极端嗜热厌氧纤维素菌的分离鉴定、系统发育分析及其酶学性质的研究

从云南高温温泉、油井等热源地区采集的大量样品中,获得了一株特殊的极端嗜热厌氧纤维素分解菌B2.分离菌株直杆,革兰氏阴性(G-),未观察到孢子,细胞单个或成对出现.菌体大小为0.4μm×(2-4)μm,严格厌氧,生长温度范围50-70℃,最适生长温度65℃.pH范围4-8,最适pH 7.0.在纤维素粉琼脂上菌落直径2-4 mm,乳白色.分离菌株能利用纤维二糖、葡萄糖、蔗糖、松子糖、淀粉、覃糖等作为碳源,分离菌株还可利用纤维素滤纸、纤维素粉、微晶纤维素、纤维素粉MN300和甘蔗渣、水稻秸杆.发酵纤维素产生乙醇、乙酸.在菌株B2的纤维素酶系中,C1酶、Cx酶和β-葡萄糖苷酶的最适温度分别为80℃、80℃和70℃,其比值为1:9:10,同时发现Cx酶具有较高的热稳定性.部分长度的16S rDNA序列分析表明,分离菌株B2与Thermoanaerobacter ethanalicus具有99.8％相似性.分离菌株B2为Thermoanaerobacter属.图5表3参21     

一株产耐热纤维素酶菌株的筛选及酶学性质

采用纤维素刚果红平板法从大田蘑菇种植场建堆的稻草样中分离得到了1株能产具有较好温度耐受性和pH稳定性的纤维素酶的放线菌DY3.综合形态、生理生化特征以及16S rDNA序列分析,将其初步鉴定为嗜热裂孢菌(Thermobifida fusca).对该菌所产纤维素酶的性质研究表明:最适催化温度为65℃,在70℃保温60 min后仍有75%以上的活力;最适催化pH为7.5,在pH 5.5～10.0之间该酶的稳定性较好,pH 10.0的条件下仍有80%的活力;最适作用底物是羧甲基纤维素钠.研究可为木质纤维素的生物预处理提供一定参考价值.     

嗜热子囊菌光孢变种内切葡聚糖酶Ⅰ基因在毕赤酵母中的表达及部分酶学性质

嗜热子囊菌是一种嗜热真菌,可以产生具有很高工业价值的内切葡聚糖酶.本研究成功表达了嗜热子囊菌内切葡聚糖酶Ⅰ基凶,并获得热稳定的重组内切葡聚糖酶.提取嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)总RNA,通过RT-PCR方法克隆出内切-β-葡聚糖酶eg1基因的成熟肽编码序列.采用基因重组的方法构建该基因的巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K-eg1,经线性化后采用电穿孔法将其导入毕赤酵母GS115中,大量筛选后获得高效表达内切葡聚糖酶Ⅰ的毕赤酵母工程菌株GpN24.该菌株采用甲醇诱导120 h后,内切葡聚糖酶Ⅰ的活力可达570.7 U/mL,最适温度为55℃,在90℃的条件下保温30 min后仍具有60%的酶活力;最适pH为5.0,在pH 3.0～5.0的条件下酶活力保持稳定.图6表2参16     

褐菖鲇肝HSC70基因的环介导等温扩增技术的建立及在石油污染剂量效应研究中的应用

从褐菖鲉肝脏中克隆了热休克蛋白HSC70基因,利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立HSC70基因的定量检测方法.为检测该方法的可行性,将褐菖鲉分别暴露于石油水溶性成分(water-soluble fraction,WSF) 20、60、180 μg·L-11d后,利用real-time PCR及LAMP技术同时测定褐菖鲉肝HSC70 mRNA表达量,两种方法测定结果基本一致,证实LAMP技术可用于褐菖鲉肝HSC70基因的定量.为更细致了解石油WSF影响褐菖鲉肝脏HSC70基因表达的剂量-效应关系,将褐菖鲉分别暴露于25、50、75、100、125、150、175 μg·L-1 WSF中,5d后采样,用LAMP技术定量检测HSC70 mRNA.结果表明,HSC70 mRNA表达量在50 μg· L-1浓度组即被显著诱导,在75μg·L-1浓度下达到最大值,这说明褐菖鲉肝HSC70基因对石油污染较敏感,有潜力作为海洋石油污染的生物标志物.     

褐菖鲉肝HSC70基因的环介导等温扩增技术的建立及在石油污染剂量效应研究中的应用

从褐菖鲉肝脏中克隆了热休克蛋白HSC70基因,利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立HSC70基因的定量检测方法。为检测该方法的可行性,将褐菖鲉分别暴露于石油水溶性成分(water-soluble fraction,WSF)20、60、180μg·L-11 d后,利用real-time PCR及LAMP技术同时测定褐菖鲉肝HSC70 mRNA表达量,两种方法测定结果基本一致,证实LAMP技术可用于褐菖鲉肝HSC70基因的定量。为更细致了解石油WSF影响褐菖鲉肝脏HSC70基因表达的剂量-效应关系,将褐菖鲉分别暴露于25、50、75、100、125、150、175μg·L-1WSF中,5 d后采样,用LAMP技术定量检测HSC70 mRNA。结果表明,HSC70 mRNA表达量在50μg·L-1浓度组即被显著诱导,在75μg·L-1浓度下达到最大值,这说明褐菖鲉肝HSC70基因对石油污染较敏感,有潜力作为海洋石油污染的生物标志物。     

不同强度冷应激对大鼠肌肉、脾脏和肝脏中HSP70表达的影响

通过两个低温温度段对健康Wistar大鼠进行冷应激试验,利用免疫印迹技术检验肌肉、脾脏和肝脏中HSP70表达的相对变化情况,探讨不同时间和强度冷应激条件下,3种组织中HSP70表达的情况以及通过HSP70表达量的变化看是否可以将其作为评估冷应激的指标.正常饲养的大鼠环境温度为(20±1)℃,而实验组大鼠分别在(0±1)℃和(10±1)℃环境下进行低温冷应激,然后在冷应激后不同时间宰杀取组织,利用Western-blotting检测HSP70的表达量.结果表明:(1)在(0±1)℃急性冷应激条件下,随着冷应激时间的延长,肌肉和脾脏中HSP70的表达持续增加,与对照组(CK)相比,差异显著(P<0.05);(2)在(10±1)℃慢性冷应激条件下,仅1wk肌肉组中HSP70的表达增加(P<0.05),其它情况下各组织中HSP70的表达与CK相比无显著差异(P>0.05).上述结果说明,相应强度的冷应激可以诱导机体的HSP70表达,并且随时间延长,HSP70的表达增加,可以考虑将HSP70作为监测大鼠应激的指标.图1表2参14     

不同适应温度对镉暴露中大型溞生理和亚细胞水平的作用

虽然水生生物大多是变温动物并且温度对化合物的毒性有非常重要的作用,但生态毒理学实验一般在实验室标准温度(20℃)或者生物最适温度下进行。我们调查了适应温度(20℃、24℃和28℃)对镉暴露(0,20和40μg·L~(-1))中大型溞生理和亚细胞水平的作用,实验终点包括镉的急性毒性(48 h-EC50)、大型溞的21 d存活率、21 d累积繁殖数(21-d CR)、第一次生育时间(time to first brood,TFB)、能量储存、电子转移活性(electron transport system activity,ETS)、热应激蛋白hsp70和金属硫蛋白(MT)。结果显示:适应温度升高时大型溞的镉耐性降低,20℃、24℃和28℃下的48 h-EC50分别为(40.2±9.9)μg·L~(-1)、(15.6±2.7)μg·L~(-1)和(10.7±1.9)μg·L~(-1);28℃下,大型溞21 d存活率分别比对照(20℃,0μg·L~(-1)镉暴露)降低23%(20μg·L~(-1)镉暴露)和87%(40μg·L~(-1)镉暴露)并且大型溞的21 d累积繁殖数(21-d CR)显著降低。大型溞的能量储存随适应温度的升高而降低,24℃和28℃时分别降低了46%和62%(0μg·L~(-1)镉暴露),48%和60%(20μg·L~(-1)镉暴露)、80%和91%(40μg·L~(-1)镉暴露)。而镉浓度只有达到40μg·L~(-1)时,对大型溞的能量储存有明显影响。适应温度和镉暴露对电子转移系统活性(ETS)没有显著影响。28℃的适应温度和40μg·L~(-1)的镉暴露都能诱导大型溞热应激蛋白hsp70和金属硫蛋白(MT)显著上升,但适应温度(20℃~28℃)和镉暴露(0~40μg·L~(-1))的结合对hsp70先诱导后抑制,MT则一直被诱导上升。总之,生物在不同温度下对毒物的反应存在差异,将不同适应温度下的毒性数据整合到生态危险性评价能够保证生物获得更充分的保护;此外,hsp70和MT的变化是环境压力的综合反应,作为某种污染物的生物标志物时需要综合考虑环境因素。     

不同强度冷应激下仔猪淋巴细胞HSP70 mRNA的转录规律

采用实时荧光定量PCR技术检测仔猪血液淋巴细胞HSP70 mRNA的转录量,探讨不同冷应激强度对淋巴细胞HSP70 mRNA转录量的影响.结果表明,仔猪在-10~-4℃冷暴露0.5h,淋巴细胞HSP70 mRNA的转录量迅速升高(P<0.05),并一直保持高水平至6h(P<0.05),随后下降,冷暴露结束后24h恢复正常.在4~10℃冷暴露前后,淋巴细胞HSP70 mRNA转录量无明显变化(P>0.05).结果提示,冷暴露温度越低,淋巴细胞中HSP70 mRNA转录越迅速,但冷暴露结束进入常温后HSP70 mRNA转录水平再次升高的时间延后;HSP70 mRNA转录呈现规律性变化,可以考虑把HSP70 mRNA优先作为评估不同冷应激的客观指标.表1图1参14     

重组植酸酶appAM8的异源表达与性质比较

为比较在不同宿主中表达的大肠杆菌植酸酶突变体app AM8的性质,将app AM8基因分别在大肠杆菌BL21(DE3)和毕赤酵母GS115中进行表达,对纯化后的植酸酶进行酶学性质分析与比较.结果显示:(1)在大肠杆菌中表达的app AM8(E-app AM8)和酵母中表达的app AM8(P-app AM8)的最适p H值均为4.5,最适反应温度均为65℃,与野生型的app A的最适p H无明显区别,比app A的最适温度高了5℃;(2)经SDS-PAGE检测,E-app AM8分子量(Mr)大小为47×103,而P-app AM8为53×103左右,由于在酵母中的糖基化修饰电泳显示为两条带;(3)在60-80℃之间,E-app AM8的热稳定性性明显下降,而且在70℃处理15 min后,E-app AM8残余活性仅为4%,而P-app AM8仍保留50%的残余活性,表明酵母表达的植酸酶经过糖基化修饰后提高了酶的热稳定性.(4)E-app AM8的Km=0.245 mmol/L,Vmax=3196 U/mg,P-app AM8的Km=0.36 mmol/L,Vmax=3333 U/mg.本研究表明,糖基化修饰对植酸酶app AM8的热稳定性起着重要作用,8个位点的突变对该植酸酶的稳定性也起到了一定作用.     

褐菖鲉肝HSC70基因的环介导等温扩增技术的建立及对石油污染的剂量效应研究

从褐菖鲉肝脏中克隆了热休克蛋白HSC70基因,利用环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)建立HSC70基因的定量检测方法。为检测该方法的可行性,将褐菖鲉分别暴露于石油水溶性成分(water-soluble fraction,WSF) 20、60、180 μg·L^-1 1 d后,利用real-time PCR及LAMP技术同时测定褐菖鲉肝HSC70 mRNA表达量,两种方法测定结果基本一致,证实LAMP技术可用于褐菖鲉肝HSC70基因的定量。为更细致了解石油WSF影响褐菖鲉肝脏HSC70基因表达的剂量－效应关系,将褐菖鲉分别暴露于25、50、75、100、125、150、175 μg·L^-1 WSF中,5天后采样,用LAMP技术定量检测HSC70 mRNA。结果表明,HSC70 mRNA表达量在50 μg·L^-1浓度组即被显著诱导,在75 μg·L^-1浓度下达到最大值,这说明褐菖鲉肝HSC70基因对石油污染较敏感,有潜力作为海洋石油污染的生物标志物。     

Rapid,recurring, structured survey versus bioblitz for generating biodiversity data and analysis with a multispecies abundance model

A bioblitz inexpensively and quickly generates biodiversity data, but bioblitzes are often conducted with haphazard, unreplicated sampling. Results tend to be taxonomically, geographically, or temporally biased, lack metadata, and consist of lists of observed taxa that do not enable further analyses or correction for imperfect detection. A rapid, recurring, structured survey (RRSS) uses a structured sampling design and temporal and spatial replication to survey randomly selected sites on a conservation property. We participated in a loosely structured bioblitz and a subsequent RRSS at Big Canoe Creek Nature Preserve in Springville (St. Clair County), Alabama (USA) to compare observed richness derived from the 2 survey approaches. The RRSS data structure enabled us to fit models that accounted for imperfect detection to estimate abundances, occupancy probabilities, and habitat associations. The loosely structured bioblitz data could not be used in such models. We present a new integrated multispecies abundance model that we applied to avian RRSS data. Our model extension enables estimation for the community, employs data augmentation to estimate the number of undetected species, and incorporates covariates. The RRSS generated a more comprehensive and less biased list of observed taxonomic richness than the loosely structured bioblitz (e.g., 73 vs. 45 bird species and 104 vs. 63 insect families from the RRSS vs. loosely structured bioblitz, respectively). Models fit to the RRSS data identified seasonal patterns in avian community composition and allowed for estimation of habitat–occupancy relationships for insect taxa. The RRSS protocol has potential for broad transferability as a standardized, quick, and inexpensive way to inventory biodiversity and estimate ecological parameters while providing an outreach opportunity.     

Compliance with and ecosystem function of biodiversity offsets in North American and European freshwaters

Land-use change via human development is a major driver of biodiversity loss. To reduce these impacts, billions of dollars are spent on biodiversity offsets. However, studies evaluating offset project effectiveness that examine components such as the overall compliance and function of projects remain rare. We reviewed 577 offsetting projects in freshwater ecosystems that included the metrics project size, type of aquatic system (e.g., wetland and creek), offsetting measure (e.g., enhancement, restoration, and creation), and an assessment of the projects’ compliance and functional success. Project information was obtained from scientific and government databases and gray literature. Despite considerable investment in offsetting projects, crucial problems persisted. Although compliance and function were related to each other, a high level of compliance did not guarantee a high degree of function. However, large projects relative to area had better function than small projects. Function improved when projects targeted productivity or specific ecosystem features and when multiple complementary management targets were in place. Restorative measures were more likely to achieve targets than creating entirely new ecosystems. Altogether the relationships we found highlight specific ecological processes that may help improve offsetting outcomes.     

A palaeo-hydrogeological model for arsenic contamination in southern and south-east Asia

An argument is presented in which areas of natural arsenic contamination of modern groundwaters throughout Asia have a common origin. Arsenic originally accumulated in oceanic ferro-manganoan sediments of the eastern Palaeo-Tethys. This was further concentrated through oceanic crustal extinction in what later became the south-east Chinese accreted mineralised terrain. Proto-Himalayan uplift of this area created the palaeo-drainage systems of the Ganges – Brahmaputra, Irrawaddy, Mekong, and Red Rivers, with consequent headwater erosion of arsenic-rich sediments. Their downstream deposition as immature and easily redistributed Neogene sandstones, silts, and iron-rich clays has created secondary and tertiary reservoirs of adsorbed and authigenic arsenic, from which the current arsenic-rich groundwaters have evolved. Considering river basins within the above palaeo-hydrogeological framework provides a basis for assessing the risk of arsenic in groundwater basins of south and south-eastern Asia.     

Cost‐effective conservation of an endangered frog under uncertainty

How should managers choose among conservation options when resources are scarce and there is uncertainty regarding the effectiveness of actions? Well‐developed tools exist for prioritizing areas for one‐time and binary actions (e.g., protect vs. not protect), but methods for prioritizing incremental or ongoing actions (such as habitat creation and maintenance) remain uncommon. We devised an approach that combines metapopulation viability and cost‐effectiveness analyses to select among alternative conservation actions while accounting for uncertainty. In our study, cost‐effectiveness is the ratio between the benefit of an action and its economic cost, where benefit is the change in metapopulation viability. We applied the approach to the case of the endangered growling grass frog (Litoria raniformis), which is threatened by urban development. We extended a Bayesian model to predict metapopulation viability under 9 urbanization and management scenarios and incorporated the full probability distribution of possible outcomes for each scenario into the cost‐effectiveness analysis. This allowed us to discern between cost‐effective alternatives that were robust to uncertainty and those with a relatively high risk of failure. We found a relatively high risk of extinction following urbanization if the only action was reservation of core habitat; habitat creation actions performed better than enhancement actions; and cost‐effectiveness ranking changed depending on the consideration of uncertainty. Our results suggest that creation and maintenance of wetlands dedicated to L. raniformis is the only cost‐effective action likely to result in a sufficiently low risk of extinction. To our knowledge we are the first study to use Bayesian metapopulation viability analysis to explicitly incorporate parametric and demographic uncertainty into a cost‐effective evaluation of conservation actions. The approach offers guidance to decision makers aiming to achieve cost‐effective conservation under uncertainty.     

Threats to intact tropical peatlands and opportunities for their conservation

Large, intact areas of tropical peatland are highly threatened at a global scale by the expansion of commercial agriculture and other forms of economic development. Conserving peatlands on a landscape scale, with their hydrology intact, is of international conservation importance to preserve their distinctive biodiversity and ecosystem services and maintain their resilience to future environmental change. We explored threats to and opportunities for conserving remaining intact tropical peatlands; thus, we excluded peatlands of Indonesia and Malaysia, where extensive deforestation, drainage, and conversion to plantations means conservation in this region can protect only small fragments of the original ecosystem. We focused on a case study, the Pastaza‐Marañón Foreland Basin (PMFB) in Peru, which is among the largest known intact tropical peatland landscapes in the world and is representative of peatland vulnerability. Maintenance of the hydrological conditions critical for carbon storage and ecosystem function of peatlands is, in the PMFB, primarily threatened by expansion of commercial agriculture linked to new transport infrastructure that is facilitating access to remote areas. There remain opportunities in the PMFB and elsewhere to develop alternative, more sustainable land‐use practices. Although some of the peatlands in the PMFB fall within existing legally protected areas, this protection does not include the most carbon‐dense (domed pole forest) areas. New carbon‐based conservation instruments (e.g., REDD+, Green Climate Fund), developing markets for sustainable peatland products, transferring land title to local communities, and expanding protected areas offer pathways to increased protection for intact tropical peatlands in Amazonia and elsewhere, such as those in New Guinea and Central Africa which remain, for the moment, broadly beyond the frontier of commercial development.     

Parasitic wasps orient to green leaf volatiles

Summary Undamaged plants emit low levels of green leaf volatiles (GLVs), while caterpillar-damaged and artificially damaged plants emit relatively higher levels of certain GLVs. Female braconid parasitoids,Microplitis croceipes, oriented to both damaged plants and to individual GLVs in no-choice tests in a wind tunnel, but seldom oriented to undamaged plants. Female ichneumonid parasitoids,Netelia heroica, also oriented to individual GLVs in a wind tunnel. Males of both wasp species failed to orient to the GLVs. These data show that leaf-feeding caterpillars can cause the release of GLVs, and that parasitic wasps can respond to these odors by flying upwind (chemoanemotactic response), which brings the wasps to their caterpillar hosts. This supports the hypothesis that plants communicate with members of the third trophic level,i.e., plants under herbivore attack emit chemical signals that guide natural enemies of herbivores to sites of plant damage. In this interaction, the GLVs serve as tritrophic plant-to-parasitoid synomones. That parasitoids from two different wasp families oriented to GLVs suggests that the response may be widespread among the Hymenoptera.Mention of a commercial or proprietary product does not constitute an endorsement by the U.S. Department of Agriculture     

Human Influence and Classical Biogeographic Predictors of Rare Species Occurrence

Biogeographic theory predicts that rare species occur more often in larger, less‐isolated habitat patches and suggests that patch size and connectivity are positive predictors of patch quality for conservation. However, in areas substantially modified by humans, rare species may be relegated to the most isolated patches. We used data from plant surveys of 81 meadow patches in the Georgia Basin of Canada and the United States to show that presence of threatened and endangered plants was positively predicted for patches that were isolated on small islands surrounded by ocean and for patches that were isolated by surrounding forest. Neither patch size nor connectivity were positive predictors of rare species occurrence. Thus, in our study area, human influence, presumably due to disturbance or introduction of competitive non‐native species, appears to have overwhelmed classical predictors of rare species distribution, such that greater patch isolation appeared to favor presence of rare species. We suggest conservation planners consider the potential advantages of protecting geographically isolated patches in human‐modified landscapes because such patches may represent the only habitats in which rare species are likely to persist. Influencia Humana y Predictores Biogeográficos Clásicos de la Ocurrencia de Especies Raras     

Seasonal dynamics of macro-algae in the South Lake of Tunis

The macro-algae communities observed in the south lake of Tunis are characterized by the predominance of nitrophilous algae which are in the order of biomass importance:Ulva, Cladophora andEnteromorpha. We have noted seasonal changes of alga distribution. The wind appears to be one of the most important factors influencing this distribution. The total biomass reaches a maximum in the spring. Rapid decomposition of the biomass leads to a severe ecological imbalance, resulting in crises of anoxia and fish death. A restoration project has already started. It aims at removal of contaminated muds and the introduction of a new circulation system. The main objectives of this work were to collect information on the distribution and biomass of the phytobenthic communities as a first step in the constitution of a database for further comparison.     

Patterns and biases of climate change threats in the IUCN Red List

International Union for Conservation of Nature (IUCN) Red List assessments rely on published data and expert inputs, and biases can be introduced where underlying definitions and concepts are ambiguous. Consideration of climate change threat is no exception, and recently numerous approaches to assessing the threat of climate change to species have been developed. We explored IUCN Red List assessments of amphibians and birds to determine whether species listed as threatened by climate change display distinct patterns in terms of habitat occupied and additional nonclimatic threats faced. We compared IUCN Red List data with a published data set of species’ biological and ecological traits believed to infer high vulnerability to climate change and determined whether distributions of climate change‐threatened species on the IUCN Red List concur with those of climate change‐threatened species identified with the trait‐based approach and whether species possessing these traits are more likely to have climate change listed as a threat on the IUCN Red List. Species in some ecosystems (e.g., grassland, shrubland) and subject to particular threats (e.g., invasive species) were more likely to have climate change as a listed threat. Geographical patterns of climate change‐threatened amphibians and birds on the IUCN Red List were incongruent with patterns of global species richness and patterns identified using trait‐based approaches. Certain traits were linked to increases or decreases in the likelihood of a species being threatened by climate change. Broad temperature tolerance of a species was consistently related to an increased likelihood of climate change threat, indicating counterintuitive relationships in IUCN assessments. To improve the robustness of species assessments of the vulnerability or extinction risk associated with climate change, we suggest IUCN adopt a more cohesive approach whereby specific traits highlighted by our results are considered in Red List assessments. To achieve this and to strengthen the climate change‐vulnerability assessments approach, it is necessary to identify and implement logical avenues for further research into traits that make species vulnerable to climate change (including population‐level threats).