (G×E) interaction for the experimental clones was found when a combined analysis of variance for both locations was carried out. However, the G×E interaction was not significant when only the 4x-parent group was considered. Clones derived from 4x European cultivars had higher GTA scores than clones derived from 4x USA cultivars. With two culling levels being set on the TTY≥4x parent group mean and the GTA≥three, about 56% (E?1) and 48% (E?2) of the clones would be retained for further evaluation. These percentages of selected clones are much higher than those reported using conventional 4x-4x crosses. Our results indicate that a USP strategy with 4x-2x (FDR) crosses would be more effective than intra-Tuberosum crosses in generating heterotic clones with a combination of desirable quantitative traits using populations with a small number of hybrid seedlings.     

De-novo identification of specific exposure biomarkers of the alternative plasticizer di(2-ethylhexyl) terephthalate (DEHTP) after low oral dosage to male volunteers by HPLC-Q-Orbitrap-MS

Context: Human exposure biomonitoring relies on the availability of specific, sensitive biomarkers. For emerging chemicals, the identification (prediction, synthesis, verification) of such biomarkers is time and cost intensive.

Objective: This study aimed to further elucidate the urinary metabolic profile of the plasticizer di(2-ethylhexyl) terephthalate (DEHTP) in search of probably additional biomarkers of exposure.

Materials and methods: Urine samples of an oral low-dose volunteer study were analysed by HPLC-Q-Orbitrap-MS combined with a commercial data mining software. Metabolite identification was based on isotopic pattern, accurate masses of product ions and excretion profiles.

Results: Nine phase I metabolites of DEHTP were tentatively identified by HPLC-Q-Orbitrap-MS. Four previously described, side chain oxidized monoester metabolites were confirmed in all samples. In addition, five previously unknown downstream metabolites were tentatively identified.

Discussion and conclusion: The excretion profiles obtained by HPLC-Q-Orbitrap-MS were in good agreement with quantitative HPLC-QqQ-MS data. For the newly discovered metabolites, plausible excretion profiles, similar to the ones of the known metabolites, were obtained. The presented approach proved to be successful for metabolite screening in urine samples after low-dose exposure and will be applied in future human metabolism studies for a fast, reliable and cost effective identification of specific biomarkers of exposure.     

Investigation of body surface temperature measured with infrared imaging and its correlation with feed efficiency in the turkey (Meleagris gallopavo)

As feed intake is an expensive trait to measure in a breeding program, this study investigated whether body surface temperature was correlated with feed efficiency in the turkey. Infrared images were captured on male turkeys in individual feeding cages at approximately 19-weeks of age. Body surface temperature was measured on the eye, head, distal metatarsus, and neck of the birds with temperatures ranging from 34 °C to 40 °C. Body weight, feed intake, and weight gain data were collected and feed efficiency was measured as residual feed intake (RFI) and feed conversion ratio (FCR). Distal metatarsus temperature showed moderate correlations with body weight (0.15), average daily gain (0.26) and feed intake (0.23). Surface temperature of the head, eye, and neck were not as strongly correlated with these production traits. The feed efficiency traits showed low correlations with eye temperature ranging from −0.05 to −0.12 and surface temperatures at the head, distal metatarsus, and neck were not correlated with feed efficiency. Surface temperature traits explained only a small proportion of variation in feed intake. While the collection of infrared images was efficient and required minimal contact with the caged birds, the low correlations indicate that the technology, as used in this study, has limited advantages for increasing the accuracy of selection for feed efficiency.     

Variation in the suitability of Pinus sylvestris to feeding by two defoliators, Diprion pini (Hym., Diprionidae) and Graellsia isabellae galliaegloria (Lep., Attacidae)

Abstract: Feeding bioassays were conducted on several Pinus sylvestris clones to establish if there were any differences in suitability for two pine defoliators: the sawfly Diprion pini L. (Hym., Diprionidae), which causes considerable damage in Europe, and the rare and protected moth Graellsia isabellae galliaegloria Oberthür (Lep., Attacidae). There were significant differences in survival, weight, sex-ratio and female fecundity of D. pini on Scots pine clones. However, sawfly survival appeared to be the most stable variable in time and three clone categories were revealed by conducting feeding bioassays on 16 clones. The survival of G. isabellae galliaegloria until the formation of the chrysalis was significantly different among clones. It varied from 35 to 75% but no clear clone category was distinguished . The two clone classifications according to insect survival showed differences. In particular, one unsuitable clone for D. pini development appeared to be favourable to moth development. The 3-carene richness of foliage was significantly linked with sawfly survival and the monoterpene composition of foliage was not linked with G. isabellae galliaegloria survival.     

A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

The association of two single nucleotide polymorphisms (SNPs) in growth hormone (GH) gene with litter size and superovulation response in goat-breeds

Two active mutations (A 781 G and A 1575 G) in growth hormone (GH) gene, and their associations with litter size (LS), were investigated in both a high prolificacy (Matou, n = 182) and a low prolificacy breed (Boer, n = 352) by using the PCR-RFLP method. Superovulation experiments were designed in 57 dams, in order to evaluate the effect of different genotypes of the GH gene on superovulation response. Two genotypes (AA and AB, CC and CD) in each mutation were detected in these two goat breeds. Neither BB nor DD homozygous genotypes were observed. The genotypic frequencies of AB and CC were significantly higher than those of AA and CD. In the third parity, Matou dams with AB or CC genotypes had significantly larger litter sizes than those with AA and CD (p < 0.05). On combining the two loci, both Matou and Boer dams with ABCD genotype had the largest litter sizes when compared to the other genotypes (p < 0.05). When undergoing like superovulation treatments, a significantly higher number of corpora lutea and ova, with a lower incidence of ovarian cysts, were harvested in the AB and CC genotypes than in AA and CD. These results show that the two loci of GH gene are highly associated with abundant prolificacy and superovulation response in goat breeds.     

过去二十年贵州黑叶猴分布与种群动态及致危因子分析(英文)

本文通过整理相关文献与政府报告数据并结合我们在贵州的最新调查与监测数据,总结过去20年间黑叶猴在贵州的分布、种群变动及其所面临的主要威胁,并在此基础上提出相关的保护管理建议。贵州黑叶猴野生种群在过去20年间增长了约10%～20%:从20世纪90年代的约109群1000只增加到现在的约132～137群1160～1200只。但同期黑叶猴已从5个原有的分布点绝迹,现仅存于大沙河、柏箐、麻阳河、宽阔水和野钟等5个保护区,总面积约912km2。现有的5个隔离分布点保存有全球黑叶猴野生种群(1800～2000)的约62%,是黑叶猴物种保存的最关键地区,并应该给于保护管理的优先考虑。偷猎压力在贵州相对较小:在过去的20年间仅有3次偷猎的官方记录。黑叶猴在贵州所面临的主要威胁是栖息地的丧失与退化。其主要表现形式有:以农作物和烟草种植为目的的耕地扩大,以做饭、取暖以及烟叶烘烤为目的的薪材需求,以及家畜(主要是山羊)的过度放牧。