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。     

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

1. Many freshwater non‐indigenous species (NISs) are stocked for recreational fishing, in some cases illegally in protected areas. In this study, fish communities were monitored using environmental DNA, electrofishing and anglers’ catches as the sources of samples in a mountainous Biosphere Reserve in Asturias (northern Spain), where stocking is forbidden.
2. Three NISs have been introduced illegally in the protected area and have shown increasing populations in the last two decades. Two species used as fishing bait, Squalius carolitertii (chub) and Phoxinus phoxinus (minnow), are expanding in running waters. Oncorhynchus mykiss (rainbow trout) was also detected and is likely to have been introduced for angling or from fish farm escapes.
3. The results suggest that sustained illegal stocking contributed to the increase of the three NISs. In contrast, Salmo trutta (brown trout) of northern European lineages, identified from *90 alleles at the LDH‐C1 locus, and formerly legally stocked for angling, is decreasing, most likely as a result of climate change. Climate change could also contribute to the expansion of the two non‐indigenous cyprinids to colder upstream areas.
4. Through the application of a social survey, it was found that unlike other population groups, anglers in the region significantly preferred stocking over environmental improvement for the management of fish populations. The results obtained suggest that raising the awareness of anglers about the importance of safeguarding native fish species could help to prevent the spread of NISs in protected areas.

     

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

1. Knowledge of species distribution is of utmost importance for conservation and management of endangered freshwater mussels. Conventional monitoring approaches are often time consuming and costly. The use of environmental DNA (eDNA) is a relatively new approach and considered as an effective tool to detect the presence of target species in aquatic environments. The aim of this study was to establish a nested PCR system to detect eDNA of Margaritifera margaritifera and to discuss the advantages and disadvantages of eDNA in mussel surveys compared with classical monitoring.
2. DNA of M. margaritifera was detected in 2 L water samples collected directly downstream (25 m) from pearl mussel populations (population size from 800–20 000), with an internal‐nested PCR approach greatly increasing the detection sensitivity (down to 10 fg target DNA). eDNA detection at greater distances downstream (500 and 1 000 m) of these populations failed, possibly due to DNA degradation or dilution processes. eDNA was also detected downstream of an extinct population, most likely resulting from overlooked mussels or the release of DNA from dead shells.
3. The eDNA approach proposed herein may be helpful in initial screening of streams that are otherwise difficult to monitor, or in the detection of buried juvenile mussels without disturbing their habitat. However, it cannot replace monitoring of population demography, and of other important information for conservation, and should thus only be seen as a supplementary tool.

Copyright © 2015 John Wiley & Sons, Ltd.     

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.     

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.     

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.     

Taxonomic and geographical representation of freshwater environmental DNA research in aquatic conservation

1. Freshwater macro‐organismic environmental DNA (eDNA) is gaining increasing popularity in detecting invasive species, assessing community assemblages, and in mapping the distribution of taxa that are rare or otherwise difficult to monitor. The objectives of this article are to review the targets of published freshwater eDNA research in relation to aquatic conservation with a focus on geographic regions covered, as well as the habitats and species investigated.
2. The analysis of 272 peer‐reviewed articles published between 2005 and 2018 revealed that 57% of the 238 primary research papers have a focus on conservation science, mostly addressing invasive and endangered species, followed by 23% papers investigating methodological developments and 11% biodiversity surveys also using eDNA metabarcoding. A strong geographical pattern emerged, with Africa, South America, and the tropics being under‐represented. Taxonomic coverage was dominated by 123 fish species, followed by 29 amphibian and 28 mollusc species. Freshwater arthropods (27 taxa) were under‐represented in relation to their estimated species richness.
3. Taxonomic bias towards certain species such as fishes observed in freshwater eDNA research is pervasive in biodiversity research and conservation sciences, and thus is not surprising. Geographical representation was biased, with a few industrialized countries from the Northern Hemisphere contributing 72% of the studies. Both findings parallel biases known from other research areas, such as marine eDNA analysis, taxonomy, or invasion biology.
4. The application of eDNA in freshwater conservation will benefit from the development of general standards and guidelines that are necessary to integrate freshwater macrobial eDNA techniques in existing monitoring frameworks. To aid future freshwater conservation, our suggestions are to harmonize eDNA methods for comparable and easier implementation worldwide, and to increase international cooperation and funding for under‐represented geographical regions and neglected taxa. This is especially crucial for the known biodiversity hotspots in developing countries where rapid changes occur to freshwater habitats and biodiversity.

     

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。