Pesticides and Ostreid Herpesvirus 1 Infection in the Pacific Oyster,Crassostrea gigas

Since 2008, mass mortality outbreaks have been reported in all French regions producing Pacific oysters, and in several Member States of the European Union. These mass mortality events of Pacific oysters are related to OsHV-1 infection. They occur during spring and summer periods leaving suspect the quality of the marine environment and the role of seasonal use of pesticides associated with the arrival of freshwater in oyster rearing areas. Pesticides have been also detected in French coastal waters, especially in areas of oyster production. Using PMA real-time PCR we showed that a mixture of 14 pesticides has no effect on the integrity of virus capsids from viral suspension in the conditions tested. A contact of oysters with this pesticide mixture was related to higher mortality rates among experimentally infected animals in comparison with control ones (no previous pesticide exposure before experimental infection). We therefore suggest that pesticides at realistic concentration can exert adverse effects on Pacific oysters and causes an increased susceptibility to the viral infection in experimental conditions.     

A Glycoprotein in Shells of Conspecifics Induces Larval Settlement of the Pacific Oyster Crassostrea gigas

Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et₂O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics.     

Extraction and Identification of the Pigment in the Adductor Muscle Scar of Pacific Oyster Crassostrea gigas

In this study, UV (ultraviolet) and IR (infrared radiation) spectral analysis were integrated to identify the pigment in the adductor muscle scar of the Pacific oyster Crassostrea gigas. The pigment was extracted from the adductor muscle scars of cleaned oyster shells that were pulverized, hydrolyzed in hot hydrochloric acid, purified with diethyl ether, and dissolved in 0.01 mL/L NaOH. The maximum absorption of the pigment in the UV absorption spectrum within the range of 190–500 nm was observed between 210–220 nm. The UV absorbance decreased with increasing wavelength which was consistent with the UV spectral absorption characteristics of melanin. In addition, Fourier transform infrared spectroscopy scanning revealed characteristic absorption peaks that emerged near 3440 cm^-1 and 1630 cm^-1, which was consistent with infrared scanning features of eumelanin (a type of melanin). This study has demonstrated for the first time that the pigment in the adductor muscle scar of the Pacific oyster is melanin, hinting that the adductor muscle could be another organ pigmenting the mollusc shell with melanin other than mantle.     

Ostreid Herpesvirus 1 Infection among Pacific Oyster (Crassostrea gigas) Spat: Relevance of Water Temperature to Virus Replication and Circulation Prior to the Onset of Mortality

A number of bivalve species worldwide, including the Pacific oyster, Crassostrea gigas, have been affected by mass mortality events associated with herpesviruses, resulting in significant losses. A particular herpesvirus was purified from naturally infected larval Pacific oysters, and its genome was completely sequenced. This virus has been classified as Ostreid herpesvirus 1 (OsHV-1) within the family Malacoherpesviridae. Since 2008, mass mortality outbreaks among C. gigas in Europe have been related to the detection of a variant of OsHV-1 called μVar. Additional data are necessary to better describe mortality events in relation to environmental-parameter fluctuations and OsHV-1 detection. For this purpose, a single batch of Pacific oyster spat was deployed in 4 different locations in the Marennes-Oleron area (France): an oyster pond (“claire”), a shellfish nursery, and two locations in the field. Mortality rates were recorded based on regular observation, and samples were collected to search for and quantify OsHV-1 DNA by real-time PCR. Although similar massive mortality rates were reported at the 4 sites, mortality was detected earlier in the pond and in the nursery than at both field sites. This difference may be related to earlier increases in water temperature. Mass mortality was observed among oysters a few days after increases in the number of PCR-positive oysters and viral-DNA amounts were recorded. An initial increment in the number of PCR-positive oysters was reported at both field sites during the survey in the absence of significant mortality. During this period, the water temperature was below 16°C.     

Evaluation of NemX,a New Cultivar of Cotton with High Resistance to Meloidogyne incognita

The level of resistance to root-knot nematode, Meloidogyne incognita, in NemX, a new cultivar of the Acala-type upland cotton, was evaluated in relation to four resistant breeding lines (N6072, N8577, N901, and N903) and four susceptible cultivars (Maxxa, SJ2, Royale, and Prema). In growth pouch tests, an average of only 4 nematode egg masses was produced on roots of NemX or the resistant lines, compared to a significantly higher average of 21 on the susceptible cultivars. In pot tests, the nematode reproduction factor (RF = Pf/Pi) in NemX and the resistant lines averaged 0.7, compared to a significantly higher average of 10 on the susceptible cultivars. Root galling in NemX or other resistant cotton averaged 15%, compared to 74% on the susceptible cultivars, in either pot or field tests. In plots with low levels of nematode infestation (Pi ≤ 150 second-stage juveniles [J2]/500 g soil), lint yield of NemX averaged 1,370 kg/ha and was less than the yield of susceptible Maxxa (1,450 k g /h a ). However, in plots with medium or high levels of nematode infestation (Pi = 151-300 or >300 J2/500 g soil, respectively), yields of NemX decreased only slightly and averaged 1,300 or 1,050 kg/ha, respectively, whereas yields of Maxxa were severely reduced to 590 or 503 kg/ha, respectively. Fusarium wih symptoms were observed on both NemX and Maxxa, and percent occurrence increased with increasing preplant nematode density. In plots with the highest nematode densities, 22% of NemX and 65% of Maxxa plants were wilted. NemX was highly effective against five M. incognita isolates and moderately effective against a sixth isolate that had been exposed to resistant cotton over several seasons. These results showed that NemX is as resistant to M. incognita as the four breeding lines, and much more resistant than the tested susceptible cultivars of cotton.     

Screening Soybeans for Resistance to Reniform Nematode Disease in the Philippines

Sixty-five soybean varieties were tested in the field for resistance to Rotylenchulus reniformis. Criteria for resistance or susceptibility were root necrosis, nematode recovery from roots and soil, and egg production. Nine varieties were resistant, 13 moderately resistant, 26 moderately susceptible, and 17 susceptible. Linear correlations between resistance rating and each assessment parameter were highly significantly positive, suggesting that any of the parameters could be used to identify resistance. There were also highly significant positive linear correlations between any two combinations of parameters, indicating that they were reciprocally related.     

Genetic Diversity,Population Structure,and Resistance to Phytophthora capsici of a Worldwide Collection of Eggplant Germplasm

Eggplant (Solanum melongena L.) is an important solanaceous crop with high phenotypic diversity and moderate genotypic diversity. Ninety-nine genotypes of eggplant germplasm (species (S. melongena, S. incanum, S. linnaeanum and S. gilo), landraces and heirloom cultivars) from 32 countries and five continents were evaluated for genetic diversity, population structure, fruit shape, and disease resistance to Phytophthora fruit rot. Fruits from each line were measured for fruit shape and evaluated for resistance to two Phytophthora capsici isolates seven days post inoculation. Only one accession (PI 413784) was completely resistant to both isolates evaluated. Partial resistance to Phytophthora fruit rot was found in accessions from all four eggplant species evaluated in this study. Genetic diversity and population structure were assessed using 22 polymorphic simple sequence repeats (SSRs). The polymorphism information content (PIC) for the population was moderate (0.49) in the population. Genetic analyses using the program STRUCTURE indicated the existence of four genetic clusters within the eggplant collection. Population structure was detected when eggplant lines were grouped by species, continent of origin, country of origin, fruit shape and disease resistance.     

AprioriGWAS,a New Pattern Mining Strategy for Detecting Genetic Variants Associated with Disease through Interaction Effects

Identifying gene-gene interaction is a hot topic in genome wide association studies. Two fundamental challenges are: (1) how to smartly identify combinations of variants that may be associated with the trait from astronomical number of all possible combinations; and (2) how to test epistatic interaction when all potential combinations are available. We developed AprioriGWAS, which brings two innovations. (1) Based on Apriori, a successful method in field of Frequent Itemset Mining (FIM) in which a pattern growth strategy is leveraged to effectively and accurately reduce search space, AprioriGWAS can efficiently identify genetically associated genotype patterns. (2) To test the hypotheses of epistasis, we adopt a new conditional permutation procedure to obtain reliable statistical inference of Pearson''s chi-square test for the contingency table generated by associated variants. By applying AprioriGWAS to age-related macular degeneration (AMD) data, we found that: (1) angiopoietin 1 (ANGPT1) and four retinal genes interact with Complement Factor H (CFH). (2) GO term “glycosaminoglycan biosynthetic process” was enriched in AMD interacting genes. The epistatic interactions newly found by AprioriGWAS on AMD data are likely true interactions, since genes interacting with CFH are retinal genes, and GO term enrichment also verified that interaction between glycosaminoglycans (GAGs) and CFH plays an important role in disease pathology of AMD. By applying AprioriGWAS on Bipolar disorder in WTCCC data, we found variants without marginal effect show significant interactions. For example, multiple-SNP genotype patterns inside gene GABRB2 and GRIA1 (AMPA subunit 1 receptor gene). AMPARs are found in many parts of the brain and are the most commonly found receptor in the nervous system. The GABRB2 mediates the fastest inhibitory synaptic transmission in the central nervous system. GRIA1 and GABRB2 are relevant to mental disorders supported by multiple evidences.     

Residues Surrounding the Glycosylphosphatidylinositol Anchor Attachment Site of PrP Modulate Prion Infection: Insight from the Resistance of Rabbits to Prion Disease

Prion diseases are a group of transmissible, invariably fatal neurodegenerative diseases that affect both humans and animals. According to the protein-only hypothesis, the infectious agent is a prion (proteinaceous infectious particle) that is composed primarily of PrP^Sc, the disease-associated isoform of the cellular prion protein, PrP. PrP^Sc arises from the conformational change of the normal, glycosylphosphatidylinositol (GPI)-anchored protein, PrP^C. The mechanism by which this process occurs, however, remains enigmatic. Rabbits are one of a small number of mammalian species reported to be resistant to prion infection. Sequence analysis of rabbit PrP revealed that its C-terminal amino acids differ from those of PrP from other mammals and may affect the anchoring of rabbit PrP through its GPI anchor. Using a cell culture model, this study investigated the effect of the rabbit PrP-specific C-terminal amino acids on the addition of the GPI anchor to PrP^C, PrP^C localization, and PrP^Sc formation. The incorporation of rabbit-specific C-terminal PrP residues into mouse PrP did not affect the addition of a GPI anchor or the localization of PrP. However, these residues did inhibit PrP^Sc formation, suggesting that these rabbit-specific residues interfere with a C-terminal PrP^Sc interaction site.Prion diseases, traditionally known as transmissible spongiform encephalopathies (TSE), are a group of invariably fatal neurodegenerative diseases that affect both humans and animals. According to the protein-only hypothesis, an abnormal isoform of the host-encoded prion protein (PrP^C), referred to as PrP^Sc, is the sole or major component of the infectious agent causing these diseases (33). These disorders affect a wide range of mammals and include diseases such as Creutzfeldt-Jakob disease (CJD), variant CJD, Gerstmann-Straüssler-Scheinker (GSS) syndrome, kuru, and fatal familial insomnia (FFI) in humans, scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE) in cattle. The term “prion” was first used to describe the unique infectious agent and was derived from “proteinaceous infectious particle” to distinguish it from conventional pathogens such as bacteria and viruses (33).To date, rabbits are one of the few mammalian species reported to be resistant to prion infection. Rabbits do not develop clinical disease after inoculation with brain tissue from individuals affected by the human prion diseases CJD and kuru, or by a number of animal forms of the disease, including scrapie and transmissible mink encephalopathy (TME) (12). In addition, mouse neuroblastoma (MNB) cells overexpressing rabbit PrP are also resistant to prion infection (45). Evidence that rabbit cells per se have the correct cellular machinery to support prion propagation has come from studies using the rabbit kidney epithelial cell line RK13. Upon transfection with appropriate PrP-expressing transgenes, these cells are a highly efficient and robust model of prion infection (6, 25, 41, 43). RK13 cells do not have detectable levels of endogenous rabbit PrP^C and are therefore ideal for studying exogenous PrP^C and the propagation of prions from different species (6). Originally, it was shown that RK13 cells overexpressing ovine PrP became susceptible to infection with scrapie (43), and more recently, RK13 cells expressing rodent PrP^C, from either the mouse or the bank vole, were readily infected by prions adapted to and propagated in these two species (6, 41). RK13 cells expressing human PrP^C, however, were resistant to infection with human prions derived directly from a patient with sporadic CJD (25). Since RK13 cells overexpressing PrP are a well-established model of prion propagation, we can therefore conclude that while these cells apparently have the appropriate cellular machinery to support prion propagation, it may be a characteristic of the rabbit prion protein itself that results in the resistance of this species to prion infection. However, the loss of a cellular cofactor may also be a contributing factor.Analysis of the rabbit PrP amino acid sequence shows that it has all the features previously described for members of the PrP protein family, including an N-terminal signal peptide, an octapeptide repeat region, and a C-terminal signal sequence (26). While amino acid sequence comparison of both mouse and rabbit PrP species reveals 87% sequence homology, there are 22 amino acid differences between the two, and several of these reside in regions of PrP known to be important in PrP^Sc formation. In scrapie-infected MNB cells, the residues Gly99 and Met108 within the N terminus, Ser173 within the central region, and Ile214 within the C terminus of rabbit PrP were shown to inhibit PrP^Sc generation when incorporated into mouse PrP, suggesting that multiple amino acid residues in rabbit PrP inhibit PrP^Sc formation (45). Approximately one-third (9/33 residues in the immature sequence) of the amino acid difference between mouse and rabbit PrPs was shown to occur at the glycosylphosphatidylinositol (GPI) anchor attachment site (see Fig. S1 in the supplemental material). As yet, studies involving this region of rabbit PrP have not been performed. Therefore, this region of rabbit PrP may provide further insight into the resistance of rabbits to prion infection.GPI anchor addition occurs via a transamination reaction in the endoplasmic reticulum (ER) following cleavage of the C-terminal signal sequence (39). There is no consensus sequence with which to identify the C-terminal cleavage site, but there are three key C-terminal elements: (i) the cleavage site, or ω site, where the GPI anchor attaches to the COOH group of the ω amino acid; (ii) a hydrophilic spacer region of 8 to 12 amino acids (ω + 1 up to ω + 10); and (iii) a hydrophobic region of 10 to 20 amino acids (ω + 11 onwards) (9). Analysis of known GPI-anchored proteins has given rise to sequence motifs in the C-terminal signal peptide allowing the prediction of the ω site of proteins. Due to the complexity of experimentally determining the ω site of GPI-anchored proteins, relatively few of the many known GPI-anchored proteins have had their ω sites determined (36 of 340 proteins in 2008) (32) The ω site of hamster PrP was determined experimentally to be at amino acid 231 (34) and is predicted to be at the same site for PrPs from all mammals, based on amino acid sequence comparison. Amino acid substitutions near the ω site of mouse PrP revealed that mouse PrP has an ω site at residue 230 (17). It was also shown that single amino acid substitutions at and near the ω site of mouse PrP affect the anchoring and conversion efficiency of PrP (17). It is therefore possible that the amino acids at the C terminus and within the GPI anchor signal sequence of rabbit PrP lead to the resistance to prion infection.To date, no protein structures containing a GPI anchor have been determined by X-ray crystallography, and although the nuclear magnetic resonance (NMR) structures of mouse and rabbit PrP have been solved, they do not contain any structural information for the residues immediately preceding the GPI anchor. We therefore created a mutant mouse PrP model containing rabbit PrP-specific amino acids at the ω site to investigate whether these residues are involved in rabbit resistance to prion infection. Here we demonstrate that the GPI anchor attachment site is an important site that controls the ability of PrP to be converted into PrP^Sc and that residues ω and ω + 1 of PrP are important modulators of this pathogenic process.     

Genetic Resistance to Bacillus thuringiensis Alters Feeding Behaviour in the Cabbage Looper,Trichoplusia ni

Evolved resistance to xenobiotics and parasites is often associated with fitness costs when the selection pressure is absent. Resistance to the widely used microbial insecticide Bacillus thuringiensis (Bt) has evolved in several insect species through the modification of insect midgut binding sites for Bt toxins, and reports of costs associated with Bt resistance are common. Studies on the costs of Bt-resistance restrict the insect to a single artificial diet or host-plant. However, it is well documented that insects can self-select appropriate proportions of multiple nutritionally unbalanced foods to optimize life-history traits. Therefore, we examined whether Bt-resistant and susceptible cabbage loopers Trichoplusia ni differed in their nutrient intake and fitness costs when they were allowed to compose their own protein:carbohydrate diet. We found that Bt-resistant T. ni composed a higher ratio of protein to carbohydrate than susceptible T. ni. Bt-resistant males exhibited no fitness cost, while the fitness cost (reduced pupal weight) was present in resistant females. The absence of the fitness cost in resistant males was associated with increased carbohydrate consumption compared to females. We demonstrate a sex difference in a fitness cost and a new behavioural outcome associated with Bt resistance.     

玉米粗缩病改良新抗源T877的抗性评价

玉米粗缩病是我国玉米产区的一种重要病害。本研究利用自然接虫法初步鉴定了41份玉米自交系对粗缩病的抗性,并对其中有代表性的10份材料进行了3个播期的试验。筛选出3份高抗、4份抗、3份中抗材料,大部分材料(占75.6%)表现为感和高感,抗性较好的材料属于PB亚群。高抗粗缩病自交系T877在不同年份、不同播期间抗性稳定,以此为亲本育成苏玉19等新品种。     

pSEUDO, a Genetic Integration Standard for Lactococcus lactis

Plasmid pSEUDO and derivatives were used to show that llmg_pseudo_10 in Lactococcus lactis MG1363 and its homologous locus in L. lactis IL1403 are suitable for chromosomal integrations. L. lactis MG1363 and IL1403 nisin-induced controlled expression (NICE) system derivatives (JP9000 and IL9000) and two general stress reporter strains (NZ9000::PhrcA-GFP and NZ9000::PgroES-GFP) enabling in vivo noninvasive monitoring of cellular fitness were constructed.     

作物抗病基因工程研究进展

控制植物病害的关键,取决于对植物与病原菌相互作用的分子机理的了解。将抗病基因、信号传导／调控基因、抗菌蛋白基因等导入拟改良的作物、选育抗病新品系,是当前作物抗病基因工程研究的主要策略。本文介绍利用植物抗病反应中系列重要基因进行作物抗病基因工程的研究进展,讨论了目前作物抗病基因工程中存在的问题及其解决的方法。     

Genetic Analysis and QTL Detection for Resistance to White Tip Disease in Rice

The inheritance of resistance to white tip disease (WTDR) in rice (Oryza sativa L.) was analyzed with an artificial inoculation test in a segregating population derived from the cross between Tetep, a highly resistant variety that was identified in a previous study, and a susceptible cultivar. Three resistance-associated traits, including the number of Aphelenchoides besseyi (A. besseyi) individuals in 100 grains (NA), the loss rate of panicle weight (LRPW) and the loss rate of the total grains per panicle (LRGPP) were analyzed for the detection of the quantitative trait locus (QTL) in the population after construction of a genetic map. Six QTLs distributed on chromosomes 3, 5 and 9 were mapped. qNA3 and qNA9, conferring reproduction number of A. besseyi in the panicle, accounted for 16.91% and 12.54% of the total phenotypic variance, respectively. qDRPW5a and qDRPW5b, associated with yield loss, were located at two adjacent marker intervals on chromosome 5 and explained 14.15% and 14.59% of the total phenotypic variation and possessed LOD values of 3.40 and 3.39, respectively. qDRPW9 was considered as a minor QTL and only explained 1.02% of the phenotypic variation. qLRGPP5 contributed to the loss in the number of grains and explained 10.91% of the phenotypic variation. This study provides useful information for the breeding of resistant cultivars against white tip disease in rice.