External Fertilization Research Articles

A test for plasticity in sperm motility activation in response to osmotic environment in an anuran amphibian.

Evolutionary theory predicts that selection will favor phenotypic plasticity in sperm traits that maximize fertilization success in dynamic fertilization environments. In species with external fertilization, osmolality of the fertilization medium is known to play a critical role in activating sperm motility, but evidence for osmotic‐induced sperm plasticity is limited to euryhaline fish and marine invertebrates. Whether this capacity extends to freshwater taxa remains unknown. Here, we provide the first test for plasticity in sperm‐motility activation in response to osmotic environment in an anuran amphibian. Male common eastern froglets (Crinia signifera) were acclimated to either low (0 mOsmol kg−1) or high (50 mOsmol kg−1) environmental osmolality, and using a split‐sample experimental design, sperm were activated across a range of osmolality treatments (0, 25, 50, 75, 100, and 200 ± 2 mOsmol kg−1). Unexpectedly, there was no detectable shift in the optimal osmolality for sperm‐motility activation after approximately 13 weeks of acclimation (a period reflecting the duration of the winter breeding season). However, in both the low and high acclimation treatments, the optimal osmolality for sperm‐motility activation mirrored the osmolality at the natural breeding site, indicating a phenotypic match to the local environment. Previously it has been shown that C. signifera display among‐population covariation between environmental osmolality and sperm performance. Coupled with this finding, the results of the present study suggest that inter‐population differences reflect genetic divergence and local adaptation. We discuss the need for experimental tests of osmotic‐induced sperm plasticity in more freshwater taxa to better understand the environmental and evolutionary contexts favoring adaptive plasticity in sperm‐motility activation.

The Development and Application of Opto-Chemical Tools in the Zebrafish.

The zebrafish is one of the most widely adopted animal models in both basic and translational research. This popularity of the zebrafish results from several advantages such as a high degree of similarity to the human genome, the ease of genetic and chemical perturbations, external fertilization with high fecundity, transparent and fast-developing embryos, and relatively low cost-effective maintenance. In particular, body translucency is a unique feature of zebrafish that is not adequately obtained with other vertebrate organisms. The animal’s distinctive optical clarity and small size therefore make it a successful model for optical modulation and observation. Furthermore, the convenience of microinjection and high embryonic permeability readily allow for efficient delivery of large and small molecules into live animals. Finally, the numerous number of siblings obtained from a single pair of animals offers large replicates and improved statistical analysis of the results. In this review, we describe the development of opto-chemical tools based on various strategies that control biological activities with unprecedented spatiotemporal resolution. We also discuss the reported applications of these tools in zebrafish and highlight the current challenges and future possibilities of opto-chemical approaches, particularly at the single cell level.

Enhanced suppression of saprotrophs by ectomycorrhizal fungi under high level of nitrogen fertilization

Ectomycorrhizal fungi (EMF) are widespread in northern conifer forests. By competing with the free-living saprotrophic fungal and bacterial communities for limited soil nitrogen, EMF are expected to suppress litter decomposition and thus drive soil carbon accumulation. The EMF may also stimulate rhizosphere microbial growth through inputs of labile plant carbon, and subsequently contribute to the soil carbon pool via microbial necromass. Here we examined the relative strength of these two potential EMF effects in a northern conifer plantation of the Saihanba Forest, the largest plantation in China. The soil fungal and bacterial biomass, as well as their respiration, were quantified within the two types of soil cores that either allow or exclude the ingrowth of EMF. We also set up a nitrogen fertilization gradient (0, 5, 10, 15 g N m–2 y–1) in this plantation to quantify the influence of external inorganic nitrogen on the EMF effects. We found evidence that EMF inhibit the overall fungal and bacteria biomass, confirming the suppression of saprotrophs by EMF. In addition, high levels of external nitrogen fertilization (15 g N m–2 y–1) might further enhance the suppression by EMF. In contrast, the presence of EMF consistently increased soil microbial respiration across all nitrogen fertilization levels, indicating that the carbon allocated to EMF could have been largely consumed by microbial respiration and contributed minimally to the accumulation of microbial biomass. Our results also indicated that the suppression of saprotrophs by EMF may play a critical role in driving continuous soil carbon accumulation in this northern pine plantation under atmospheric nitrogen deposition.

The influence of salinity on the toxicity of chemical UV-filters to sperms of the free-spawning mussel Mytilus galloprovincialis (Lamark, 1819)

Marine-coastal systems have been increasingly exposed to multiple stressors, including anthropogenic pollution and variations of Climate Change (CC) related factors, whose coexistence could create associated environmental and ecotoxicological risks. Among emergent stressors, 4-methylbenzylidenecamphor (4-MBC) and benzophenone-3 (BP-3) UV-filters are compounds widely used in increasing consumer products, resulting in their ubiquity in aquatic environments and possible pressing challenges on gamete susceptibility. Since most marine invertebrates reproduce by external fertilization, after spawning, gametes may be exposed to several pressures, affecting reproductive success and outcome. The present study focuses on the spermiotoxicity of the environmentally relevant UV-filters 4-MBC and BP-3 combined with salinity shifts, as potential modulators of their harmful effects. For this, Mytilus galloprovincialis male gametes were exposed in vitro to environmentally relevant and slightly higher concentrations (1, 10 and 100 µg/L) of 4-MBC or BP-3 under three different salinities (S 20, 30 and 40). Sperm quality endpoints associated with oxidative status, viability, motility, kinetics, and genotoxicity were evaluated. Similarities and differences in sperm responses among all conditions were highlighted by principal coordinates analysis (PCO). Results showed that salinity acting alone posed greater sperms impairments at the lowest (20) and highest (40) tested levels. When salinity acts as a co-varying stressor, salinity-dominant interactive effects resulted evident, especially for 4-MBC at S 40 and BP-3 at S 20. These findings were pointed out as the worst exposure conditions for M. galloprovincialis sperms, since caused major toxicological effects in terms of: (I) oxidative stress, sperm structural impairments, motility and kinetic alterations in 4-MBC-exposed sperms; (II) DNA damage, compromised mitochondrial activity and hyperactivation in BP-3-exposed ones. Overall, it stands out that salinity influences UV-filter toxicological pathways and, thereby, the potential environmental risk of these contaminants on M. galloprovincialis male gametes, especially in an expected salinity stress scenario.

The Need for a Framework Addressing the Temporal Aspects of Fish Sperm Motility Leading to Community-Level Standardization.

Motility is a widely available parameter that can be used to assess sperm quality of aquatic species. Sperm from fishes with external fertilization usually undergo a dynamic and short-lived period of motility after activation. The common practice of assigning a single value at an arbitrary peak of motility presents challenges for reproducibility, community-level standardization, and comparisons across studies. This study aimed to explore statistical approaches to standardize motility reporting, and to develop an initial framework for community-level standards. Sperm samples from 14 zebrafish (Danio rerio) with a total of 21,705 cells were analyzed by use of computer-assisted sperm analysis with data collection starting at 10 s after activation at 5-s intervals for 50 s. Four common motility variables were selected for analyses: curvilinear velocity, straight-line velocity, beat cross frequency, and amplitude of lateral head displacement. Cluster analysis was used to evaluate sperm subpopulations within and among males over time, least-square means was used to explore temporal aspects, and the first derivative of the regression equations was used to calculate the rate of change for the motility parameters. Cluster analysis proved informative, but overlapping ephemeral clusters were not valuable for providing standardization options. Analysis of temporal aspects and rate of change indicated opportunities for standardization by reporting the overall motility-time functions or reporting during stable time windows instead of peak motility or at random times. These approaches could minimize the inconsistencies caused by male-to-male variation and dynamic changes of subpopulations while providing comparable information. An overall temporal framework was identified for motility reporting along the collection-processing-cryopreservation-thawing sequence to provide a basis to support efforts of community-level standardization.

Broodstock exposure to warming and elevated pCO2 impairs gamete quality and narrows the temperature window of fertilisation in Atlantic cod.

Impacts of global warming and CO2 -related ocean acidification (OA) on fish reproduction may include chronic effects on gametogenesis and gamete quality, as well as acute effects on external fertilisation. Here, temperature thresholds and OA-sensitivity of gametogenesis and fertilisation were investigated in Atlantic cod, Gadus morhua. Three broodstock groups of farmed cod (FC 1-3) were exposed for 3 months to three maturation conditions (FC 1: control, 6°C/400 μatm CO2 ; FC 2: warming, 9.5°C/400 μatm; FC 3: warming and OA, 9.5°C/1100 μatm). In addition, a broodstock group of wild cod (WC) was kept at control conditions to compare the acute temperature window of fertilisation with that of farmed cod (FC 1). Fertilisations were conducted in a temperature-gradient table at 10 temperatures (between -1.5 and 12°C) and two CO2 levels (400/1100 μatm). In FC 1 and WC, fertilisation success was relatively high between 0.5°C and 11°C (TRange of c. 10.5°C), indicating similar gamete quality in farmed and wild broodstocks kept at control conditions. Exposure of farmed broodstocks to warming (FC 2) and the combination of warming and OA (FC 3) impaired gamete quality, causing a reduction in fertilisation success of -20% (FC 2) and - 42% (FC 3) compared to FC 1. The acute temperature window of fertilisation narrowed from FC 1 (TRange =10.4°C) to FC 2 (TRange =8.8°C) and FC 3 (TRange =5.9°C). Acute effects of CO2 on fertilisation success were not significant. This study demonstrates potential climate change impacts on gametogenesis and fertilisation in Atlantic cod, suggesting the loss of spawning habitat in the coming decades.

Contrasting reproductive strategies of two Hawaiian Montipora corals

Sessile invertebrates often engage in synchronized spawning events to increase likelihood of fertilization. Although coral reefs are well studied, the reproductive behavior of most species and the relative influence of various environmental cues that drive reproduction are not well understood. We conducted a comparative examination of the reproduction of the well-studied Hawaiian coral Montipora capitata and the relatively unknown reproduction of its congener, Montipora flabellata. Both are simultaneous hermaphroditic broadcast spawners that release egg-sperm bundles with external fertilization. Montipora capitata had a distinct reproductive pattern that resulted in coordinated gamete maturation and the synchronized release of thousands of egg-sperm bundles across two spawning pulses tightly coupled to consecutive new moon phases in June and July. Montipora flabellata exhibited a four month reproductive season with spawning that was four-fold less synchronous than M. capitata; its spawning was aperiodic with little linkage to moon phase, a broadly distributed release of only dozens or hundreds of bundles over multiple nights, and a spawning period that ranged from late June through September. The reproductive strategy of M. flabellata might prove detrimental under climate change if increased frequency and severity of bleaching events leave it sparsely populated and local stressors continue to degrade its habitat.

CRISPR/Cas Genome Editing—Can It Become a Game Changer in Future Fisheries Sector?

Fisheries and aquaculture are the fastest-growing food-producing sector and rapidly becoming an important element for the global food security since they are the primary source of seafood and high animal protein in the human diet. Genome editing offers new possibilities such as the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) technology, which has the potential to accelerate the sustainable genetic improvement in fisheries and aquaculture. The CRISPR/Cas9 system has four key components, namely, target DNA, Cas9, the protospacer adjacent motif sequence, and the guide RNA or single-guide RNA. CRISPR/Cas is cheaper, easier, and more precise than the other genome editing technologies and can be used as a new breeding technology in fisheries and aquaculture to solve the far-reaching challenges. The attributes like high fecundity, external fertilization, short generation interval, the established method of breeding, and the larval rearing of most aquaculture species have advantages for CRISPR/Cas9 genome editing applications. CRISPR/Cas9 has recently been applied to the traits valued in some aquaculture species (almost >20 species), targeting the main traits of traditional genetic improvement initiatives like growth, disease resistance, reproduction, sterility, and pigmentation. Genome editing can fast forward the breeding process with precision where changes occur in the targeted genes. The probability of desired changes occurring and passing the trait in the next generation is high, so it takes 1-3 generations to establish a breed. Moreover, CRISPR/Cas genome editing rapidly introduces favorable changes by disrupting genes with targeted minor changes, in contrast to transgenesis, which introduces foreign genes into the host genome and thereby alleviates major public concerns on safety. Although the CRISPR/Cas technology has a tremendous potential, there are several technical challenges and regulatory and public issues concerning the applications in fisheries and the aquaculture breeding sector. Nonetheless, the exciting point in the CRISPR/Cas9 genome editing is that two CRISPR-edited fish, namely, red sea bream and tiger puffer developed by the Kyoto-based startup got approval and are now on the market for sale, and another fish, FLT-01 Nile tilapia developed by the AquaBounty, is not classified under genetically modified organism regulatory. However, there is still a way to go before it revolutionizes and becomes viable in commercial aquaculture as the new breeding technology for aquaculture-important traits and species.

Effect of Maize High Density Planting on Cluster Bean Performance under Maize-cluster Bean Cropping Sequence

Background: Cluster bean was cultivated as a catch crop without external fertilization, solely to recharge the soil fertility and to harvest the residual nutrient after maize crop; clusterbean had been included as a succeeding crop. Doubling the fertilizer for maize would leave more residues. Even though maize is an exhaustive crop, since the nutrient uptake was need based, the excess fertilizers were effectively utilized by cluster bean. Methods: A field research was carried out at Tamil Nadu Agricultural University, Coimbatore under irrigated conditions. Experimental trials were adopted to estimate the effect of high density with nutrient management practices on maize which influences the performance of succeeding cluster bean under maize-cluster bean cropping sequence during Rabi seasons of 2017-18 and 2018-19 by using cultivar Pusa Naubahar. No separate treatments were adapted to cluster bean crop. Result: The results of cluster bean sown after the high density of 1,33,333 plants ha-1 maize with nutrient level of 200 per cent RDF produced taller plants, higher drymatter production (636 and 607 kg/ha), increased yield attributes, higher yield (6.58 and 6.81 t/ha) and better economics (B:C ratio -1.87 and 1.94) in both the seasons. Post-harvest available nutrient status revealed that the higher population by closer spacing with increased fertilizer levels (T7) maintained its superiority during both the years. Moreover, this proved to be the most viable practice for production and residual nutrient uptake, by including pulse (cluster bean) as succeeding crop and soil health was effectively maintained by fixing atmospheric nitrogen by cluster bean.

Bateman gradients from first principles

In 1948, Angus Bateman presented experiments and concepts that remain influential and debated in sexual selection. The Bateman gradient relates reproductive success to mate number, and Bateman presented this as the cause of intra-masculine selection. A deeper causal level was subsequently asserted: that the ultimate cause of sex differences in Bateman gradients is the sex difference in gamete numbers, an argument that remains controversial and without mathematical backup. Here I develop models showing how asymmetry in gamete numbers alone can generate steeper Bateman gradients in males. This conclusion remains when the further asymmetry of internal fertilisation is added to the model and fertilisation is efficient. Strong gamete limitation can push Bateman gradients towards equality under external fertilisation and reverse them under internal fertilisation. Thus, this study provides a mathematical formalisation of Bateman’s brief verbal claim, while demonstrating that the link between gamete number and Bateman gradients is not inevitable nor trivial.

The taxonomic position of brooding limpets of the genera Erginus and Rhodopetala (Patellogastropoda)

Limpets of the genera Erginus Jeffreys, 1877 (previously in Lottiidae) and Rhodopetala Dall, 1921 (previously in Acmaeidae), unlike other patellogastropods, brood larvae and juveniles in the modified pallial cavity. A phylogenetic analysis using three mitochondrial gene markers (cytochrome c oxidase subunit I, 16S rRNA, and 12S rRNA) has shown that Rhodopetala rosea (Dall 1872) and four Erginus species form a highly supported clade that cannot be assigned to Acmaeidae or Lottiidae. The transition from external fertilization and pelagic development to internal fertilization with brooded larvae and juveniles in the special pallial cavity occurred in the evolution of patellogastropods only once. The genera Erginus and Rhodopetala belong to Rhodopetalidae Lindberg, 1981 stat. nov. (= Erginidae Chernyshev 2018 syn. nov.) elevated from subfamily Rhodopetalinae to family status. Erginus puniceus Lindberg, 1988a is restored from synonym of Erginus sybariticus (Dall 1871) for both typical purple forms and the ‘sybariticus’ forms with small shell.

Mating system in a small pelagic spawner: field case study of the mandarinfish, Synchiropus splendidus

Mating systems in fishes are extremely diverse, ranging from monogamy to multiple polygamous forms, some of which include alternative male mating tactics within species. The major reproductive mode across approximately 33,000 bony fishes is pelagic-spawning with external fertilization. Despite the dominance of this mode, it is relatively little studied in the field because the greater mobility of larger species typically makes individuals difficult to follow for extensive periods, or to catch and mark. However, smaller, more sedentary pelagic spawners provide excellent opportunities to examine mating system variability in response to environmental conditions. The mandarinfish, Synchiropus splendidus, is a small sedentary pelagic spawner which is accessible to divers. Its complex body markings are distinctive and variable allowing individuals to be identified and followed repeatedly without the need for capturing and marking fish. In over 400 h of underwater surveys, we recorded movements, inter- and intra-sexual behaviors, sex ratios, and the timing and duration of spawning activity of focal fish in predetermined study areas. We concluded that the mandarinfish has a polygamous mating system with pair-spawning. Females are sequentially polyandrous, spawning once nightly or not at all, while males are polygynous, spawning up to 8 times a night. Males and females use loosely defined spawning sites on a daily basis and show flexibility in their tactics through differential movements according to dispersion of individuals, distances between spawning sites and temporally limited daily female receptivity. These results reveal the mandarinfish to be an excellent model species for studying small pelagic spawners in the field.

Managing Land Carrying Capacity: Key to Achieving Sustainable Production Systems for Food Security

Many previous studies have estimated the carrying capacity and feasible planetary boundaries for humankind. However, less attention has been given to how we will sustainably feed 9 billion people in 2050 and beyond. Here, we review the major natural resources that limit food production and discuss possible options, measures, and strategies to sustainably feed a human population of 9 billion in 2050 and beyond. Currently, food production greatly depends on external inputs, e.g., irrigation water and fertilizers, but these approaches are not sustainable. Due to the unbalanced distribution of global natural resources and large regional differences, urbanization expansion causes important areas to face more serious arable land resource shortages. Hence, sustainably feeding 9 billion people in 2050 and beyond remains an immense challenge for humankind, and this challenge requires novel planning and better decision-making tools. Importantly, the measures and strategies employed must be region-/country-specific because of the significant differences in the socioeconomic characteristics and natural environmental carrying capacity in different parts of the world. Considering the impact of unexpected extreme events (e.g., a global pandemic and war) in the future, the food trade and translocation of goods will also face challenges, and the strategies and decision-making processes employed must consider the possible influences at both regional and global scales.

Reproductive adaptations of sea urchins with planktotrophic larvae

The brief review summarizes the authors' original data on the role of environmental factors in triggering and synchronization of spawning in natural populations of two species of sea urchins with planktotrophic larvae. It is shown that both species exhibit similar reproductive adaptations aimed at enhancing reproductive success. The high sensitivity of sea urchins, primarily males, to environmental signal(s), most likely to phytoplankton, can be considered as a large-scale adaptation characteristic of many marine invertebrates with external fertilization and planktotrophic larvae and promoting the development of mass spawning events. Night time, as well as the new moon and full moon phases, are modulating factors that increase the likelihood of mass spawning. Longer duration of spawning in males compared to females, longer duration of sperm release during mass spawning compared to that during solitary spawning, longer duration of spawning in males living in an environment with higher level of phytoplankton, and approach of males and females during mass spawning can be considered as small-scale adaptations that increase the likelihood of fertilization.

The impact of time and environmental factors on the mitochondrial vesicle and subsequent motility of amphibian sperm

For amphibian species that display external fertilization in an aquatic environment, hypoosmotic shock to sperm cells can quickly result in damage to cellular structure and function. This study sought to determine how fertilization media osmolality, temperature, and time impact the stability of the mitochondrial vesicle's association with the sperm head and thus motility and quality of forward progression. The presence of the mitochondrial vesicle and its relationship with sperm motility and quality of forward progression were analyzed in sperm samples from the Fowler's toad (Anaxyrus fowleri) (n = 10) when held for six hours under two temperatures and four osmolalities. Results indicated that the presence of the mitochondrial vesicle is needed for sperm motility over time (p < 0.001), where higher osmolalities (p < 0.001) and lower temperatures (p < 0.001) correlated with maintaining the vesicle attachment to the spermatozoa. The higher osmolality of spermic urine was the most important factor for maintaining higher quality of forward progressive motility (p < 0.01) of spermatozoa. Sperm samples held at 4 °C and 40 mOsm/kg had the longest half-life for motility (540 min) and quality of forward progression (276 min), whereas sperm held at 22 °C and 2.5 mOsm/kg had the shortest half-life for motility (7 min) and quality of forward progression (18 min). Special attention should be given to the osmolality and temperature of fertilization solutions, or breeding tank water, when developing cold storage protocols for toad sperm or reproducing animals to ensure the retention of the mitochondrial vesicle for maximum fertilization capability.

Developmental life stages of the Pickersgill's reed frog (Hyperolius pickersgilli) in an ex-situ environment at Johannesburg Zoo's captive breeding facility, South Africa.

Globally, the threats of habitat loss and disease on amphibian survival have necessitated the creation of ex-situ insurance populations as a conservation tool. We initiated a captive breeding project to create an insurance population for the endangered Pickersgill's reed frog (Hyperolius pickersgilli Raw, 1982) at the Johannesburg Zoo from parents collected from KwaZulu-Natal Province, South Africa, in 2017. We found that this species has seven developmental life stages, each with unique management requirements. The quiescent tadpoles hatched 6-8 days after the eggs were laid and remained at this stage for 2 days. The next stage, the developing tadpoles, showed no form of cannibalism or carrion feeding. The external appearance of the first leg (the right hind) occurred 5-6weeks after the tadpoles hatched, and the metamorph stage was reached after 7-8 weeks. The metamorph stage lasted 3-5 days, after which tail resorption was complete and the froglet stage reached. Froglets could not be sexed externally, although body color changed based on the amount of light present at the resting place. Sub-adults were 6 months and older with adult coloration and sex differentiation visible even with color change. Adults were older than 18 months and fully developed and sexually mature, displaying amplexus, oviposition, and external fertilization. A greater understanding of Pickersgill's reed frog's developmental stages and physiological and environmental needs can improve captive breeding and subsequent release of the frogs, facilitate captive breeding elsewhere, and improve the species' conservation status.

Atypical Centriolar Composition Correlates with Internal Fertilization in Fish.

The sperm competition theory, as proposed by Geoff Parker, predicts that sperm evolve through a cascade of changes. As an example, internal fertilization is followed by sperm morphology diversification. However, little is known about the evolution of internal sperm structures. The centriole has an ancient and evolutionarily conserved canonical structure with signature 9-fold, radially symmetric microtubules that form the cell’s centrosomes, cilia, and flagella. Most animal spermatozoa have two centrioles, one of which forms the spermatozoan flagellum. Both are delivered to the egg and constitute the embryo’s first two centrosomes. The spermatozoa of mammals and insects only have one recognizable centriole with a canonical structure. A second sperm centriole with an atypical structure was recently reported in both animal groups and which, prior to this, eluded discovery by standard techniques and criteria. Because the ancestors of both mammals and insects reproduced by internal fertilization, we hypothesized that the transition from two centrioles with canonical composition in ancestral sperm to an atypical centriolar composition characterized by only one canonical centriole evolved preferentially after internal fertilization. We examined fish because of the diversity of species available to test this hypothesis–as some species reproduce via internal and others via external fertilization–and because their spermatozoan ultrastructure has been extensively studied. Our literature search reports on 277 fish species. Species reported with atypical centriolar composition are specifically enriched among internal fertilizers compared to external fertilizers (7/34, 20.6% versus 2/243, 0.80%; p < 0.00001, odds ratio = 32.4) and represent phylogenetically unrelated fish. Atypical centrioles are present in the internal fertilizers of the subfamily Poeciliinae. Therefore, internally fertilizing fish preferentially and independently evolved spermatozoa with atypical centriolar composition multiple times, agreeing with Parker’s cascade theory.

Defaunação de peixes em reservatórios do baixo rio Paranapanema, Brasil

Abstract Until now no study has used a defaunation index to quantify the decline of Neotropical freshwater fishes in environments fragmented by dams and reservoirs. So, we applied this index to 143 native fish in five reservoirs in the Lower Paranapanema River, that is situated in one of the Brazilian aquatic environments most impacted by anthropic degradation. Fish species were classified according to their functional groups, which were selected according to the biological characteristics that may reflect in defaunation events. The biggest reservoir in area with more tributaries and forest cover showed lowest defaunation index. The functional groups of fishes more affected by defaunation included species characterized by periphytivores, invertivores and algivores, non-migratory habit, with external fertilization, and parental care. Although reservoirs have different characteristics, this method can be tested in any other hydrographic basin. The results suggested continued conservation efforts to preserve the integrity of tributaries and the native fishes in reservoirs and pointed out the importance of maintaining native vegetation cover and fish restocking programs in the reservoirs with the highest defaunation values. Our finding can be use as the first data source for future studies using this defaunation index.

Illegal use of fertilized eggs outside the womb (A comparative study)

Illegal use of fertilized eggs outside the womb (A comparative study)

Environmental DNA detects a possible Japanese eel spawning event near a video-recorded anguillid eel in the open ocean

Facultative catadromous eels migrate back to the sea to reproduce, but their spawning behavior and locations have remained elusive. Using environmental DNA (eDNA), we identified a likely spawning site location and time of spawning of the Japanese eel. We detected Japanese eel eDNA at 400 and 600 m and recorded a likely sighting of this species at about 220 m using a deep-tow camera system 6 d before the new moon. A strong eDNA signal was obtained at 400 m from the apparent spawning event the morning after the estimated peak of eel spawning, 3 d before the new moon. These findings indicate that Japanese eels were already within the area where they were going to spawn at least 6 d before the new moon and then may have spawned near the strong eDNA station 3 d before the new moon. We concluded that the eDNA analysis is useful in searching for spawning sites and determining the timing of spawning of aquatic organisms with external fertilization that causes a temporary surge in eDNA, although prior knowledge of likely spawning sites is needed.