Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.     

Promotion of initial swimbladder inflation in Pacific bluefin tuna,Thunnus orientalis (Temminck and Schlegel), larvae

Initial swimbladder inflation (ISI) of Pacific bluefin tuna (PBT), Thunnus orientalis, larvae was studied to increase the survival of cultured larvae. Experiment 1 was conducted to explore promotion and inhibition of ISI under different water surface conditions; including the use of surface skimmer to remove autogenous surface substances (SS), covering the water surface with liquid‐paraffin‐layer (LP) and oil film (OF), and a control (non‐treatment, NT). Significantly higher inflation frequency was observed in SS (62.2%) than NT (11.9%), LP (2.7%) and OF (3.9%). This indicates that ISI in PBT larvae can be promoted by removal of surface substances on rearing water which inhibit larval air gulping. Experiment 2 aimed to elucidate proper day of larval age to start skimming for promoting ISI with four different periods of oil film removal: from 3 to 8 (SF3D), 4 to 8 (SF4D), 5 to 8 (SF5D), 6 to 8 (SF6D) days‐post‐hatch (dph). Significant improvement in ISI frequency was observed in SF3D (80.2%) but the frequency was very poor in SF4D, SF5D, and SF6D (17.8–7.5%). This implies the need of oil film removal without missing a narrow window, 1 day of 3 dph, to promote ISI in practical PBT larviculture.     

Optimal periods of night‐time lighting in the sea cage culture of Pacific bluefin tuna,Thunnus orientalis,juvenile (Temminck and Schlegel)

We have previously reported that night‐time lighting prevents the mass death of Pacific bluefin tuna (PBT) juvenile due to collision and/or contact with the walls of sea cages, immediately after transfer to the cages, and that night‐time lighting does not negatively impact fish stress levels. However, the period of night‐time lighting should be limited to minimize negative impacts on the surrounding environment and aid management. Therefore, we investigated the optimal period of night‐time lighting by evaluating the whole‐body cortisol and glucose levels as stress parameters, growth performance and survival of PBT juvenile in four cages with different periods of night‐time lighting (i.e. unlit, 4‐day, 8‐day and 12‐day lighting). The results showed that almost all fish were died 1 day after transfer to the unlit cage. In comparison, the other groups (4‐day, 8‐day and 12‐day lighting) had high survival rates (92.5–96.0%) without significant difference. However, in the 4‐day‐lighting group, an obvious stress response was recorded on day 5, and growth performance was significantly lower. In the 8‐day‐lighting group, whole‐body cortisol levels were slightly elevated on day 9; however, significantly elevation was not recorded on day 12. These results indicate that the recommended lighting period of night‐time lighting in sea cages is 8–12 days.     

The bioavailability of magnesium from Wakame ( Undaria pinnatifida) and Hijiki (Hijikia fusiforme) and the effect of alginic acid on magnesium utilization of rats

The bioavailability of magnesium from Wakame and Hijiki, and the effects of alginic acid on absorption of dietary magnesium were examined in five groups of rats fed either control, Wakame, Hijiki, AW (containing the same amount of alginate as in the Wakame) and AH (containing the same amount of alginate as in the Hijiki) diets, and animals fed a low magnesium diet (LMg) (twentieth amount of magnesium in the original mineral mixtures as the control). Food intake and body weight gain were decreased by adding sodium alginate to the diets. A large amount of calcium accumulated only in the kidneys of the rats fed the LMg diet. Serum magnesium concentration decreased only in the LMg group. The magnesium content in the defatted left femurs did not differ between the control and Wakame fed animals and also among the animals eating Wakame, Hijiki and AW diets. The breaking force of the right femurs did not differ among all the groups except the LMg group. The ratio of apparent magnesium absorption (%) of the control, LMg, Wakame, Hijiki, AW and AH groups was 82.2, 72.7, 66.9, 50.8, 69.3 and 54.2 in the first experimental period, and was 75.3, 52.1, 57.7, 46.9, 62.6 and 60.5 in the second experimental period, respectively. It was clear that the bioavailability of magnesium in the Wakame fed rats was higher than in those eating the Hijiki. Large amounts of sodium alginate lowered magnesium absorption from the diet.     

Effects of application of certain herbicides on soil nematodes and aquatic invertebrates in rice paddy fields in Japan

As representatives of herbicides which are used in rice fields in Japan, paraquat (Pa), chlormethoxynil (Ch), a mixture of thiobencarb and simetryne (BS), and oxadiazon (Ox) were chosen for investigation of their effects on invertebrates in soil and flood water of rice paddies. With regard to nematodes, plant-parasitic species from rice roots, mainly Hirschmaniella oryzae, were the most abundant in B plots. All herbicide plots yielded more plant-parasitic nematodes from the soil than did the hand-weeded control plots. Predaceous mononchids, which mostly live in the surface stratum (oxidation zone) were drastically decreased in B and Ch plots. The vertical distribution of the nematode population showed similar patterns between control plots and those to which Pa was applied 2 months before flooding, and also between Ch and B plots, both of which herbicides were applied after flooding. Measurement of the similarity values of the nematode community confirmed these results. Invertebrates in the flood water were not adversely affected in Pa plots, but other herbicides brought about a ripid decrease in arthropods. However, water fleas (excluding copepods) were restored to their usual population levels within a month. The similarity values of the insect community were high between the Ch and B plots (as found for nematodes). Pond snails increased in herbicide-treated plots, especially in Ox plots which had a dense growth of green algae.     

Allele frequency distribution of the canine dopamine receptor D4 gene exon III and I in 23 breeds

Various canine breeds are remarkably different from each other not only in their sizes and shapes but also in behavioral traits, suggesting that some of them are under genetic control. Although dopaminergic neurotransmission system is considered to affect animal behavior, little is known about related genes in canine. Relations between specific alleles in polymorphic regions of the dopamine receptor D4 gene (DRD4) and personality or psychiatric disorders have been reported in humans, and we first found polymorphism in exon III region of the gene in 4 canine breeds. In this study we surveyed allele frequency distribution in 23 breeds including a total of 1,535 unrelated individuals. In exon III, 8 alleles including a novel allele were identified. A group of breeds in which the alleles 447b, 498 and 549 were frequent tended toward high scores in aggression-related behavioral traits than that with frequent alleles 435 and 447a. Moreover, a polymorphism based on 24 bp insertion/deletion was found in exon I region for the first time in dogs. This information may be of use for candidate gene studies of behavioral variation in dogs.     

Birth of normal offspring from mouse eggs activated by a phospholipase Czeta protein lacking three EF-hand domains

Sperm-specific phospholipase C, PLCzeta, is a candidate for the Ca(2+) oscillation-inducing factor that is introduced into the ooplasm upon sperm-egg fusion. In addition to the 647-residue full-length PLCzeta, s-PLCzeta lacking the N-terminal 110 amino acids is known to be present in the mouse testis. In this study, we attempted to obtain full-term offspring from s-PLCzeta-activated eggs by round spermatid injection. Metaphase II-arrested eggs injected with a high RNA concentration of s-PLCzeta RNA normally developed to blastocysts. When the round spermatid nucleus was injected into telophase II-stage eggs previously activated by s-PLCzeta RNA, three live offspring were successfully obtained by transfer of the developed 4-cell embryos to pseudopregnant mice. These three offspring all grew to be normal adults and reproduced healthy second-generation mice.     

基于SRY基因分析梅花鹿的遗传多样性及父系类型

试验旨在研究梅花鹿SRY基因的遗传多样性及其父系类型。选取东北亚种、北海道亚种、本州亚种、指名亚种、屋久岛亚种5个亚种的144个个体,利用试剂盒和传统酚/仿(1∶1)法进行DNA的提取,采用PCR扩增和直接测序法分析梅花鹿的核苷酸多样性(Pi)、单倍型多样性(Hd)、遗传距离及系统进化关系。结果显示,试验所获序列长度为1 613bp,在第47、62、602、1 148、1 569、1 604bp处共检测到6个SNPs多态位点,占核苷酸总数的0.3%,碱基替换以碱基转换为主。通过6个SNPs多态位点确立了6种单倍型:Hap-1、Hap-2、Hap-3、Hap-4、Hap-5和Hap-6,其中Hap-4、Hap-5和Hap-6为新发现单倍型。遗传多样性由高至低依次为:指名亚种、屋久岛亚种、东北亚种、本州亚种、北海道亚种。各亚种间遗传距离最小为北海道亚种与本州亚种的距离(0.000056),最大为东北亚种与指名亚种的距离(0.001733)。基于单倍型利用邻接法构建系统进化树,结果显示,与日本梅花鹿相比,东北亚种与马鹿关系更近,东北亚种存在两大分支,日本梅花鹿各亚种间无明显分支,其他单倍型均是由Hap-3进化而来,单倍型最小跨度网络图与系统进化树一致。结果表明,东北梅花鹿存在两大父系类型,日本梅花鹿存在一个父系类型,单倍型Hap-3是日本梅花鹿的原始单倍型。