Accurate methods of DNA extraction and PCR-based genotyping for single scallop embryos/larvae long preserved in ethanol

Marine scallops are sessile as adult but have a long planktonic larval phase showing great possibility to migrate in marine realm lacking of obvious barriers. Genetic analysis of scallop embryos/larvae based on molecular markers is very essential to clarify the spatial and temporal gene flow and the unique population and community structure. However, the technical challenges, such as single embryos/larvae isolation and low quantity and poor quality of DNA extracted, make genotyping for a single embryo/larva long preserved in ethanol to be a really difficult task. In this study, we analysed the factors that might affect the DNA quantity and quality for simple sequence repeat‐based genotyping for single embryos/larvae. Based on the factors analysed, we developed a LoTEPA buffer‐based method, of which the accuracy, stability and reproducibility were evaluated by controlled inter‐ and intraspecies and self‐fertilized scallop families. The genotyping results showed the high success rate of more than 90% in total for embryos/larvae preserved in ethanol for 1–5 years. Furthermore, the successful genotyping for the larvae sampled from a natural habitat well demonstrated the potential use of this method in practical ecological analysis.     

Microsatellite DNA markers for the Chinese wood frog (Rana chensinensis) and tests for their cross-utility in 15 ranid frog species

We developed 22 microsatellite markers for the Chinese wood frog (Rana chensinensis) to study the impact of landscape features on its population structure. Thirty‐four individuals from one breeding site were examined and 14 loci were polymorphic. The number of alleles, expected heterozygosity and observed heterozygosity varied from two to 14, from 0.0833 to 0.9118, and from 0.1376 to 0.8667, respectively. Cross‐species amplification was tested for 15 ranid frog species. The Plateau brown frog, Rana kukunoris (n = 23), was successfully amplified at 18 loci, and 15 were polymorphic with number of alleles varying from two to 18. Ten other species were also amplified at a limited number of loci.     

Electron and light microscopy of neutrophil responses in mice vaccinated and challenged with third-stage infective hookworm (Ancylostoma caninum) larvae

The role of neutrophils in mediating host inflammation was examined in mice vaccinated with living third-stage infective hookworm larvae (L₃). Mice were vaccinated by oral immunization with 500 L₃ (Ancylostoma caninum) once every 2 weeks for a total of three immunizations. The vaccinated mice were then challenged intraperitoneally with 2000 L_3, 1 week after the final immunization. To stimulate peritoneal production of neutrophils, 2 ml of 2% glycogen were injected intraperitoneally at 16 h prior to the challenge infection. Neutrophils were found to comprise 85% of the peritoneal cell population. L₃ from the challenge infection were collected and then examined at timed intervals by inverted light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Greater than a fivefold increase in the total numbers of peritoneal cells was noted in the vaccinated mice as compared to unvaccinated mice. In the peritoneal cavity of vaccinated mice, the neutrophils adhered to the L₃ within 2 h, and over 55% of the L₃ were surround by clusters of neutrophils to form a sausage-like sheath 4 h later. At 24–72 h after challenge, almost all of the L₃ recovered from the vaccinated mice were covered with thick clusters of cells. Both SEM and TEM demonstrated extensive ultrastructural damage to the L_3. In contrast, the L₃ recovered from the unvaccinated mice appeared to be unaffected by neutrophils. These studies suggest that neutrophils, like macrophages, can have an important role as effector cells in L₃-vaccinated mice.     

大肠杆菌NAD+合成关键酶的克隆表达及发酵优化

【目的】烟酰胺腺嘌呤二核苷酸(NAD~+)在细胞基因表达、氧化还原反应、能量代谢以及调控细胞生命周期中具有重要的作用,其细胞内含量是能量效率的关键因素。强化辅因子合成策略,获得高产NAD~+菌株,对于NAD~+依赖型氧化还原反应的速率和调节相关生化合成途径的代谢流具有重要意义。【方法】首先通过内源性调节,对代谢途径中的关键酶基因进行强化,过量表达和共表达NAD~+合成途径中的关键酶基因pncB、nadD和nadE;其次,通过外源调节增加NAD~+前体物,优化诱导条件提高发酵过程中关键酶的表达量,增加NAD~+的合成量;最后在单因素优化试验的基础上,以NAD~+含量为响应值,采用Box-Bohnken试验设计方法,研究3个显著性影响因素相互作用对NAD~+积累量的影响,确定最佳的优化条件。【结果】根据关键酶基因强化策略,构建了7株重组菌,其中重组菌E.coli BL21/p ET-21a-nad E-pncB胞内NAD~+含量相比初始菌株E.coli BL21/pET-21a提高了405.2%。通过对该菌株诱导条件和NAD~+合成前体的优化,使用Design Expert 8.0分析实验数据,得出该重组菌株的最佳发酵条件为:诱导温度控制在15–20 oC,OD_(600)为0.6–0.8时添加IPTG 0.63 mmol/L、烟酸15.8 mg/L、诱导时长控制在24 h。NAD~+含量在最优条件下实验验证值可达43.16μmol/g DCW,与优化前相比提高了123.6%,与初始菌株相比提高了1029.8%。【结论】在大肠杆菌中共表达关键酶基因pncB和nadE,胞内NAD~+合成量明显增加,前体物以及诱导条件的外源调节使NAD~+积累量达到最佳优化值。实现了提高NAD~+含量的目标,胞内辅因子浓度的增加为提高生物催化效率奠定了可行性基础。     

Forced hot-air treatment against <Emphasis Type="Italic">Bactrocera papayae</Emphasis> (Diptera: Tephritidae) in papaya

The Asian papaya fruit fly, Bactrocera papayae Drew and Hancock, was treated with hot-water immersion and forced hot air to develop a phytosanitary heat treatment schedule. Hot-water immersion tests were conducted with 12- and 24-h-old eggs and with first and third instar larvae to compare the relative thermotolerances of this fruit fly among these life stages. The 24-h-old eggs, the most thermotolerant among the four life stages tested, were subjected to time and temperature tests using cage-infested papaya fruits in a forced hot-air chamber. Heating the papayas to a minimum core temperature of 47.7 °C (95% confidence interval 47.2–48.3 °C) was estimated to induce probit-nine mortality based on a probit analysis of the data. Confirmatory tests in which papayas infested with 24-h-old eggs were heated to a minimum fruit core temperature of 47.2 °C that was maintained for 0–30 min followed by hydrocooling to a fruit core temperature of ≈25 °C resulted in the complete mortality of an estimated treated population of 43,425 eggs aged 24 h (99.9931% mortality at the 95% confidence level). Therefore, heating the papaya fruits to a core temperature of 47.2 °C for a minimum dwell time of 30 min, which was the longest dwell time in the confirmatory tests, may serve as a phytosanitary heat treatment schedule for the control of B. papayae in papaya fruits.     

Epigenetic divergence of key genes associated with water temperature and salinity in a highly invasive model ascidian

Invasive species offer good models for studying mechanisms of response to rapid changing environments in the wild. DNA methylation is considered to be one of crucial drivers for rapid local adaptation. However, the extent of epigenetic variation and the factors driving such variation remain largely unexplored in biological invasions. Here we used direct bisulfite sequencing to investigate DNA methylation patterns of five key genes corresponding to two important environmental factors in marine ecosystems, water temperature and salinity, in a model invasive ascidian Ciona robusta (=C. intestinalis spA). Our results clearly showed that DNA methylation mainly occurred in gene bodies, rather than promoters, at regions with low values of CpG O/E. We detected significant variation of DNA methylation among populations in two genes (heat shock protein 90 and Na⁺-K⁺-2Cl^? cotransporter). Interestingly, significant correlation was detected between methylation levels and the two environmental factors at some CpGs in these two genes. When the data of all CpGs was subjected to principal component analysis, individuals were assigned back to their population orgins. All the results suggest that environmental factors likely contribute, at least partially, to the observed DNA methylation variation. Such variation, either by some loci alone or through gene networks, might be involved in rapid local adaptation during biological invasions.