Vitrification protocol for immature Brycon orbignyanus ovarian tissue as an extinction escape strategy.

Piracanjunba (Brycon orbignyanus) is an endangered South American fish, and ovarian tissue cryopreservation is an alternative method for preserving maternal germplasm and genetic diversity. Therefore, our aim was to test a vitrification protocol for ovarian tissue containing primary growth (PG) oocytes of B. orbignyanus as a strategy to avoid the threat of extinction. Two vitrification solutions were evaluated (VS1: 1.5 M methanol + 4.5 M propylene glycol and VS2: 1.5 M methanol + 5.5 M Me2SO) and compared using control/fresh ovarian tissue. After vitrification, the following factors were analyzed: membrane integrity using trypan blue, morphology using a histological assessment, oxidative stress (total reactive antioxidant potential (TRAP) and reduced thiol [-SH]), mitochondrial activity using MTT, and DNA damage using a comet assay. The vitrified oocytes (VS1 = 24.3 ± 0.49% and VS2 = 24.8 ± 0.69%) showed higher DNA damage than the control group (control = 20.7 ± 1.03%) (P = 0.004). In contrast, in most evaluations (membrane integrity, membrane damage, oxidative stress, and mitochondrial activity), there were no discernible differences between the control group and the vitrified samples. In addition, oocyte (P = 0.883) and nuclear diameter (P = 0.118) did not change after vitrification. VS2 treatment resulted in higher nuclear damage (15.7 ± 1.45%) than in the control treatment (3.5 ± 1.19%); however, VS1 treatment did not result in significantly more damage (9.5 ± 3.01%) than in the control (P = 0.015). Therefore, the protocol for ovarian tissue vitrification tested in this study resulted in high maintenance of PG oocyte cell integrity, making it a promising alternative for B. orbignyanus maternal genome preservation.

Developmental Staging and Salinity Tolerance in Embryos of the Delta Smelt, Hypomesus transpacificus.

Delta smelt (Hypomesus transpacificus) is a critically endangered species endemic to the San Francisco Bay Delta (SFBD). Important for the conservation of this species is understanding the physiological and ecological impacts contributing to their population decline, and current studies lack information on embryonic development. Changes in patterns of salinity across the SFBD may be a particularly important environmental stressor contributing to the recruitment and survival of the species. Throughout their ontogeny, delta smelt may exhibit unique requirements and tolerances to environmental conditions including salinity. Here, we describe 22 stages of embryonic development of H. transpacificus that characterize early differentiation from the fertilized egg until hatching, allowing the identification of critical morphological features unique to this species. Additionally, we investigated aspects of physiological tolerance to environmental salinity during development. Embryos survived incubation at salinity treatments between 0.4 and 20 ppt, yet had lower hatch success at higher salinities. Prior to hatching, embryos exposed to higher salinities had increased osmolalities and reduced fractions of yolk implying that the elevated external salinity altered the physiology of the embryo and the environment internal to the chorion. Lastly, egg activation and fertilization appear to also be impacted by salinity. Altogether, we suggest that any potential tolerance to salinity during embryogenesis, a common feature in euryhaline teleost species, impacts life cycle transitions into, and out of, embryonic development. Results from this investigation should improve conservation and management practices of this species and further expand our understanding of the intimate relationship between an embryo and its environment.

Out of the pot and into the fire: Explaining the vulnerability of an endangered small headwater stream fish to black-bass Micropterus spp. invasion.

Introduced predatory fishes have had consistently severe consequences for native fishes in stream environments around the world, although the drivers of these effects are often unclear. In the Swartkops River headwaters in South Africa, native Eastern Cape redfin Pseudobarbus afer were always absent from sites occupied by non-native black basses Micropterus salmoides and Micropterus dolomieu, but generally co-occurred with the native predators Anguilla marmorata and Anguilla mossambica. A natural experiment provided by flood-mediated recolonization of black-bass occupied sites by P. afer demonstrated depletion in black-bass invaded sites. Field behavioural observations of P. afer indicated that they foraged among benthic cover during the day, but suspended in open water at night. As the nocturnal A. marmorata and A. mossambica foraged actively within structural cover at night and M. dolomieu and M. salmoides are diurnal or crepuscular predators, P .afer is thus optimized to avoid predation by native anguillid predators and not the functionally unique predatory black basses. The integration of distributional, temporal population dynamics and behavioural data suggests that the severe effects of Micropterus spp. are probably a consequence of prey naïveté and behaviour evolved to evade native predators.

Successful vitrification of whole juvenile testis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens).

Sperm cryopreservation is a valuable conservation method for endangered fish species. Here we report an easy and efficient cryopreservation method for juvenile whole testis by vitrification and successful sperm production from the vitrified whole testis via in vitro spermatogenesis in the critically endangered cyprinid honmoroko (Gnathopogon caerulescens). Juvenile testis (approximately 10 mm in length and 1 mm in width), consisting predominantly of spermatogonia, were aseptically dissected out and adherent fatty and non-testicular tissues were subsequently removed. Then, the testes were rapidly cooled on a nylon mesh by direct immersion in liquid nitrogen after serial exposures to pretreatment solution (PS), containing 2 M ethylene glycol (EG) and 1 M dimethyl sulfoxide (DMSO), for 20 or 30 min and vitrification solution (VS), containing 3 M EG, 2 M DMSO, and 0.5 M sucrose, for 5, 10, or 20 min. The highest survival rate of testicular cells (84.0%) was obtained from testes vitrified by immersion in PS for 20 min and in VS for 10 min. Spermatogonia were recovered from the vitrified testis by dissociation and cell culture produced many haploid sperm. Fertility and developmental competence were confirmed by in vitro fertilization assays. These results indicate that the vitrification of juvenile whole testis provides a new strategy to preserve the genetic resources of endangered fishes without affecting their reproductive population.

Habitat use of the European mudminnow Umbra krameri and association with other fish species in a disconnected Danube side arm.

Fish assemblages along the longitudinal course of an old, disconnected and modified side arm of the Danube floodplain downstream of Vienna, Austria, as well as habitat structure, hydro-morphological and hydro-chemical factors, were investigated in order to analyse the key environmental determinants of the European mudminnow Umbra krameri. Generally, U. krameri was the most abundant species in the system. It occurred in disconnected ditches, ponds and pools with dense reed belts and comparatively low nutrient content, indicating its natural association with marsh habitats. At infrequently disturbed sites it was associated with a small group of stagnophilious and highly specialized species with adaptations to strong oxygen fluctuations. At frequently flooded sites, the species was absent or occurred in low abundances, indicating its adaptation to water bodies in older successional stages and its low competitive power in permanently connected floodplain habitats.

Effects of water temperature and discharge on natural reproduction time of the Chinese sturgeon, Acipenser sinensis, in the Yangtze River, China and impacts of the impoundment of the Three Gorges Reservoir.

Chinese sturgeon, Acipenser sinensis, is a critically endangered anadromous fish species spawning in the Yangtze River of China during October and November. In this study, we analyzed the effects of hydrological factors, such as water temperature and discharge, on the natural reproduction time of the Chinese sturgeon and evaluated the impact of the impoundment of the Three Gorges Reservoir (TGR) based on our survey data from 1998 to 2011. The results showed that the first spawning dates were significantly (P < 0.05) correlated with the date of water temperature reaching 20°C (20°C WT dates), October mean discharge (Oct. discharge), and the discharge change from October to November (Oct-Nov Δdischarge). Regression analysis suggested that one day delay of 20°C WT dates could postpone the first spawning date by 0.673 days. A discharge increase of 1000 m(3)s(-1) in October could bring forward the first spawning date by two days. Our results also indicated that the impoundment of the TGR had delayed the first spawning time due to water temperature lag and flow regulation. We suggest the following ecological regulations in order to facilitate conservation of the Chinese sturgeon: to eliminate water temperature lag by regulating water temperature downstream of the Three Gorges Dam (TGD), to enhance water discharge downstream in October, and to complete impoundment before October.

Aliphatic hydrocarbons in fin spines of adult sturgeon (Acipenser stellatus) and their relationship with potentially toxic elements in the northern and southern regions of the Caspian Sea.

Currently, the pollution of the Caspian Sea by the oil industry is one of the highest problems in this area. Critically endangered species inhabit this sea, such as sturgeons, whose ecological value is incalculable. Thus, we aimed to evaluate the level of contamination of aliphatic hydrocarbons of petroleum and its relation with several toxic elements directly on sturgeons spines. A total of 40 adult starry sturgeons (Acipenser stellatus) were obtained within a repopulation programme in the northern and southern coastal waters of the Caspian Sea. The marginal pectoral fin was extracted from each fish to determine aliphatic hydrocarbons, arsenic, cadmium, mercury, nickel, lead, and vanadium. Subsequently, the sturgeons were released. Clearly, the presence of hydrocarbons was evidenced in all the sampled areas finding higher concentrations in the northern areas (N1 = 1.35 ± 0.4; N2 = 1.65 ± 0.46; N3 = 1.27 ± 0.40; S1 = 0.61 ± 0.22; S2 = 0.85 ± 0.43 mg/kg). Furthermore, to a greater or lesser extent, some toxic elements, mainly Hg and As, have been linked to aliphatic hydrocarbons.

Whole-genome resequencing confirms the genetic effects of dams on an endangered fish Hemibagrus guttatus (Siluriformes: Bagridae): A case study in a tributary of the Pearl River.

Dam construction in riverine ecosystems has fragmented natural aquatic habitats and has altered environmental conditions. As a result, damming has been demonstrated to threaten aquatic biodiversity by reducing species distribution ranges and hindering gene exchange, leading to the inability to adapt to environmental changes. Knowledge of the contemporary genetic diversity and genetic structure of fish populations that are separated by dams is vital to developing effective conservation strategies, particularly for endangered fish species. We chose the Lianjiang River, a tributary of the Pearl River, as a case study to assess the effects of dams on the genetic diversity and genetic structure of an endangered fish species, Hemibagrus guttatus, using whole-genome resequencing data from 63 fish samples. The results indicated low levels of genetic diversity, high levels of inbreeding and decreasing trend of effective population size in fragmented H. guttatus populations. In addition, there were significant genetic structure and genetic differentiation among populations, suggesting that the dams might have affected H. guttatus populations. Our findings may benefit management and conservation practices for this endangered species that is currently suffering from the effects of dam construction.

Toxicity prediction and risk assessment of per- and polyfluoroalkyl substances for threatened and endangered fishes.

Per- and polyfluoroalkyl substances (PFASs) are severely polluted in aquatic environments and can harm aquatic organisms. Due to the limitation of conducting toxicity experiments directly on threatened and endangered (T&E) species, their toxicity data is scarce, hindering accurate risk assessments. The development of computational toxicology makes it possible to assess the risk of pollutants to T&E fishes. This study innovatively combined machine learning models, including random forest (RF), artificial neural network (ANN), and XGBoost, and the QSAR-ICE model to predict chronic developmental toxicity data of PFASs to T&E fishes. Among these, the XGBoost model exhibited superior performance, with R2 of 0.95 and 0.81 for the training and testing sets, respectively. Internal and external validation further confirmed that the XGBoost model is robust and reliable. Subsequently, it was used to predict chronic developmental toxicity data for seven priority PFASs to T&E fishes in the Yangtze River. Acipenseridae fishes (e.g., Acipenser dabryanus and Acipenser sinensis) showed high sensitivity to PFASs, possibly due to their unique lifestyle and physiological characteristics. Based on these data, the predicted no-effect concentration (PNEC) of individual PFASs was calculated, and the risk for T&E fishes in the Yangtze River was assessed. The results indicated that the risk of PFASs to T&E fishes is low (3.85 × 10-9∼8.20 × 10-4), with perfluorohexanoic acid (PFHxA) and perfluorooctanoic acid (PFOA) as the high-risk pollutants. The risk in the middle and lower reaches of the river is higher than in the upper reaches. This study provides a new approach for obtaining chronic toxicity data and conducting risk assessments for T&E species, advancing the protection of T&E species worldwide.

The Impact of Weir Construction in Korea's Nakdong River on the Population Genetic Variability of the Endangered Fish Species, Rapid Small Gudgeon (Microphysogobio rapidus).

Microphysogobio rapidus, an endemic cyprinid fish species found exclusively in Korea, has been identified in only two tributaries of the Nakdong River. The species predominantly occupies the near-gravel bottom waters within shallow sections of the middle and lower reaches of the river, characterized by swift currents. M. rapidus is currently recognized as a critically endangered species due to its distinct habitat preference, as well as the negative impacts of stream dam development and water environment pollution. In this study, we used 10 microsatellite markers to examine the genetic diversity of M. rapidus in the upper Nam (UN), lower Nam (LN), and Deokcheon Rivers (DC) in Korea, with a specific focus on assessment of the impact of dam development. Fish sampled from the UN and LN showed a greater average number of alleles and allelic richness (A = 18.3-18.4, AR = 13.8) compared to those from DC (A = 11.8, AR = 11.5). The observed heterozygosity among the fish examined ranged from HO = 0.748 (LN) to 0.766 (DC). All three fish groups exhibited a significant departure from Hardy-Weinberg equilibrium (HWE) (p < 0.05). Despite having the largest effective population size (Ne = 175 and 157, respectively), the fish sampled from UN and LN showed the highest inbreeding coefficients (FIS = 0.056-0.053, respectively), which were highly significant (p < 0.01). In contrast, the fish sampled from DC exhibited the smallest effective population size (Ne = 61) and showed an inbreeding coefficient close to zero (p > 0.05). BOTTLENECK analysis and estimated M-ratio values (0.341-0.372) revealed indications of past population size reduction in all fish groups examined. No significant genetic differentiation (FST < 0.05) was detected using the DAPC, STRUCTURE, and AMOVA among the fish studied. However, pairwise comparisons of FST between fish sampled from the Nam and Deokcheon Rivers revealed significant values (p < 0.001) ranging from 0.013 to 0.014. In addition, the closest genetic distance (0.026) was observed between UN and LN, while the greatest distance (0.087) was found between UN and DC. Analysis of gene flow rates among the fish examined indicated asymmetrical gene exchange within the Nam River, which was 31.51% in the downstream direction (from UN to LN), with a minimal gene flow rate (0.41%) in the upstream (from LN to UN) direction. The opposite trend was recorded between DC and LN, with a higher gene flow rate (29.74%) in the upstream direction compared to the downstream direction (0.12%). Our study highlighted the importance of implementing long-term conservation efforts focused on maintaining river integrity by removing water barriers such as weirs that impede fish migration and implementing active protection measures, such as aquaculture breeding and reasonable stocking practices, to preserve M. rapidus in the study area.

An Evolutionary Strategy for Spawning Habitat Selection by Pseudopungtungia tenuicorpa.

In this study, we hypothesized that the limited geographical distribution of Pseudopungtungia tenuicorpa in South Korea is closely related to the local abundance of their preferred spawning habitats. Eggs of P. tenuicorpa were most frequently observed in cracks smaller than 5 cm, followed by the nests of other fish, such as Coreoperca herzi. Although eggs of P. tenuicorpa were also found in cracks larger than 6 cm, most of them were consumed or damaged by other fish. However, the number of P. tenuicorpa eggs spawned in cracks smaller than 5 cm remained similar to the number of eggs at the time of initial spawning, even after 9 days. The strong preference of P. tenuicorpa for cracks smaller than 5 cm was found to influence the geographical distribution of P. tenuicorpa in South Korea. In the upper reaches of the Han River, the ratio of cracks smaller than 5 cm is higher than that in the Geum River and in the Nakdong River, which contributes to the stable population growth of P. tenuicorpa there. In the future, management strategies to maintain/restore cracks smaller than 5 cm should be reviewed for the preservation of this endemic fish in South Korea.

Detection of the Endangered Siamese Bat Catfish (Oreoglanis siamensis Smith, 1933) in Doi Inthanon National Park Using Environmental DNA.

Siamese bat catfish (Oreoglanis siamensis Smith, 1993) has been listed as an endangered species, and its abundance has been severely declining due to habitat degradation and overfishing. To establish an appropriate management strategy, it is crucial to gain information about the distribution of this endangered species. As O. siamensis live under rocks in streams, detecting their presence is difficult. Recently, environmental DNA (eDNA)-based detection has been demonstrated to be a valid tool for monitoring rare species, such as O. siamensis. Therefore, this study developed an eDNA assay targeting a 160 bp fragment of the COI region to detect the presence of this species in its natural habitat. An amount of 300 mL of water samples (0.7 µm filtered) were collected from 15 sites in the Mae Klang sub-basin, where this fish species was visually detected at two locations. O. siamensis eDNA was detected at 12 of the 15 sites sampled with varying concentrations (0.71-20.27 copies/mL), including at the sites where this species was visually detected previously. The developed O. siamensis eDNA assay was shown to be effective for detecting the presence of this endangered species in the Klang Phat and Klang Rivers within the Doi Inthanon National Park.

Outbreak of Parasitic Dinoflagellate Piscinoodinium sp. Infection in an Endangered Fish from India: Arulius Barb (Dawkinsia arulius).

Freshwater velvet disease is caused by the dinoflagellate parasite, Piscinoodinium sp. This parasite has been reported in tropical and subtropical fishes, and it can cause devastating losses. Moreover, Piscinoodinium sp. is identified as one of the least studied finfish parasites, and the available molecular information about this parasite is meager. Recently, Piscinoodinium sp. was responsible for the 100% cumulative mortality of the captive-bred F1 generation of Arulius barb (Dawkinsia arulius), an endangered freshwater fish native to India. The trophont stages of the parasite were observed in the skin and gills of the affected fish. The total DNA was extracted from the trophonts collected from the affected Arulius barb and the partial nucleotide sequence of the rDNA complex region (2334 bp) was amplified using PCR. The amplified PCR product exhibited a high sequence identity (97.61%) with Piscinoodinium sp. In the phylogenetic analysis of the SSU rDNA, Piscinoodinium sp. emerged as a separate clade from other dinoflagellate species. This is the first report of the infection of Piscinoodinium sp. in Arulius barb and the molecular information generated from this study can serve as a baseline to study the diversity of the parasite in India. Furthermore, the impact of this parasite among wild fish stock is not known, and this parasite needs further research focus to generate more molecular information and to understand the host-pathogen interaction.

The complete mitochondrial genome of the endangered freshwater fish Odontobutis obscurus from Korea.

We report the first complete mitochondrial genome of Odontobutis obscurus, which consists of 17,038 bp harboring 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a control region (D-loop). The overall base composition of the complete genome is A (30.63%), C (28.72%), T (25.70%), G (14.95%). The complete mitogenome of Odontobutis obscurus, most closely related to congeners in the Bayesian inference and maximum likelihood tree, provides a better understanding of the phylogeny of the genus Odontobutis.

Management of Genetic Variation in the Gamete Bank of the Endangered Lake Minnow Eupallasella percnurus, Using Genassemblage 2.2 Software.

The management of genetic resources deposited in gene banks requires knowledge of the genetic profiles of the gamete donors and bioinformatics tools to process this information. In this work, we show how to use Genassemblage 2.2 software in managing the genetic variation deposited in a bank of cryopreserved semen. Our demonstration was based on the leuciscid fish species, lake minnow Eupallasella percnurus, which is designated as endangered in Poland. The semen samples (n = 192) were taken from four Polish lake minnow populations and frozen in the gene bank. Fin clips were taken and DNA extracted. Across 13 investigated microsatellite loci, 21-53 alleles were identified in each population and 66 in the entire group of samples. The module "Management of genetic variation in gamete bank" of Genassemblage 2.2 software was used to find the set of samples that will preserve 100% of the detected allelic diversity in the next generation. Our results showed that a small group of 4-19 semen samples was enough to transfer all alleles detected across this set of samples. We, therefore, recommend Genassemblage 2.2 as a convenient tool for the detection of genetic differences between donors, the construction of optimal sets of samples for conservation of genetic variation, and for managing genetic variation deposited in gamete banks. Consequently, it can be used in breeding human-dependent populations and to optimize the use of genetic diversity in samples in the gamete banks. It can be especially useful for conserving populations of species characterized by low genetic variation, such as the lake minnow.

The complete mitochondrial genome of the endangered freshwater fish Kichulchoia brevifasciata in Korea.

Kichulchoia brevifasciata is an endangered fish that is distributed across the Goheung Peninsula, South Korea. This is the first report of the complete mitochondrial genome of K. brevifasciata, which consists of 16,646 bp with 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region (D-loop). The overall base composition of the complete genome is A (29.57%), T (28.08%), G (16.54%), and C (25.80%), with a high A + T content of 57.65%. Phylogenetic analysis showed that K. brevifasciata was most closely related to K. multifasciatas.

Isolated reefs support stable fish communities with high abundances of regionally fished species.

Anthropogenic impacts at isolated and inaccessible reefs are often minimal, offering rare opportunities to observe fish assemblages in a relatively undisturbed state. The remote Rowley Shoals are regarded as one of the healthiest reef systems in the Indian Ocean with demonstrated resilience to natural disturbance, no permanent human population nearby, low visitation rates, and large protected areas where fishing prohibitions are enforced. We used baited remote underwater video systems (BRUVS) to quantify fish assemblages and the relative abundance of regionally fished species within the lagoon, on the slope and in the mesophotic habitat at the Rowley Shoals at three times spanning 14 years and compared abundances of regionally fished species and the length distributions of predatory species to other isolated reefs in the northeast Indian Ocean. Fish assemblage composition and the relative abundance of regionally fished species were remarkably stable through time. We recorded high abundances of regionally fished species relative to other isolated reefs, including globally threatened humphead Maori wrasse (Cheilinus undulatus) and bumphead parrotfish (Bolbometopon muricatum). Length distributions of fish differed among habitats at the Rowley Shoals, suggesting differences in ontogenetic shifts among species. The Cocos (Keeling) Islands typically had larger-bodied predatory species than at the Rowley Shoals. Differences in geomorphology, lagoonal habitats, and fishing history likely contribute to the differences among remote reefs. Rowley Shoals is a rare example of a reef system demonstrating ecological stability in reef fish assemblages during a time of unprecedented degradation of coral reefs.

Knowledge Gaps in the Definition of Threats for the Red List Assessment of European Freshwater-Dependent Fish Species.

Freshwater ecosystems are disproportionally important for biodiversity conservation, as they support more than 9% of known animal species while representing less than 1% of the Earth's surface. However, the vast majority of the threats (99%, or 826 out of 837) identified by the International Union for Conservation of Nature Red List of Threatened Species known to affect the 434 known freshwater-dependent fish and lampreys of Europe are not supported by validated published scientific knowledge. This general lack of information about freshwater-dependent fish and lamprey species may have deleterious effects on species conservation, and additional funding is required to fill baseline knowledge gaps.

What are the best upland river characteristics for glass eel restocking practice?

The fitness of restocked European eel (Anguilla anguilla), an endangered fish species, was studied in relation to the environmental variables of habitats in six upland rivers that are typologically different in terms of their hydromorphological and physicochemical characteristics, food resources and fish communities. These rivers received a total of 76,370 imported glass eels in 2017. During a three-year period, we monitored eels with respect to total length, annual growth rate, condition factor and density using capture-mark-recapture experiments to understand the effects of the characteristics of receiving rivers on restocking success levels. Our results showed the survival of the restocked eels in the six rivers and revealed significant differences between them in terms of total length, condition factor and density. Better performance in eel yield variables was observed in a eutrophic alkaline river with greater roughness of riverbed substrates, dominant pool- and riffle-type flow facies and lower brown trout density. The variables conductivity and total hardness had higher explanatory power and were strongly associated with increased eel density. This study suggests that a well-selected habitat/river in a restocking programme can be beneficial for the species and recommends restocking practice as a management tool to achieve eel conservation goals.

Extinction of one of the world's largest freshwater fishes: Lessons for conserving the endangered Yangtze fauna.

The mega river ecosystem of the Yangtze River was once home to diverse aquatic megafauna but is increasingly affected by various anthropogenic stressors that have resulted in continuous loss of biodiversity, such as the probable extinction of Yangtze River Dolphin. The Chinese paddlefish, Psephurus gladius, was one of only two extant members of a relict lineage that was most diverse and widespread 34-75 million years ago. It is also one of the largest freshwater fish species, reaching up to 7 m in length. The Chinese paddlefish was once common in the Yangtze River, with c.25 t being harvested per annum during the 1970s. Populations have, however, declined drastically since the late 1970s as a result of overfishing and habitat fragmentation. Here, a basin-wide capture survey during 2017-2018 found 332 fish species, but did not find a single specimen of Chinese paddlefish. Furthermore, 140 historically reported fish species have not been found and most of them are considered highly endangered. Based on 210 sightings of Chinese paddlefish during the period 1981-2003, we estimated the timing of extinction to be by 2005, and no later than by 2010. In addition, the paddlefish probably became functionally extinct (i.e. it was unable to reproduce) by 1993, before it went extinct. It is likely that the lack of reproduction was among the major causes of extinction. As no individuals exist in captivity, and no living tissues are conserved for potential resurrection, the fish should be considered extinct according to the IUCN Red List criteria. The delayed extinction of Chinese paddlefish resulted from multiple threats, suggesting that optimizing conservation efforts on endangered Yangtze fauna is urgently needed.