Comparison of traditional and environmental DNA survey methods for detecting rare and abundant freshwater fish

1. Detecting rare species is often a necessity for conservation and management, yet challenging for many field survey methods. Environmental DNA (eDNA) is a highly promising solution that has been shown to outperform many established survey methods.
2. Macquarie perch (Macquaria australasica) is an endangered native species that has declined significantly in range and abundance. Detection of M. australasica was compared with an abundant alien fish species (Oncorhynchus mykiss) using eDNA and three conventional survey methods: gill nets, electrofishing and fyke nets.
3. eDNA occupancy estimates for both fish species were compared using four different models to investigate what effect these differences have on false positives and false negatives for the rare and common fish species. These models used unadjusted eDNA detections in water samples, eDNA detections that have been screened using a limit of detection method to remove potential false positives, eDNA data supplemented with a second survey method, or eDNA data augmented with sequencing of positive polymerase chain reaction replicates.
4. eDNA surveying as a single detection method was found to be more efficient and sensitive compared with each capture method separately and combined. Occupancy estimates for the common and rare species did not vary significantly between the four site occupancy-detection models, suggesting that supplementary data may not have as much effect on occupancy estimates compared with other approaches such as temporal or spatial sampling.
5. We conclude that eDNA outperforms the three established survey methods for both a rare and common freshwater fish species. Although there was no significant effect of augmenting eDNA survey methods with other survey data, additional data may improve confidence in detection, and provide confirmatory evidence for unexpected or new detections of a species.

     

检测cDNA芯片中差异表达基因的非转换方法

cDNA芯片已经成为生物学试验中一种重要试验手段,与其相应的检测差异表达基因的统计方法也在快速地完善。目前通用的方法是将数据进行对数比转换,并相应进行标准化处理。鉴于对数转换的缺点,本研究提出一种非转换方法,即背景校正后直接进行数据标准化。研究表明:利用这种非转换方法,可以更有效地剔除试验中的“噪音,”提高检测的准确率。本研究利用Apo AI试验的芯片数据进行了效率验证,结果表明:与对数转换方法相比,非转换方法能检测出更多的差异表达基因,可以作为cDNA芯片数据分析的一种简便高效方法。     

Simultaneous analysis of the intestinal parasites and diet through eDNA metabarcoding

Agricultural expansion and intensification are having a huge impact on plant and arthropod diversity and abundance, affecting food availability for farmland birds. Difficult food access, in turn, can lead to immunosuppression and a higher incidence of parasites. In the studies designed to examine changes in the diet of birds and their parasites, metabarcoding is proving particularly useful. This technique requires mini-barcodes capable of amplifying the DNA of target organisms from fecal environmental DNA. To help to understand the impact of agricultural expansion on biodiversity, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the feces of farmland birds. The capacity to identify diet and parasites of 2 existing and 3 newly developed mini-barcodes was tested “in silico” in relation to the behavior of a reference eukaryotic barcode. Among the newly designed mini-barcodes, MiniB18S_81 showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa. Moreover, when it was tested on fecal samples from 5 different steppe bird species, MiniB18S_81 showed high taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes, and Apicomplexa at the order level. Thus, the mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasites of birds, essential for conservation and management.     

烤烟品种K326茎叶消减文库的构建及质量分析

利用抑制性消减杂交(SSH)技术,构建烤烟品种K326的茎叶抑制性消减cDNA文库.结果表明,文库的滴度为1.76×106cfu.mL-1,重组率为75%,随机扩增480个克隆,显示插入片段长度在200-1000 bp之间,符合建库的质量标准.     

Abstracts of Nippon Dojohiryogaku Zasshi

Abstract

The humus composition was analyzed and the humic acid characterized by UV and visible absorption spectroscopy in order to investigate the rotting and maturing process of city refuse compost according to the method of Kumada et al. During the composting process, the following findings were obtained: (1) the HT value was almost constant, but the HE/HT ratio varied somewhat, (2) HA increased with decrease in FA, and the PQ value so increased clearly, (3) the shoulder-like absorption at a wavelength near 270 nm weakened, and (4) the RF value of humic acid increased, whereas the Δ log K value seldom varied.

The IR spectrum of humic acid gradually changed as follows: (1) the absorption band in the 1700-1600 cm^-1 region and in the 1550-1500 cm^-1 region increased slightly, (2) the band in the 1100-1000 cm^-1 region decreased, and (3) the bands at 835 and 710 cm^-1 com pletely disappeared. On the whole, the shape of the IR spectrum of the city refuse compost became featureless. These changes were probably due to the oxidation which occurred in the composting process.     

Environmental DNA as a monitoring tool for the endangered freshwater pearl mussel (Margaritifera margaritifera L.): a substitute for classical monitoring approaches?

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Non‐indigenous fish in protected spaces: Trends in species distribution mediated by illegal stocking

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甘蔗果糖-1，6-二磷酸酯酶的cDNA片段克隆及序列分析

植物生长发育所需要的光合产物大部分以蔗糖的形式供应和运输，其中果糖-1，6-二磷酸酯酶（fructose-1,6-bisphosphatase，FBPase）水解果糖-1，6-二磷酸（FBP）形成F6P和无机磷。根据GenBank登录的甘蔗细胞质型FBPase基因（GenBank登录号x89006）的序列设计引物．从甘蔗叶片cDNA文库和茎中扩增出FBPase基因的cDNA片段sfbp1和sfbp2，并构建到pMD18-T载体进行序列测定和分析。结果表明，sfbp1和sfbp2长均是1180bp，包含完整的开放读码框．编码332个氨基酸。sfbp1核苷酸序列及其推演的氨基酸序列与甘蔗FBPase基因进行序列比对，相似性均是99％。采用DNAMAN比对分析sfbp1和sfbp2的cDNA序列及其推演的氨基酸序列，结果表明，相似性分别是99．93％和99．7％。采用ExPASy软件预测推演的sfbp1的分子量大小为36．074kD，理论等电点为5．83。     

Improved detection sensitivity using an optimal eDNA preservation and extraction workflow and its application to threatened sawfishes

1. Pressures on coastal ecosystems are increasing and aquatic species that are restricted to these habitats are facing the threat of extinction. However, the true extent of many threatened and rare aquatic species, especially elasmobranchs, remains unclear due to high levels of data deficiency and poor efficacy of traditional survey methods. Sawfishes (Pristidae), a family of shark-like rays, are among the most threatened and rare elasmobranch species and are difficult to detect in turbid, coastal habitats. Reliable cost-effective tools to detect these species are urgently needed to increase their conservation potential.
2. Characterization of environmental DNA (eDNA) extracted from water samples has garnered significant appeal for detection of rare and threatened species. To assist conservation and monitoring efforts for sawfishes using eDNA, species-specific TaqMan quantitative polymerase chain reaction assays were developed and validated to detect 1.25–5 copies of a 12S rRNA gene fragment. Filter samples were collected in Northern Territory, Australia to assess the utility of the developed eDNA assays and compare the efficacy of preservation and extraction workflows for detecting rare species.
3. Dwarf sawfish (Pristis clavata) were detected in three of 20 sites, and there was a significant effect of preservation and extraction workflow on total eDNA yield and subsequent detection success. Longmire's preserved samples extracted using glycogen-aided precipitation yielded a significantly higher concentration of total eDNA (n = 60; β = 1.27, t(95) = 8.172, P < 0.0001) and yielded positive P. clavata eDNA detections compared to ethanol preserved samples extracted using QIAGEN DNeasy kit, which did not yield any positive detections.
4. The optimized eDNA assays were developed to support monitoring efforts for endangered sawfishes. Importantly, this study demonstrates that choice of preservation and extraction workflow requires careful consideration, especially when detection of rare or threatened species can have important management and conservation outcomes.