Environmental DNA quantification in a spatial and temporal context: a case study examining the removal of brook trout from a high alpine basin

Analysis of aquatic environmental DNA (eDNA) is a promising tool to determine species distribution, abundance, and biomass. Understanding how the amount of eDNA collected is affected by spatial and temporal processes needs to become better understood before eDNA quantification can be used in species management. In this study, we analyzed how the amount of eDNA changed across space and time in a high mountain basin where nonnative fish were being removed. We sampled from restoration (sites with fish removal activities; n?=?6) and control sites (sites with no fish removal activities where fish were present; n?=?3) and found the number and biomass of fish removed were related to the quantities of DNA collected and not related to site position within the drainage. Our results indicate that the amount of eDNA collected in an open system can provide an index of population size despite inherent complications of analyzing a spatially connected and temporally dynamic watershed. However, there are complications when applying these methods in species management: (1) small increases in eDNA density corresponded to large increases in trout density; (2) eDNA and traditional field techniques disproportionately target certain life stages, complicating comparisons between techniques; and (3) eDNA index values may need to be calibrated when sampling different species, life stages, environments, and habitats. We call for further research before this process can be used in a management context.     

Performance of eDNA assays to detect and quantify an elusive benthic fish in upland streams

The sensitivity and specificity of eDNA-based monitoring, coupled with its potential utility to estimate population density or biomass, makes it a useful tool in invasive species management. In this study, we investigated the potential of the eDNA method to improve the detection of the elusive invasive fish, oriental weatherloach (Misgurnus anguillicaudatus), in a river system where a density gradient of the species occurs. We compared detection rates between eDNA and conventional monitoring methods and examined the relationship between eDNA and abundance in a flowing environment. The eDNA method had a higher site detection rate than conventional methods (63 vs. 38%). Weatherloach eDNA was detected at all sites where the fish has been previously caught and none of the sites where the species has not been caught for the past 7 years. There was an increasing density trend going downstream based on long-term conventional monitoring, but the eDNA concentration in water samples reflected this trend only in a continuous section of the river where impoundments were absent. We did not find a positive relationship between eDNA concentration and contemporary abundance estimates in our study area. A high eDNA concentration was recorded at a site (DVC) which was designated a low density site based on long-term catch data. This discrepancy was a likely result of physical habitat characteristics which influenced the efficiency of the conventional methods used. This study highlighted the challenges of inferring density from eDNA data in flowing water because habitat features may confound results, necessitating careful consideration for results to be useful to management.     

Environmental DNA analysis for estimating the abundance and biomass of stream fish

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Fishing-induced changes in predation pressure by perch (<Emphasis Type="Italic">Perca fluviatilis</Emphasis>) regulate littoral benthic macroinvertebrate biomass,density, and community structure

We aimed to study whether the varying changes in predation pressure by perch (Perca fluviatilis) reflect the biomass, density, and community structure of the benthic macroinvertebrates. Prey preference is size-dependent, and overall predation pressure is density dependent, and thus the size structure of the P. fluviatilis population should affect the structure of the macroinvertebrate community, and the population density of P. fluviatilis should reflect the overall density of benthic macroinvertebrates. We sampled the littoral benthic community in a boreal lake that had been divided into two parts that were subjected to two different fishing procedures during 2007–2012 period and analyzed the macroinvertebrate diet of fish. The benthic macroinvertebrate community reflected the predation pressure. Total macroinvertebrate biomass increased during the study period in the lake division with a non-size-selective fishing procedure (NSF), i.e., all invertivorous perch size-classes targeted, but decreased in the section with negatively size-selective fishing procedure (SSF), i.e., large invertivorous individuals ≥ 16 cm were not targeted. This difference was a result of the increase in large-sized species, such as Odonata, for the NSF procedure and decrease in the SSF procedure. In contrast to total biomass, total macroinvertebrate density did not show a response to predator size structure but rather total macroinvertebrate density decreased with increasing fish density. The study demonstrates the effect of predation pressure of P. fluviatilis on benthic communities, thus highlighting the keystone predator role of the species in boreal lakes and gives more insight on the multiple effects of fish predation on littoral benthic communities.     

The Release Rate of Environmental DNA from Juvenile and Adult Fish

The environmental DNA (eDNA) technique is expected to become a powerful, non-invasive tool for estimating the distribution and biomass of organisms. This technique was recently shown to be applicable to aquatic vertebrates by collecting extraorganismal DNA floating in the water or absorbed onto suspended particles. However, basic information on eDNA release rate is lacking, despite it being essential for practical applications. In this series of experiments with bluegill sunfish (Lepomis macrochirus), we examined the effect of fish developmental stage on eDNA release rate. eDNA concentration reached equilibrium 3 days after the individual fish were introduced into the separate containers, enabling calculation of the eDNA release rate (copies h⁻¹) from individual fish on the assumption that the number of eDNA released from the fish per unit time equals total degradation in the container (copies h⁻¹). The eDNA release rate was 3–4 times higher in the adult (body weight: 30–75 g) than in the juvenile group (0.5–2.0 g). Such positive relationship between fish size and eDNA release rate support the possibility of biomass rather than density estimation using eDNA techniques. However, the eDNA release rate per fish body weight (copies h⁻¹ g⁻¹) was slightly higher in the juvenile than the adult group, which is likely because of the ontogenetic reduction in metabolic activity. Therefore, quantitative eDNA data should be carefully interpreted to avoid overestimating biomass when the population is dominated by juveniles, because the age structure of the focal population is often variable and unseen in the field. eDNA degradation rates (copies l⁻¹ h⁻¹), calculated by curve fitting of time-dependent changes in eDNA concentrations after fish removal, were 5.1–15.9% per hour (half-life: 6.3 h). This suggests that quantitative eDNA data should be corrected using a degradation curve attained in the target field.     

Genetic variability in anadromous fishes,chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis> (Walbaum, 1792), and Sakhalin taimen <Emphasis Type="Italic">Parahucho perryi</Emphasis> (Brevoort, 1856) from the Northwestern Pacific as a reflection of paleoclimate oscilations

The genetic variability distribution of two mtDNA segments of chum salmon (Oncorhynchus keta) (Walbaum, 1792) and Sakhalin taimen (Parahucho perryi) (Brevoort, 1856) was examined in populations of the Sea of Japan and the Sea of Okhotsk. The values of haplotype and nucleotide variability in these species are, in general, of the same level. The dating of the divergence time of species haplotypes revealed four evolutionary periods in Sakhalin taimen and three in chum salmon. In the taimen, the first divergence time occurred approximately 430 thousand years (kyr) ago, the second 220 kyr ago, and the third 70 kyr ago. In the chum salmon, the first divergence time corresponds to 220 kyr; the second is approximately 100 kyr ago. In both species, the main portion of presently revealed haplotypes evolved over the past 50–10 kyr. Certain glacioeustatic sea level fluctuations influenced each stage of evolution history of species, contributing to their geographic isolation. Demographic population history research found that the initial stage of population growth in the taimen occurred at the time period of approximately 12 kyr ago and was apparently associated with the end of the Last Glacial Maximum. In the chum salmon, this period began somewhat earlier, 30–35 kyr ago; it has accelerated in the past 10–15 kyr. The last glaciation to a lesser extent impacted the demographics of chum salmon, probably due to the greater eurythermity and to the larger range of this species.     

On catches of low-latitude fish at Sakhalin coast in summer 2014

In summer 2014, catches of three tropical and subtropical fish species were registered at Sakhalin coast. The Japanese sea bass Lateolabrax japonicus was detected at Sakhalin coast for the first time, while the Japanese amberjack Seriola quinqueradiata was first detected in the Sea of Okhotsk waters sweeping the Sakhalin coast. The catch of the great white shark Carcharodon carcharias is the second confirmed case of this fish occurrence near the Sakhalin coast for the last 10 years.     

Morphological Traits of Two Seed-Feeding Beetle Species and the Relationship to Resource Traits

Morphological traits are useful to investigate insect sex-related differences in body size and to reveal differences in resource use. It has been suggested that as the resource increases, so does the body size of organisms interacting with the resource, highlighting the crucial role of resource quality and quantity in determining the morphological traits of organisms interacting with the resource. Here, we describe morphological traits of two species of Bruchinae, Merobruchus terani (Kingsolver 1980) and Stator maculatopygus (Pic 1930), consuming seeds of Senegalia tenuifolia (Fabaceae: Mimosoideae). We evaluated the influence of monthly sample and sampling sites on tibia and femur length and biomass. In addition, we tested two predictions in which body size related to resource amount and body size related to longevity. Males of M. terani were heavier than females, whereas the two sexes of S. maculatopygus did not differ in biomass. Both species had larger body sizes in the late ripe-fruit stage. With respect to sampling sites, biomass of M. terani did not differ, whereas S. maculatopygus did differ in biomass. Merobruchus terani showed a positive relationship with seed traits, whereas S. maculatopygus showed no relationship. At the same time, fruit traits showed a negative effect on morphological traits for both beetle species. The longevity experiment, performed using only M. terani, showed an equal longevity and seed consumption rate for both sexes. Our study indicates that different species, interacting in the same system and performing similar functional behaviors, respond differently to the same resource.     

Distribution and habitat associations of the California moray (<Emphasis Type="Italic">Gymnothorax mordax</Emphasis>) within Two Harbors,Santa Catalina Island,California

While kelp forests are some of the best-surveyed ecosystems in California, information on cryptic inhabitants and their role within the community are lacking. Kelp itself provides overall structure to the habitat; however the rocky reef to which the kelp attaches is known to provide additional structure for cryptic species. Gymnothorax mordax, the California moray, is an elusive predatory species that is considered abundant in the waters around Catalina Island. However, no life history data exists for this species. We examined habitat composition, relative abundance, size pattern distributions, and biomass of G. mordax within Two Harbors, Catalina Island. Habitats were sampled using a combination of baited trap collection and transect surveys using SCUBA. A total of 462 G. mordax were captured, primarily in shallow (< 10 m) waters. Individuals of G. mordax were associated with mostly boulder and cobble substrates. Measurements of relative abundance and density indicate that G. mordax is more prevalent than reported in previous studies. We also discovered that the 6 trapping sites from which all morays were collected, differed in size structuring and density while the relatively high biomass did not change across sites. In general, southern facing sites exhibited higher densities of morays, while northern facing sites showed more size structuring. We show how the structural complexity of the rocky reef habitat in an already diverse kelp forest ecosystem, can support a high biomass of a cryptic elongate predatory fish.     

Disentangling the stream community impacts of <Emphasis Type="Italic">Didymosphenia geminata</Emphasis>: How are higher trophic levels affected?

Human activities frequently result in either intentional or unintentional introductions of species to new locations, and freshwater environments worldwide are particularly vulnerable to species invasions. An introduced freshwater diatom, Didymosphenia geminata, was first discovered in New Zealand in 2004 but there was limited research available to predict the drivers of D. geminata biomass and how biomass variability might influence higher trophic levels (e.g. invertebrates and fish). We examined the effect of D. geminata biomass on benthic invertebrates, invertebrate drift and fish communities in 20 rivers in New Zealand with variable hydrology, physical habitat and water chemistry. Variation in D. geminata biomass was best explained by a model that showed D. geminata biomass increased with time since the last flow event exceeding three times the median annual discharge and decreasing concentration of dissolved reactive phosphorus. Analyses of biotic responses showed that high D. geminata biomass did not affect either invertebrate or fish diversity but altered the structure of benthic communities, changed the composition of drifting invertebrate communities and reduced fish biomass by 90 %, particularly trout. A partial least squares path model was used to disentangle both direct and indirect effects of D. geminata on fish communities and showed D. geminata had a significant negative direct effect on fish communities. This is the first study to show how the potential effects of the introduced diatom D. geminata can impact fish communities and has shown that D. geminata impacts fish both directly and indirectly through changes in their invertebrate prey community.     

Fish assemblage structure in an estuary of the Atlantic Forest biodiversity hotspot (southern Brazil)

We described the fish assemblage in the estuary of the Guaraguaçu River (one of the largest tributaries of the Paranaguá Bay Estuary, located within Brazil’s Atlantic Forest Biosphere Reserve) from June 2005 to May 2006, and assessed the seasonal and spatial effects of abiotic environmental attributes on the fish assemblage structure. Despite some oscillations in salinity, the upper and lower estuaries had year-round persistent oligohaline and polyhaline conditions, respectively. Despite high species richness (55 species), the Guaraguaçu River Estuary fish community contains a few dominant taxa; 11% of the richness accounts for >60% of its density and biomass. The most abundant species (in terms of both biomass and density) was Atherinella brasiliensis. Species whose densities were most strongly associated with the upper estuary were Centropomus parallelus, Ctenogobius schufeldti, Eucinostomus melanopterus, Platanichthys platana, Trinectes paulistanus, and Eugerres brasilianus. Those whose densities were most strongly associated with the lower estuary were A. brasiliensis, Sphoeroides greeleyi, Eucinostomus argenteus, Sphoeroides testudineus, Diapterus rhombeus, and Harengula clupeola. Throughout the year, canonical correspondence analysis identified: (1) the pattern of horizontal stratification of salinity along the river as being the most important variable for explaining most of the fish fauna structure; and (2) a strong relationship between the fish fauna and the salinity gradient along the estuary. Analysis of similarity further confirmed that each estuarine zone supports a year-round persistent and relatively homogeneous fish species assemblage. Total mean density and biomass remained constant over time in each estuarine habitat, but density shifted in the most abundant species, which appears related to recruitment patterns. Such species and abundance persistence likely occurs because seasonal rainfall-induced changes in river discharge are not sufficient to significantly shift runoff and salinity and thus fish assemblage structure (species composition, density and biomass) along the estuary. Such a lack of seasonal fish fauna movement as a response to changes in river flow contrasts with other estuarine systems around the world.     

Environmental DNA as a ‘Snapshot’ of Fish Distribution: A Case Study of Japanese Jack Mackerel in Maizuru Bay,Sea of Japan

Recent studies in streams and ponds have demonstrated that the distribution and biomass of aquatic organisms can be estimated by detection and quantification of environmental DNA (eDNA). In more open systems such as seas, it is not evident whether eDNA can represent the distribution and biomass of aquatic organisms because various environmental factors (e.g., water flow) are expected to affect eDNA distribution and concentration. To test the relationships between the distribution of fish and eDNA, we conducted a grid survey in Maizuru Bay, Sea of Japan, and sampled surface and bottom waters while monitoring biomass of the Japanese jack mackerel (Trachurus japonicus) using echo sounder technology. A linear model showed a high R² value (0.665) without outlier data points, and the association between estimated eDNA concentrations from the surface water samples and echo intensity was significantly positive, suggesting that the estimated spatial variation in eDNA concentration can reflect the local biomass of the jack mackerel. We also found that a best-fit model included echo intensity obtained within 10–150 m from water sampling sites, indicating that the estimated eDNA concentration most likely reflects fish biomass within 150 m in the bay. Although eDNA from a wholesale fish market partially affected eDNA concentration, we conclude that eDNA generally provides a ‘snapshot’ of fish distribution and biomass in a large area. Further studies in which dynamics of eDNA under field conditions (e.g., patterns of release, degradation, and diffusion of eDNA) are taken into account will provide a better estimate of fish distribution and biomass based on eDNA.     

Summer-autumn ichthyoplankton of the Sea of Okhotsk and the Sea of Japan and special traits of feeding of fish larvae and fry in 2003–2004

In summer-autumn of 2003–2004, the ichthyoplankton of the Sea of Okhotsk comprised 35 species. In this period the most widely distributed and numerous were larvae of the lord Hemilepidotus gilberti, the Pacific stout sand lance Ammodytes hexapterus, and the Sakhalin dab Limanda sakhalinensis. The maximum catches of fish larvae were attributed to coastal waters off eastern Sakhalin and to the shelf of the northern part of the Sea of Okhotsk. In November of 2003, the ichthyoplankton of the Sea of Japan was represented by fish larvae belonging mainly to the boreal ichthyocomplex. The catches consisted predominantly of larvae of the arabesque greenling Pleurogrammus azonus, the ronquil Bathymaster derjugini, and the rockfish Sebastes owstoni. Fish larvae and fry in the northwestern part of the Sea of Japan were caught principally within 43°–45° N and 137°–139° E above the depth 1500–2000 m. The food spectrum of fish larvae in the Sea of Okhotsk and the Sea of Japan comprised over 20 plankters of various size belonging to seven taxa. Irrespective of fish species, the food items common of all fish were copepods Pseudocalanus minutus and Oithona similis. The daily rations were calculated for mass species (Hemilepidotus gilberti, Ammodytes hexapterus, Hexagrammos stelleri, Pleurogrammus azonus, Bathymaster derjugini, and Sebastes owstoni). The larvae of all considered species in the Sea of Japan and in the Sea of Okhotsk fed predominantly in the light period of the day.     

Phylogenetic relationships of Sakhalin taimen <Emphasis Type="Italic">Parahucho perryi</Emphasis> inferred from PCR-RFLP analysis of mitochondrial DNA

RFLP analysis of three amplified mtDNA fragments (Cytb/D-loop, ND1/ND2, and ND3/ND4L/ND4) was performed in the following taxa: Parahucho perryi, Hucho taimen, Brachymystax lenok, B. tumensis, Salmo salar, Salvelinus leucomaenis, and S. levanidovi. For mtDNA of P. perryi, a substantial decrease in the haplotype and nucleotide diversity was observed as a result of random genetic drift, caused by a reduction in the effective population size. Nucleotide divergence estimates between the mtDNA haplotypes were determined. Sakhalin taimen P. perryi was found to be approximately equally diverged from S. salar and from the charrs of the genus Salvelinus, by 11.0 and 10.0%, respectively. The divergence between P. perryi and H. taimen constituted 14.6%, between P. perryi and lenoks of the genus Brachymystax, 14.2%, and between H. taimen and Brachymystax, 7.7%. The analysis of possible phylogenetic relationships of the mtDNA from P. perryi among the group of taxa examined confirmed validity of the genus Parahucho. Phylogenetic reconstructions performed showed that robustness of the trees constructed for the complex of phylogenetically informative characters over three mtDNA fragments was considerably higher than that of the trees constructed for individual genes.     

Changes in the structural parameters of the summer zooplankton in the pelagic zone of Lake Sevan (Armenia) during an increase in fish abundance and biomass

Features of the composition and structure of the summer zooplankton in the pelagic zone of Lake Sevan in years that were characterized by different abundances and biomasses of fish are described. It is shown that the species diversity of the community and the specific number of species of zooplankteurs, the quantity of Copepods in the total abundance and biomass of zooplankton, as well as the value of the Shannon index and the trophic coefficient, increased upon an increase in the fish abundance. At the same time, the total biomass of zooplankton and biomass of cladocerans decreased, among which the density and biomass of the larger and more colored Daphnia (Ctenodaphnia) magna Straus decreased, but the density and biomass of D. (Daphnia) hyalina Leydig and Diaphаnosoma brachyurum Lievin increased.     

Effect of water temperature and fish biomass on environmental DNA shedding,degradation, and size distribution

Environmental DNA (eDNA) analysis has successfully detected organisms in various aquatic environments. However, there is little basic information on eDNA, including the eDNA shedding and degradation processes. This study focused on water temperature and fish biomass and showed that eDNA shedding, degradation, and size distribution varied depending on water temperature and fish biomass. The tank experiments consisted of four temperature levels and three fish biomass levels. The total eDNA and size‐fractioned eDNA from Japanese Jack Mackerels (Trachurus japonicus) were quantified before and after removing the fish. The results showed that the eDNA shedding rate increased at higher water temperature and larger fish biomass, and the eDNA decay rate also increased at higher temperature and fish biomass. In addition, the small‐sized eDNA fractions were proportionally larger at higher temperatures, and these proportions varied among fish biomass. After removing the fish from the tanks, the percentage of eDNA temporally decreased when the eDNA size fraction was >10 µm, while the smaller size fractions increased. These results have the potential to make the use of eDNA analysis more widespread in the future.     

Comparative Analysis of Content of Omega-3 Polyunsaturated Fatty Acids in Food and Muscle Tissue of Fish from Aquaculture and Natural Habitats

Two fish species reared in aquaculture (pink salmon Oncorhynchus gorbuscha and whitefish Coregonus lavaretus) and ten fish species from natural habitats (whitefish C. lavaretus, tugun Coregonus tugun, broad whitefish Coregonus nasus, least cisco Coregonus sardinella, vendace Coregonus albula, boganid charr Salvelinus boganidae, charr Salvelinus alpinus complex, northern pike Esox lucius, sharp-snouted lenok Brachymystax lenok, and taimen Hucho taimen) have been studied. The content of two long-chain polyunsaturated omega-3 fatty acids (PUFAs), eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA), in the muscle tissue of the fish and in their food (intestine contents) are compared. In the aquacultures of whitefish and pink salmon, the total content of EPA and DHA is significantly higher in feed than in the muscle tissue of the fish, which indicates losses of PUFA in the two-link food chain of the aquaculture during their transfer to the upper trophic level. EPA and DHA losses in aquaculture, which are confirmed by numerous literature data, mean an inefficient usage of the available sources of PUFAs and the aggravation of the global deficit of these biochemicals in the human diet. A study of natural fish populations reveals the accumulation of EPA and DHA in their biomass compared to food in many cases, although opposite phenomena are also observed. An assumption on the presence of an optimal, physiologically adequate species-specific level of PUFA in the fish muscle tissue has been made based on our data and literature data. If the level of PUFAs in the muscles is lower than optimal, their accumulation (bioaccumulation) from food and/or de novo synthesis are observed. When the optimal level is exceeded, the content of EPA and DHA in biomass approaches maximum species-specific values; however, part of these PUFAs entering from food is not digested or is catabolized. According to the obtained data, the species of the order Salmoniformes have an optimal level of 2 to 6 mg/g of wet weight. It has been found that in aquaculture approaching to maximum values of EPA + DHA content was accompanied by their losses (scattering) in the food chains, while in natural ecosystems the maximum values of PUFA content in the fish biomass are achieved by their accumulation from the lower trophic level. Boganid charr S. boganidae had the highest content of EPA + DHA in the muscle tissue among all known fish species (32.78 mg/g of wet weight).     

Exposure to waterborne copper and high temperature induces the formation of reactive oxygen species and causes mortality in the Amazonian fish <Emphasis Type="Italic">Hoplosternum littorale</Emphasis>

Hoplosternum littorale is an Amazon fish that lives in urban areas surrounded by polluted igarapés, where elevated copper concentrations eventually occur. The central goal of this study was to evaluate the associated effects of high temperature and copper contamination on survival time and biochemical responses of the Amazonian fish species H. littorale. We exposed fish to two nominal dissolved copper concentrations (50 and 500 µg l^?1) and combined temperatures of 28 and 34°C. Our findings showed that the combination of these variables affects the survival time of this species. The activity of the biotransformation enzymes ethoxyresorufin-O-deethylase and glutathione-S-transferase showed no alterations in fish within all treatments. The increase of reactive oxygen species and the decrease in potential total antioxidant capacity promoted the imbalance in the antioxidant system. An induction in superoxide dismutase activity occurred in fish exposed to copper concentrations of 50 and 500 µg l^?1 at both temperatures, suggesting liver impairments. Thus, we suggest that H. littorale is sensitive to copper, and this sensitivity is increased further with exposure to high temperatures, particularly in the survival time and reactive oxygen species formation of this fish species.