舟山近海水样环境DNA获取方法的建立

以曼氏无针乌贼(Sepiella japonica)为研究对象, 通过绝对定量技术建立和优化了舟山近海高浊度水样环境DNA(Environmental DNA, eDNA)的获取方法。研究结果如下: (1)同体积水样采用乙醇沉淀法获得的eDNA产量是滤膜抽滤法的1.76—2.53 倍, 但在实际应用中由于受到采样体积、处理方式、配套设备的限制, 乙醇沉淀法难以发挥出优势; (2)滤网对泥沙等杂质无过滤作用, 添加滤网并不能过滤掉泥沙及增加抽滤体积; (3)滤膜孔径的大小对少量水样的eDNA产量有很大影响, 但对大体积水样eDNA的产量无影响; (4)水样静置处理有可能会增大eDNA产量, 但也有可能会增大eDNA结果的波动性, 使生物量评估结果误差较大; (5)阳离子表面活性剂对eDNA降解有明显的抑制作用; (6)去膜法效果优于碎膜法, 建议使用去膜法进行eDNA消化, 使用时增加离心时间; (7)酚抽除沙法虽然不能提高eDNA产量, 但能明显提高产物纯度。研究首次建立了舟山近海水样大生物eDNA最适获取方法, 为相似水域的水样采集及eDNA提取提供了借鉴参考。     

Diversity and metabolism of marine bacteria cultivated on dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Diversity and Metabolism of Marine Bacteria Cultivated on Dissolved DNA

Dissolved DNA (dDNA) is a potentially important source of energy and nutrients in aquatic ecosystems. However, little is known about the identity, metabolism, and interactions of the microorganisms capable of consuming dDNA. Bacteria from Eel Pond (Woods Hole, MA) were cultivated on low-molecular-weight (LMW) or high-molecular-weight (HMW) dDNA, which served as the primary source of carbon, nitrogen, and phosphorus. Cloning and sequencing of 16S rRNA genes revealed that distinct bacterial assemblages with comparable levels of taxon richness developed on LMW and HMW dDNA. Since the LMW and HMW dDNA used in this study were stoichiometrically identical, the results confirm that the size alone of dissolved organic matter can influence bacterial community composition. Variation in the growth and metabolism of isolates provided insight into mechanisms that may have generated differences in bacterial community composition. For example, bacteria from LMW dDNA enrichments generally grew better on LMW dDNA than on HMW dDNA. In contrast, bacteria isolated from HMW dDNA enrichments were more effective at degrading HMW dDNA than bacteria isolated from LMW dDNA enrichments. Thus, marine bacteria may experience a trade-off between their ability to compete for LMW dDNA and their ability to access HMW dDNA via extracellular nuclease production. Together, the results of this study suggest that dDNA turnover in marine ecosystems may involve a succession of microbial assemblages with specialized ecological strategies.     

Extracellular DNA in single- and multiple-species unsaturated biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.     

Alternating current voltammetric determination of DNA damage

The conditions for alternating current (a.c.) voltammetric DNA determinations have been investigated with respect to its use with alkaline filter elution techniques at low DNA concentrations. In inorganic electrolyte solutions three current peaks can be distinguished: peak I around -1.1 V caused by the reorientation or desorption of DNA segments; peak II around -1.2 V caused by the native DNA (nDNA) form; peak III caused by denatured DNA (dDNA) at -1.4 V. Sonication of nDNA increases the peak current, however not with dDNA. Both dDNA and nDNA give linear peak current increments with DNA increments, their regression lines cutting the concentration axis at the origin. In filter elution techniques organic bases are often used. Adding ethanolamine (EA) elution buffer decreases the peak amplitude of DNA. It turns out that an unknown substance, perhaps a protein or RNA, elutes from the filters and gives rise to a current peak at about -1.3 V. This substance can interfere with the dDNA by competing for electrode surface area, since it diffuses much faster than the large molecules of the DNA. Since however, dDNA has a higher affinity for the electrode surface, after enough time, usually few minutes, the dDNA increasingly displaces the substance and occupies the surface. The same is valid for other organic molecules and thus also for EA. It is therefore remarkable that the unknown substance can be altered by ultrasonication, so that it will no longer interfere with dDNA, in contrast to EA. EA, on the other hand, can be "titrated". When EA is present at short accumulation times it prevents dDNA adsorption. By adding dDNA, the EA can be scavanged and further addition will adsorb and thus increase peak current in proportion to the concentration of the DNA present. The conditions for voltammetric DNA determination have been investigated obeying the recognized interactions. Avoiding organic bases and using inorganic ones would simplify the determination procedure. The reproducibility of the procedure in the range of 50-60 ng DNA/ml has been found to be +/- 6%.     

Optimizing Phytoplasma DNA purification for genome analysis.

Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.     

Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.     

Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.     

Effects of water pH and proteinase K treatment on the yield of environmental DNA from water samples

Environmental DNA (eDNA) analysis has recently been applied to the study of aquatic macroorganisms. In most studies, sample water was filtered and the extracted DNA from the residues on the filter used for the following molecular analysis to detect species of interest. This quick, new biomonitoring method has received broad attention, but some unknowns remain, such as the eDNA yield in relation to water quality. Previous studies suggest that eDNA is composed of various forms, such as the free-floating naked form and in organelles and cells. Therefore, the eDNA yield in the filtration and extraction steps might change depending on the composition of eDNA. Especially the filtration efficiency of free-floating DNA would be affected by the electrical effect of water pH. In this study, not only the free-floating naked DNA, but also all DNA fragments released from the organisms and contained in the water were defined as eDNA, including cells and organelles. We examined (1) the effect of water pH on the eDNA yield at filtration and (2) the effect of proteinase K treatment on the extraction efficiency of DNA from filter samples, with consideration of the variety of the eDNA forms in water. In a laboratory experiment using the purified DNA of common carp (Cyprinus carpio carpio) spiked into ultrapure water, the water pH and DNA yield showed a negative relationship within the pH range of 5–9, that is, the DNA yield was higher in acidic conditions, plausibly because of pH-dependent adsorption onto the glass fiber filter at the filtration step. In case the field water contained eDNA derived from the inhabiting common carp and the purified DNA of ayu (Plecoglossus altivelis altivelis) spiked in the sample as an internal standard, adjustment of the pH to 5 prior to filtration did not increase the eDNA yield of common carp, and the spiked ayu DNA was not detected at all. During the DNA extraction step, a standard protocol including proteinase K treatment marked higher DNA yield than that without proteinase K treatment. Overall, the present results indicate successful collection of eDNA using filters without any special attention to the pH of the sample water, and a conventional protocol with proteinase K treatment is appropriate for eDNA recovery.     

Nutrient supplementation experiments with saltern microbial communities implicate utilization of DNA as a source of phosphorus

All environments including hypersaline ones harbor measurable concentrations of dissolved extracellular DNA (eDNA) that can be utilized by microbes as a nutrient. However, it remains poorly understood which eDNA components are used, and who in a community utilizes it. For this study, we incubated a saltern microbial community with combinations of carbon, nitrogen, phosphorus, and DNA, and tracked the community response in each microcosm treatment via 16S rRNA and rpoB gene sequencing. We show that microbial communities used DNA only as a phosphorus source, and provision of other sources of carbon and nitrogen was needed to exhibit a substantial growth. The taxonomic composition of eDNA in the water column changed with the availability of inorganic phosphorus or supplied DNA, hinting at preferential uptake of eDNA from specific organismal sources. Especially favored for growth was eDNA from the most abundant taxa, suggesting some haloarchaea prefer eDNA from closely related taxa. The preferential eDNA consumption and differential growth under various nutrient availability regimes were associated with substantial shifts in the taxonomic composition and diversity of microcosm communities. Therefore, we conjecture that in salterns the microbial community assembly is driven by the available resources, including eDNA.Subject terms: Metagenomics, Microbial ecology     

Environmental DNA analysis shows high potential as a tool for estimating intraspecific genetic diversity in a wild fish population

Environmental DNA (eDNA) analysis has recently been used as a new tool for estimating intraspecific diversity. However, whether known haplotypes contained in a sample can be detected correctly using eDNA‐based methods has been examined only by an aquarium experiment. Here, we tested whether the haplotypes of Ayu fish (Plecoglossus altivelis altivelis) detected in a capture survey could also be detected from an eDNA sample derived from the field that contained various haplotypes with low concentrations and foreign substances. A water sample and Ayu specimens collected from a river on the same day were analysed by eDNA analysis and Sanger sequencing, respectively. The 10 L water sample was divided into 20 filters for each of which 15 PCR replications were performed. After high‐throughput sequencing, denoising was performed using two of the most widely used denoising packages, unoise3 and dada2 . Of the 42 haplotypes obtained from the Sanger sequencing of 96 specimens, 38 (unoise3 ) and 41 (dada2 ) haplotypes were detected by eDNA analysis. When dada2 was used, except for one haplotype, haplotypes owned by at least two specimens were detected from all the filter replications. Accordingly, although it is important to note that eDNA‐based method has some limitations and some risk of false positive and false negative, this study showed that the eDNA analysis for evaluating intraspecific genetic diversity provides comparable results for large‐scale capture‐based conventional methods. Our results suggest that eDNA‐based methods could become a more efficient survey method for investigating intraspecific genetic diversity in the field.     

A comparative evaluation of methods for isolation of RNA-directed DNA polymerase from cells in a reconstituted system.

We have compared the relative merits of several procedures for the isolation of RNA-directed DNA polymerase (EC 2.7.7.7.) from cells using a reconsituted model system consisting of a mixture of woolly monkey (simian) sarcoma virus and a cultured human lymphoblastoid cell line, NC-37. When the cell-virus mixture was gently disrupted and fractionated by differential centrifugation, most of the added polymerase was recovered associated with a particulate fraction obtained from the post-mitochondrial supernatant. Purification of the polymerase was best achieved starting from this fraction. The particulate fraction itself can be purified by gel filtration through a Sepharose 2 B column. This procedure did not significantly alter the composition of viral and cellular DNA polymerases. Whereas as little as 7.5 - 10(5) viral particles were sufficient for the detection of RNA-directed DNA polymerase activity, a minimum of about 10(11) particles were necessary for the isolation and unequivocal characterization of the enzyme from the cell-virus mixture by subcellular fractionation and chromatographic separation from cellular DNA polymerases. Purified RNA-directed DNA polymerase had the same primer-template characteristics, sedimentation properties, and immunological cross reactivity as the enzyme purified from density gradient-banded virions of simian sarcoma virus. Methods involving total extraction of the cell-virus mixture either by repeated freezing and thawing followed by detergent treatment or by Dounce homogenization and treatment with high salt and detergent failed to provide RNA-directed DNA polymerase free of cellular DNA polymerases. Because of this, low levels of cellular RNA-directed DNA polymerase may be missed when these approaches are used.     

Evaluating environmental DNA‐based quantification of ranavirus infection in wood frog populations

A variety of challenges arise when monitoring wildlife populations for disease. Sampling tissues can be invasive to hosts, and obtaining sufficient sample sizes can be expensive and time‐consuming, particularly for rare species and when pathogen prevalence is low. Environmental DNA (eDNA)‐based detection of pathogens is an alternative approach to surveillance for aquatic communities that circumvents many of these issues. Ranaviruses are emerging pathogens of ectothermic vertebrates linked to die‐offs of amphibian populations. Detecting ranavirus infections is critical, but nonlethal methods have the above issues and are prone to false negatives. We report on the feasibility and effectiveness of eDNA‐based ranavirus detection in the field. We compared ranavirus titres in eDNA samples collected from pond water to titres in wood frog (Lithobates sylvaticus; n = 5) tadpoles in sites dominated by this one species (n = 20 pond visits). We examined whether ranavirus DNA can be detected in eDNA from pond water when infections are present in the pond and if viral titres detected in eDNA samples correlate with the prevalence or intensity of ranavirus infections in tadpoles. With three 250 mL water samples, we were able to detect the virus in all visits with infected larvae (0.92 diagnostic sensitivity). Also, we found a strong relationship between the viral eDNA titres and titres in larval tissues. eDNA titres increased prior to observed die‐offs and declined afterwards, and were two orders of magnitude higher in ponds with a die‐off. Our results suggest that eDNA is useful for detecting ranavirus infections in wildlife and aquaculture.     

Individual haplotyping of whale sharks from seawater environmental DNA

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied “eDNA haplotyping” to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.     

Variation of Japanese eel eDNA in sequentially changing conditions and in different sample volumes

Environmental DNA (eDNA) surveys have been conducted to evaluate the distribution and abundance of Japanese eels. However, various environmental and biological factors may influence eDNA concentrations. An experiment was conducted using three water sample replicates (50, 100 and 200 ml) of the same group of eels in a tank that were exposed to sequential nonfeeding/feeding and low/high temperature conditions. Slightly higher concentrations occurred at higher temperature (22–23°C) with nonfeeding, and the highest concentrations occurred when feeding started even though it was in the lower temperature (16–17°C) condition, but sample volume had no effect.     

Integration of progeny simian virus 40 DNA into the host cell genome

A procedure was developed for the separation of cellular DNA of productively infected monkey kidney cells from free simian virus 40 DNA. The application of this procedure allowed the investigation of progeny viral DNA integration into the host cell DNA by nucleic acid hybridization techniques. The purification consisted of precipitation of the cellular DNA by Hirt's (1967) method, velocity centrifugation in alkaline sucrose gradients, equilibrium centrifugation in ethidium bromide/CsCl solution, and an additional velocity centrifugation in an alkaline sucrose gradient. The efficiency of each step of the procedure was determined by monitoring the amount of contaminating free viral DNA. Purified cellular DNA, isolated from cells late after infection, contained approximately 0/sd006% free viral DNA, but as much as 2% integrated simian virus 40 DNA. This corresponds to more than 20,000 integrated virus genome equivalents per cell, as determined by DNA-DNA reassociation kinetics. Integration of simian virus 40 DNA into the cellular DNA became detectable at 24 hours after infection, and increased with the increase in the rate of viral DNA synthesis.     

Optimal extraction methods for the simultaneous analysis of DNA from diverse organisms and sample types

Using environmental DNA (eDNA) to assess the distribution of micro‐ and macroorganisms is becoming increasingly popular. However, the comparability and reliability of these studies is not well understood as we lack evidence on how different DNA extraction methods affect the detection of different organisms, and how this varies among sample types. Our aim was to quantify biases associated with six DNA extraction methods and identify one which is optimal for eDNA research targeting multiple organisms and sample types. We assessed each methods’ ability to simultaneously extract bacterial, fungal, plant, animal and fish DNA from soil, leaf litter, stream water, stream sediment, stream biofilm and kick‐net samples, as well as from mock communities. Method choice affected alpha‐diversity for several combinations of taxon and sample type, with the majority of the differences occurring in the bacterial communities. While a single method performed optimally for the extraction of DNA from bacterial, fungal and plant mock communities, different methods performed best for invertebrate and fish mock communities. The consistency of methods, as measured by the similarity of community compositions resulting from replicate extractions, varied and was lowest for the animal communities. Collectively, these data provide the first comprehensive assessment of the biases associated with DNA extraction for both different sample types and taxa types, allowing us to identify DNeasy PowerSoil as a universal DNA extraction method. The adoption of standardized approaches for eDNA extraction will ensure that results can be more reliably compared, and biases quantified, thereby advancing eDNA as an ecological research tool.     

Evidence of compositional differences between the extracellular and intracellular DNA of a granular sludge biofilm

Aims: Extracellular polymeric substances (EPS) are an important component of microbial biofilms, and it is becoming increasingly apparent that extracellular DNA (eDNA) has a functional role in EPS. This study characterizes the eDNA extracted from the novel activated sludge biofilm process of aerobic granules. Methods and Results: Exposing the sludge to cation exchange resin (CER) was used for the extraction of eDNA and intracellular DNA (iDNA) from aerobic granules. This was optimized for eDNA yield while causing minimal cell lysis. We then compared the DNA composition of these extractions using randomly amplified polymorphic DNA (RAPD) fingerprinting and PCR‐based denaturing gradient‐gel electrophoresis (DGGE). Upon the analysis of the genomic DNA and the 16S rRNA genes, differences were detected between the sludge biofilm eDNA and iDNA. Conclusions: Different bacteria within the biofilm disproportionally release DNA into the EPS matrix of the biofilm. Significance and Impact of the Study: The findings further the idea that eDNA has a functional role in the biofilm state, which is an important conceptual information for industrial application of biofilms.     

Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus type 1 Angelotti DNA

It has been shown earlier that the reiterated regions TRS and IRS bracketing the Us segment of herpes simplex virus type 1 Angelotti DNA are heterogeneous in size by stepwise insertion of one to six copies of a 550-base-pair nucleotide sequence. Considerably higher amplification of this sequence was observed in defective viral DNA: up to 14 copies were detected to be inserted in the repeat units of a major class of defective herpes simplex virus type 1 Angelotti DNA, dDNA1, which originated from noncontiguous sites located in UL and the inverted repeats of the S component of the parental genome. Physical maps were established for the cleavage sites of KpnI, PstI, XhoI, and BamHI restriction endonucleases on the repeats of dDNA1. The map position of the insertion sequence was determined. It was demonstrated that the amplified inserts were not distributed at random among or within the repeats. A given total population of dDNA1 molecules consisted of different homopolymers, each of which contained a constant number of inserts in all of its repeats. Assuming that a rolling-circle mechanism is involved in the generation of full-length defective herpes simplex virus type 1 Angelotti DNA from single repeat units, these data suggest that the 550-base-pair sequence is amplified in the repeats before the replication process.     

Rapid degradation of longer DNA fragments enables the improved estimation of distribution and biomass using environmental DNA

The advent of environmental DNA (eDNA) analysis methods has enabled rapid and wide‐range ecological monitoring in aquatic ecosystems, but there is a dearth of information on eDNA degradation. The results of previous studies suggest that the decay rate of eDNA varies depending on the length of DNA fragments. To examine this hypothesis, we compared temporal change in copy number of long eDNA fragments (719 bp) with that of short eDNA fragments (127 bp). First, we isolated rearing water from a target fish species, Japanese Jack Mackerel (Trachurus japonicus), and then quantified the copy number of the long and short eDNA fragments in 1 L water samples after isolating the water from the fish. Long DNA fragments showed a higher decay rate than short fragments. Next, we measured the eDNA copy numbers of long and short DNA fragments using field samples, and compared them with fish biomass as measured by echo intensity. Although a previous study suggested that short eDNA fragments could be overestimated because of nontarget eDNA from a nearby fish market and carcasses, the eDNA concentrations of long fragments were correlated with echo intensity. This suggests that the concentration of longer eDNA fragments reflects fish biomass more accurately than the previous study by removing the effects of the fish market and carcasses. The length‐related differences in eDNA have a substantial potential to improve estimation of species biomass.