Fluctuating interaction network and time-varying stability of a natural fish community.

Ecological theory suggests that large-scale patterns such as community stability can be influenced by changes in interspecific interactions that arise from the behavioural and/or physiological responses of individual species varying over time. Although this theory has experimental support, evidence from natural ecosystems is lacking owing to the challenges of tracking rapid changes in interspecific interactions (known to occur on timescales much shorter than a generation time) and then identifying the effect of such changes on large-scale community dynamics. Here, using tools for analysing nonlinear time series and a 12-year-long dataset of fortnightly collected observations on a natural marine fish community in Maizuru Bay, Japan, we show that short-term changes in interaction networks influence overall community dynamics. Among the 15 dominant species, we identify 14 interspecific interactions to construct a dynamic interaction network. We show that the strengths, and even types, of interactions change with time; we also develop a time-varying stability measure based on local Lyapunov stability for attractor dynamics in non-equilibrium nonlinear systems. We use this dynamic stability measure to examine the link between the time-varying interaction network and community stability. We find seasonal patterns in dynamic stability for this fish community that broadly support expectations of current ecological theory. Specifically, the dominance of weak interactions and higher species diversity during summer months are associated with higher dynamic stability and smaller population fluctuations. We suggest that interspecific interactions, community network structure and community stability are dynamic properties, and that linking fluctuating interaction networks to community-level dynamic properties is key to understanding the maintenance of ecological communities in nature.

Post-settlement dynamics of Japanese flounder Paralichthys olivaceus in Tango Bay: Seasonal patterns in growth, mortality, and recruitment potential.

Japanese flounder Paralichthys olivaceus is one of the most valuable coastal flatfish species in East Asia. To investigate post-settlement growth and mortality, juveniles were sampled in Tango Bay (Japan) weekly throughout the settlement period in 2007 and 2008. Otolith (lapillus) microstructure analysis enabled the categorization of juveniles into six biweekly cohorts each year. Later cohorts exhibited higher growth rates possibly because of higher water temperatures. A key observation was the direct relationship between high mortality and high density in mid-season cohorts in both years, pointing to density-dependent mortality. This increased mortality may be attributed to predation, including cannibalism by earlier cohorts. Furthermore, growth-selective mortality was evident soon after settlement, underscoring the vulnerability of slow growers to predation during the early juvenile stage. Although earlier and later cohorts were less abundant but showed promising recruitment potential, the prospective contribution of mid-season cohorts to the adult population remained uncertain. The results clearly highlight the importance of density-dependent mortality in population regulation in post-settlement Japanese flounder.

Analysis of the Persistence and Particle Size Distributional Shift of Sperm-Derived Environmental DNA to Monitor Jack Mackerel Spawning Activity.

Environmental DNA (eDNA) analysis holds great promise as an efficient and noninvasive method to monitor not only the distribution of organisms but also their spawning activity. In eDNA analysis-based monitoring of spawning activity, the detection of sperm-derived eDNA is a key point; however, its characteristics and dynamics are completely unknown. The present study focuses on the persistence and particle size distribution (PSD) of eDNA derived from the sperm of Japanese jack mackerel. First, we investigated the time-dependent degradation and the PSD of sperm-derived eDNA by artificially adding sperm to seawater. Next, we kept fish in tanks and examined the changes in eDNA concentration and PSD before and after spawning. The results of two experiments showed that the degradation of sperm-derived eDNA proceeded rapidly, with PSD shifting to a smaller size regardless of the DNA region (Cyt b or ITS1). Additionally, it was shown that the nuclei and mitochondria released from sperm through degradation had a size distribution that was not simply dependent on each organelle size. These results will contribute to elucidating the characteristics and dynamics of eDNA specifically during the spawning season and to further developing eDNA analysis as a powerful tool for the monitoring of spawning activity.

Estimating fish population abundance by integrating quantitative data on environmental DNA and hydrodynamic modelling.

Molecular analysis of DNA left in the environment, known as environmental DNA (eDNA), has proven to be a powerful and cost-effective approach to infer occurrence of species. Nonetheless, relating measurements of eDNA concentration to population abundance remains difficult because detailed knowledge on the processes that govern spatial and temporal distribution of eDNA should be integrated to reconstruct the underlying distribution and abundance of a target species. In this study, we propose a general framework of abundance estimation for aquatic systems on the basis of spatially replicated measurements of eDNA. The proposed method explicitly accounts for production, transport and degradation of eDNA by utilizing numerical hydrodynamic models that can simulate the distribution of eDNA concentrations within an aquatic area. It turns out that, under certain assumptions, population abundance can be estimated via a Bayesian inference of a generalized linear model. Application to a Japanese jack mackerel (Trachurus japonicus) population in Maizuru Bay revealed that the proposed method gives an estimate of population abundance comparable to that of a quantitative echo sounder method. Furthermore, the method successfully identified a source of exogenous input of eDNA (a fish market), which may render a quantitative application of eDNA difficult to interpret unless its effect is taken into account. These findings indicate the ability of eDNA to reliably reflect population abundance of aquatic macroorganisms; when the "ecology of eDNA" is adequately accounted for, population abundance can be quantified on the basis of measurements of eDNA concentration.

Angling gear avoidance learning in juvenile red sea bream: evidence from individual-based experiments.

Angling gear avoidance learning is a possible factor that contributes to the vulnerability of caught-and-released fish to angling. Whereas past studies suggested angling gear avoidance learning, they were based on large-scale experiments on groups of fish and unable to verify learning accurately. Details of avoidance learning are also unclear. The present study investigated angling gear avoidance learning through a series of individual-based experiments using red sea bream (Pagrus major) juveniles. Fish avoided angling gear after only one or two catches while showing feeding motivation for pellets, representing avoidance learning for angling gear. Most of the experienced fish avoided krill attached to a fishing line, but not krill alone or pellets presented near the angling gear. Experienced fish were less vulnerable to angling than control fish. Approximately half of the experienced fish kept the memory of angling gear 2 months after learning. The learning effect through the catch-and-release procedure would reduce catchability and the value of fishery-dependent stock assessments.

Freshwater entry behaviour of a non-migratory stenohaline marine fish Takifugu snyderi.

We conducted salinity choice trials with the stenohaline marine species Takifugu snyderi to test their freshwater (FW) entry frequency in relation to starvation. The fish preferred to enter non-natal FW rather than remain in seawater. No relationship was detected between starvation and FW entry behaviour. Our results provide new empirical evidence of a stenohaline fish entering a non-natal osmotic environment. Further research on the entry of stenohaline species such as this one into lethal environments may help determine if this might help promote the evolution of diadromous life histories.

Particle Size Distribution of Environmental DNA from the Nuclei of Marine Fish.

Environmental DNA (eDNA) analyses have enabled a more efficient surveillance of species distribution and composition than conventional methods. However, the characteristics and dynamics of eDNA (e.g., origin, state, transport, and fate) remain unknown. This is especially limited for the eDNA derived from nuclei (nu-eDNA), which has recently been used in eDNA analyses. Here, we compared the particle size distribution (PSD) of nu-eDNA from Japanese Jack Mackerel (Trachurus japonicus) with that of mt-eDNA (eDNA derived from mitochondria) reported in previous studies. We repeatedly sampled rearing water from the tanks under multiple temperatures and fish biomass levels, and quantified the copy numbers of size-fractioned nu-eDNA. We found that the concentration of nu-eDNA was higher than that of mt-eDNA at 3-10 µm size fraction. Moreover, at the 0.8-3 µm and 0.4-0.8 µm size fractions, eDNA concentrations of both types increased with higher temperature and their degradation tended to be suppressed. These results imply that the production of eDNA from large to small size fractions could buffer the degradation of small-sized eDNA, which could improve its persistence in water. Our findings will contribute to refine the difference between nu- and mt-eDNA properties, and assist eDNA analyses as an efficient tool for the conservation of aquatic species.

Memory retention capacity using two different training methods, appetitive and aversive learning, in juvenile red sea bream Chrysophrys major.

Memory retention based on appetitive and aversive learning was studied in juvenile red sea bream Chrysophrys major. The fish were individually trained via appetitive and aversive learning. In general, they retained appetitive memories for 30 days, but not for 60 days. Conversely, aversive memory endured for 1 day, but not for 3 days or longer. Analyses at the individual level revealed that some fish retained appetitive memories for 60 days, whereas others lost it within 3 days; this suggests considerable variability in memory retention capacity among individual fish. The memory duration for aversive learning was remarkably short, which should be considered when releasing trained fish into the wild for stock enhancement. Furthermore, the high inter-individual variability suggests that evaluating memory retention capacity through group experiments might lead to overestimation of fishes' ability.

Biomass-dependent emission of environmental DNA in jack mackerel Trachurus japonicus juveniles.

Environmental DNA (eDNA) from juvenile jack mackerel Trachurus japonicus was detected in tanks with 1, 3, 10, or 30 individuals per tank. Quantitative PCR using a set of species-specific primers and a probe revealed that the concentration of eDNA increased almost linearly with the density of fish. The coefficient of determination (R2 ) in the linear regression was lower than values previously reported for freshwater fishes in similar settings.

Left-right pigmentation pattern of Japanese flounder corresponds to expression levels of melanocortin receptors (MC1R and MC5R), but not to agouti signaling protein 1 (ASIP1) expression.

Body coloration in flatfish is one of the most distinctive asymmetries in the animal kingdom, although the fundamental molecular mechanism of the pigmentation is unclear. In the dorso-ventral coloration (countershading) of other teleost fishes, ventral-specific expression of agouti signaling protein 1 (ASIP1), an endogenous antagonist of melanocortin 1 receptor (MC1R), has been reported to play a pivotal role. Contribution of ASIP1 is also suggested in the asymmetrical pigmentation of flatfish. In order to confirm the contribution of ASIP1 and further examine receptor function in the body coloration of Japanese flounder, expression levels of asip1, mc1r, melanocortin 5 receptor (mc5r), and melanin-concentrating hormone receptor 2 (mchr2) were measured in the normally pigmented area of the left side, the normally non-pigmented area of the right side, and the abnormally pigmented (exhibiting hypermelanosis) area of the right side. Measurement was also carried out under conditions of hypermelanosis stimulated by cortisol and during the transition from non-pigmentation to pigmentation in areas of hypermelanosis. Contrary to our expectations, no difference was detected in asip1 expression between pigmented and non-pigmented areas. There was also no difference between normal and hormonally stimulated pigmented conditions in areas of hypermelanosis or during the transition process. Instead, the expression levels of mc1r, mc5r, and mchr2 were consistently higher in pigmented areas, and were especially increased under hormonally stimulated conditions. In addition, expressions of these receptor genes increased prior to pigmentation in areas of future hypermelanosis. Our results suggest that MC1Rand MC5R, but not necessarily ASIP1, contribute to pigmentation and hypermelanosis in Japanese flounder. We propose a yet unknown molecular mechanism for asymmetrical pigmentation in flatfish that is distinct from that of countershading in other vertebrates.

A mixed-species shoal including fewer heterospecifics is preferred by jack mackerel Trachurus japonicus juveniles.

Mixed-species shoals of fishes were reported from various environments such as coral reefs and temperate lakes. The driving mechanism of forming mixed-species shoal can be either haphazard or a preferential choice by an individual. Here, we tested if jack mackerel Trachurus japonicus, a temperate marine pelagic fish, could choose a shoal composition using vision based on a choice experiment. Shoal preference of jack mackerel juveniles was evaluated by presenting shoals comprising one to four heterospecifics (half-lined cardinal Ostorhinchus semilineatus) out of four shoal members to the focal fish. When both conspecific and heterospecific shoals were presented to a single jack mackerel, the focal fish showed a preference for the conspecific rather than heterospecific shoal, indicating that they can choose a conspecific shoal based on the visual information. However, when a focal fish encountered both conspecific and mixed-species shoals including fewer heterospecifics (one out of four individuals), the latter was preferred. The result implies that jack mackerel can judge a preferable composition of shoal using visual information. The inclusion of fewer heterospecifics may be exploiting oddity effects, whereby minority species are exposed to relatively high vulnerability to predators, as well as resulting in reduced competition for food while maintaining predator vigilance in a shoal.

Dynamics-based characterization and classification of biodiversity indicators.

Various biodiversity indicators, such as species richness, total abundance, and species diversity indices, have been developed to capture the state of ecological communities over space and time. As biodiversity is a multifaceted concept, it is important to understand the dimension of biodiversity reflected by each indicator for successful conservation and management. Here we utilized the responsiveness of biodiversity indicators' dynamics to environmental changes (i.e., environmental responsiveness) as a signature of the dimension of biodiversity. We present a method for characterizing and classifying biodiversity indicators according to environmental responsiveness and apply the methodology to monitoring data for a marine fish community under intermittent anthropogenic warm water discharge. Our analysis showed that 10 biodiversity indicators can be classified into three super-groups based on the dimension of biodiversity that is reflected. Group I (species richness and community mean of latitudinal center of distribution (cCOD)) showed the greatest robustness to temperature changes; Group II (species diversity and total abundance) showed an abrupt change in the middle of the monitoring period, presumably due to a change in temperature; Group III (species evenness) exhibited the highest sensitivity to environmental changes, including temperature. These results had several ecological implications. First, the responsiveness of species diversity and species evenness to temperature changes might be related to changes in the species abundance distribution. Second, the similar environmental responsiveness of species richness and cCOD implies that fish migration from lower latitudes is a major driver of species compositional changes. The study methodology may be useful in selecting appropriate indicators for efficient biodiversity monitoring.

Temperature sensitivity of the interspecific interaction strength of coastal marine fish communities.

The effects of temperature on interaction strengths are important for understanding and forecasting how global climate change impacts marine ecosystems; however, tracking and quantifying interactions of marine fish species are practically difficult especially under field conditions, and thus, how temperature influences their interaction strengths under field conditions remains poorly understood. We herein performed quantitative fish environmental DNA (eDNA) metabarcoding on 550 seawater samples that were collected twice a month from 11 coastal sites for 2 years in the Boso Peninsula, Japan, and analyzed eDNA monitoring data using nonlinear time series analytical tools. We detected fish-fish interactions as information flow between eDNA time series, reconstructed interaction networks for the top 50 frequently detected species, and quantified pairwise, fluctuating interaction strengths. Although there was a large variation, water temperature influenced fish-fish interaction strengths. The impact of water temperature on interspecific interaction strengths varied among fish species, suggesting that fish species identity influences the temperature effects on interactions. For example, interaction strengths that Halichoeres tenuispinis and Microcanthus strigatus received strongly increased with water temperature, while those of Engraulis japonicus and Girella punctata decreased with water temperature. An increase in water temperature induced by global climate change may change fish interactions in a complex way, which consequently influences marine community dynamics and stability. Our research demonstrates a practical research framework to study the effects of environmental variables on interaction strengths of marine communities in nature, which would contribute to understanding and predicting natural marine ecosystem dynamics.

The world's oceans are home to tens of thousands of fish species, many of which live in nutrient-rich coastal waters. Different species living in a particular environment interact with each other in many ways. For example, a predatory fish may prey on some species of small fish but avoid feeding on others that help it by removing parasites from its skin. Rising ocean temperatures caused by global climate change could affect how different fish species interact with one another and, as a result, impact their communities. One of the first steps to understanding how fish interact with each other in nature typically requires researchers to count the number of different species present and observe how they behave, which is time-consuming and labor-intensive. An alternative is to use an emerging technique in which researchers extract DNA from water, soil or air ­ known as environmental DNA ­ and analyze it to identify the species present and estimate their numbers. Ushio et al. analyzed hundreds of samples of seawater that had been collected over a two-year period from the Boso Peninsula in Japan. Statistical methods were used to quantify how strongly fish species interact with each other and determine whether the temperature of the water influenced how different species of fish interacted over time. The findings showed that water temperature had a significant but complex effect on how strongly pairs of fish species interacted, with both positive and negative effects depending on the conditions. The impact of water temperature on the strength of the interactions varied between species, for example, Japanese anchovy and largescale blackfish interacted less strongly with other fish species in warmer water, whereas the Stripey and a species of wrasse interacted with other fish species more strongly. The findings provide new insights into how water temperature affects the communities of fish living in coastal areas. Alongside complementing existing knowledge in the field, refining the research framework used in this work will benefit those working in fishery science by providing valuable insights into how natural and commercially important fish species respond to climate change.

Intraspecific genetic variability and diurnal activity affect environmental DNA detection in Japanese eel.

Environmental DNA (eDNA) analysis with species-specific primer/probe sets is promising as a tool to quantify fish abundance and distribution. Nevertheless, several factors could reduce the accuracy of this method. Here, we aimed to analyze whether intraspecific variability and diel activity rhythm affect eDNA detection in Japanese eels (Anguilla japonica). For this purpose, we performed tank experiments focusing on two points. First, we assessed the effects of base pair sequences with probe region polymorphism on eDNA detection. Next, we evaluated the influences of diel rhythm, activity, and individual differences in eDNA release rate on eDNA concentration. We examined the base pair sequences of the probe regions of 20 individuals and found genetic mismatches in two of them. The eDNA concentration was estimated to be much lower in these variants than it was in the other individuals. We conducted a rearing experiment on four non-variant individuals to explore the influences of diel activity and inter-individual differences in eDNA detection. Nocturnal eel activity was reflected in the eDNA detection but the inter-individual differences remained large. The observed weak positive correlations between eDNA concentration and activity suggest that eDNA emission is highly dependent on basal metabolism. The present study suggests that consideration of polymorphic sites at the probe region and diel activity rhythms should improve the accuracy and precision of abundance estimation through eDNA. Such fine-tuning is applicable not only for eels but also for other fishes to be targeted by eDNA technology.

Environmental DNA preserved in marine sediment for detecting jellyfish blooms after a tsunami.

Environmental DNA (eDNA) can be a powerful tool for detecting the distribution and abundance of target species. This study aimed to test the longevity of eDNA in marine sediment through a tank experiment and to use this information to reconstruct past faunal occurrence. In the tank experiment, juvenile jack mackerel (Trachurus japonicus) were kept in flow-through tanks with marine sediment for two weeks. Water and sediment samples from the tanks were collected after the removal of fish. In the field trial, sediment cores were collected in Moune Bay, northeast Japan, where unusual blooms of jellyfish (Aurelia sp.) occurred after a tsunami. The samples were analyzed by layers to detect the eDNA of jellyfish. The tank experiment revealed that after fish were removed, eDNA was not present in the water the next day, or subsequently, whereas eDNA was detectable in the sediment for 12 months. In the sediment core samples, jellyfish eDNA was detected at high concentrations above the layer with the highest content of polycyclic aromatic hydrocarbons, reflecting tsunami-induced oil spills. Thus, marine sediment eDNA preserves a record of target species for at least one year and can be used to reconstruct past faunal occurrence.

Tropical fishes vanished after the operation of a nuclear power plant was suspended in the Sea of Japan.

Thermal discharge from a nuclear power plant (NPP) provides an opportunity to foresee changes in faunal communities that may be induced by ocean warming. I assessed these changes by identifying characteristics of the fish community near the thermal discharge from a NPP and by recording temporal changes that occurred after the suspension of the NPP. Underwater visual censuses were conducted near Takahama NPP in the Sea of Japan, and fish assemblages were compared to those in two other sites: a site with discharge from a coal-fired power plant and a control site. During the surveyed period (8 years) when the NPP was in operation, the sea water temperature at the site near the NPP was warmer, had a significantly higher fish abundance, and a higher species richness, including tropical fishes, than the other two sites. However, once the NPP was suspended, tropical fishes dramatically decreased near the NPP. This abrupt change in fish assemblage may be due to the lowest lethal temperatures of tropical fishes being only slightly higher than the winter temperature in this area. Relatively poor ecosystem structure in the local warming area may also have contributed to low resilience of tropical fish species to this temperature change.

Selective collection of long fragments of environmental DNA using larger pore size filter.

Environmental DNA (eDNA) can exist in water with various sizes and states. Among them, relative to extra-cellular DNA, intra-cellular DNA such as cell and tissue fragments can mainly be detected at larger size fractions, and may be protected from enzymatic DNA degradation processes. Here, we verified the hypothesis that the selective collection of such large-sized eDNA enhances the efficiency of capturing less-degraded eDNA, based on a tank experiment using Japanese Jack Mackerel (Trachurus japonicus) as a model species. We concentrated different volumes of rearing water using the filters with different pore sizes (0.7 µm and 2.7 µm), and quantified the copy number of short and long mitochondrial and short nuclear DNA fragments of target species in water samples. As a result, the ratio of long to short eDNA concentrations was higher in the larger pore size filter, which would support our stated hypothesis. In addition, the ratio of nuclear to mitochondrial eDNA was lower in the larger pore size filter. These results imply a difference in the persistence of nuclear and mitochondrial DNA between intra- and extra-cellular environments. Moreover, larger filter pore size did not necessarily decrease the yields of eDNA, and there was little difference in yields in smaller filtration volumes. The findings of this study indicate the potential to select information from eDNA signals by focusing on eDNA of specific size and state, which may contribute to efficient utilization of the information on species taxonomy and physiology in water samples.

Comparing the efficiency of open and enclosed filtration systems in environmental DNA quantification for fish and jellyfish.

Water sampling and filtration of environmental DNA (eDNA) analysis have been performed by several different methods, and each method may yield a different species composition or eDNA concentration. Here, we investigated the eDNA of seawater samples directly collected by SCUBA to compare two widely used filtration methods: open filtration with a glass filter (GF/F) and enclosed filtration (Sterivex). We referred to biomass based on visual observation data collected simultaneously to clarify the difference between organism groups. Water samples were collected at two points in the Sea of Japan in May, September and December 2018. The respective samples were filtered through GF/F and Sterivex for eDNA extraction. We quantified the eDNA concentration of five fish and two cnidarian species by quantitative polymerase chain reaction (qPCR) using species-specific primers/probe sets. A strong correlation of eDNA concentration was obtained between GF/F and Sterivex; the intercepts and slopes of the linear regression lines were slightly different in fish and jellyfish. The amount of eDNA detected using the GF/F filtration method was higher than that detected using Sterivex when the eDNA concentration was high; the opposite trend was observed when the eDNA concentration was relatively low. The concentration of eDNA correlated with visually estimated biomass; eDNA concentration per biomass in jellyfish was approximately 700 times greater than that in fish. We conclude that GF/F provides an advantage in collecting a large amount of eDNA, whereas Sterivex offers superior eDNA sensitivity. Both filtration methods are effective in estimating the spatiotemporal biomass size of target marine species.

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.