Global freshwater fish invasion linked to the presence of closely related species.

In the Anthropocene, non-native freshwater fish introductions and translocations have occurred extensively worldwide. However, their global distribution patterns and the factors influencing their establishment remain poorly understood. We analyze a comprehensive database of 14953 freshwater fish species across 3119 river basins and identify global hotspots for exotic and translocated non-native fishes. We show that both types of non-native fishes are more likely to occur when closely related to native fishes. This finding is consistent across measures of phylogenetic relatedness, biogeographical realms, and highly invaded countries, even after accounting for the influence of native diversity. This contradicts Darwin's naturalization hypothesis, suggesting that the presence of close relatives more often signifies suitable habitats than intensified competition, predicting the establishment of non-native fish species. Our study provides a comprehensive assessment of global non-native freshwater fish patterns and their phylogenetic correlates, laying the groundwork for understanding and predicting future fish invasions in freshwater ecosystems.

Biotic resistance to fish invasions in southern China: Evidence from biomass, habitat, and fertility limitation.

Understanding the mechanisms underlying the invasion success or failure of alien species can help to predict future invasions and cope with the invaders. The biotic resistance hypothesis posits that diverse communities are more resistant to invasion. While many studies have examined this hypothesis, the majority of them have focused on the relationship between alien and native species richness in plant communities, and results have often been inconsistent. In southern China, many rivers have been invaded by alien fish species, providing an opportunity to test the resistance of native fish communities to alien fish invasions. Using survey data for 60,155 freshwater fish collected from five main rivers of southern China for 3 years, we assessed the relationships between native fish richness and the richness and biomass of alien fishes at river and reach spatial scales, respectively. Based on two manipulative experiments, we further examined the impact of native fish richness on habitat selection and the reproductive ability of an exotic model species Coptodon zillii. We found no apparent relationship between alien and native fish richness, whereas the biomass of alien fish significantly decreased with increasing native fish richness. In experiments, C. zillii preferred to invade those habitats that had low native fish richness, given evenly distributed food resources; reproduction of C. zillii was strongly depressed by a native carnivorous fish Channa maculata. Together, our results indicate that native fish diversity can continue to provide biotic resistance to alien fish species in terms of limiting their growth, habitat selection, and reproduction when these aliens have successfully invaded southern China. We thus advocate for fish biodiversity conservation, especially for key species, to mitigate against the population development and ecological impact of alien fish species.

Environmental-related variation of stoichiometric traits in body and organs of non-native sailfin catfishes Pterygoplichthys spp.

Intraspecific variation in stoichiometric traits was thought to be an adaptive response to reduce the elemental imbalance between organism and diet in the habitat. Studying the spatial variation of stoichiometric traits of non-native species and the factors contributing to the variation could help to better understand the invasion mechanism of non-native fish. In this study, stoichiometric traits (i.e. carbon [C], phosphorus [P], calcium [Ca] and their ratios) variation in the body and organs of non-native sailfin catfishes Pterygoplichthys spp. were investigated across 13 river sections in the main river basins of Guangdong province. The relationships between environmental factors and stoichiometric traits were analyzed using a general linear model and an information-theoretic approach. A manipulated feeding experiment was conducted to investigate the impact of food quality on the stoichiometry of sailfin catfishes in a greenhouse. Sailfin catfishes exhibited considerable variability in body and organ elemental composition. Site identity was the main factor contributing to the variation, which could be explained by a combination of environmental factors including climate, diet quality, fish species richness and trophic status in the invaded rivers. Water chemistry (i.e. total nitrogen and phosphorus, ammonia nitrogen and soluble reactive phosphorus) contributed to the most variation of stoichiometric traits. Imbalances of P and Ca between sailfin catfishes and food resources varied among sampling sites, reflecting the spatial heterogeneity of nutrients limitation. Juvenile sailfin catfishes exhibited stoichiometric homeostasis (0 < 1/H < 0.25) for all elemental contents and ratios in the feeding experiment. These findings suggested variation in stoichiometric traits of sailfin catfishes might be attributed to the changes in elemental metabolism to cope with context-specific environments. This study provided heuristic knowledge about environmental-related variation in stoichiometric traits, which could enhance the understanding of the non-native species' adaptation to resource fluctuation in the invaded ecosystems.

FishPIE: A universal phylogenetically informative exon markers set for ray-finned fishes.

Understanding the evolutionary history of the highly diverse ray-finned fishes has been challenging, and the development of more universal primers for phylogenetic analyses may help overcoming these challenges. We developed FishPIE, a nested PCR primer set of 82 phylogenetically informative exon markers, and tested it on 203 species from 31 orders of Actinopterygii. We combined orthologous sequences of the FishPIE markers obtained from published genomes and transcriptomes and constructed the phylogeny of 710 species belonging to 190 families and 60 orders. The resulting phylogenies had topologies comparable to previous phylogenomic studies. We demonstrated that the FishPIE markers could address phylogenetic questions across broad taxonomic levels. By incorporating the newly sequenced taxa, we were able to shed new light on the phylogeny of the highly diverse Cypriniformes. Thus, FishPIE holds great promise for generating genetic data for broad taxonomic groups and accelerating our understanding of the fish tree of life.

Exotic fishes that are phylogenetically close but functionally distant to native fishes are more likely to establish.

Since Darwin's time, degree of ecological similarity between exotic and native species has been assumed to affect the establishment success or failure of exotic species. However, a direct test of the effect of exotic-native similarity on establishment of exotics is scarce because of the difficulty in recognizing failures of species to establish in the field. Here, using a database on the establishment success and failure of exotic fish species introduced into 673 freshwater lakes, we evaluate the effect of similarity on the establishment of exotic fishes by combining phylogenetic and functional information. We illustrate that, relative to other biotic and abiotic factors, exotic-native phylogenetic and functional similarities were the most important correlates of exotic fish establishment. While phylogenetic similarity between exotic and resident fish species promoted successful establishment, functional similarity led to failure of exotics to become established. Those exotic species phylogenetically close to, but functionally distant from, native fishes were most likely to establish successfully. Our findings provide a perspective to reconcile Darwin's naturalization conundrum and suggest that, while phylogenetic relatedness allows exotic fish species to pre-adapt better to novel environments, they need to possess distinct functional traits to reduce competition with resident native fish species.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning.

OBJECTIVES: To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning. METHODS: Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated. RESULTS: The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05). CONCLUSIONS: MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Preparation of monoclonal antibodies against KHV and establishment of an antigen sandwich ELISA for KHV detection.

Infections with koi herpesvirus (KHV) in carp are still a severe problem worldwide. Detection and elimination of infected fish are necessary for control of the Koi herpesvirus disease (KHVD). Serum is an excellent specimen for KHV testing because of high survivability of KHV in serum and ease of collection, storage, and handling. The direct detection of fish viruses based on the sandwich ELISA has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against purified KHV. By using hybridoma-monoclonal antibody technology, two hybridoma cell lines secreting MAbs against the KHV were established. By Western blot and IFAT analysis, the secreted MAbs from cell line IB7IB4 and cell line 7C72F7 recognized proteins of KHV. The result demonstrated that the MAbs were highly specific and sensitive to the KHV, and can be used for monitoring the virus quantification of carp, for example, the direct KHV diagnosis by sandwich enzyme-linked immunosorbent assay(ELISA). An antigen sandwich ELISA applying the biotin-avidin system was established using the biotinylated MAb IB7IB4 and 7C72F7 to detect virus in koi sera. These MAbs did not react with any of the tested other viruses by ELISA except KHV. The detection limit of the test was 3.923ng/ml KHV. Thus, this antigen sandwich ELISA is suitable for recognition of KHV.

The complete mitochondrial genomes of two freshwater snails provide new protein-coding gene rearrangement models and phylogenetic implications.

BACKGROUND: Mitochondrial (mt) genome sequences are widely used for species identification and to study the phylogenetic relationships among Gastropoda. However, to date, limited data are available as taxon sampling is narrow. In this study we sequenced the complete mt genomes of the freshwater gastropods Radix swinhoei (Lymnaeidae) and Planorbarius corneus (Planorbidae). Based on these sequences, we investigated the gene rearrangement in these two species and the relationships with respect to the ancestral gene order and assessed their phylogenetic relationships. METHODS: The complete mt genomes of R. swinhoei and P. corneus were sequenced using Illumina-based paired-end sequencing and annotated by comparing the sequence information with that of related gastropod species. Putative models of mitochondrial gene rearrangements were predicted for both R. swinhoei and P. corneus, using Reishia clavigera mtDNA structure as the ancestral gene order. The phylogenetic relationships were inferred using thirteen protein sequences based on Maximum likelihood and Bayesian inference analyses. RESULTS: The complete circular mt genome sequences of R. swinhoei and P. corneus were 14,241 bp and 13,687 bp in length, respectively. Comparison of the gene order demonstrated complex rearrangement events in Gastropoda, both for tRNA genes and protein-coding genes. The phylogenetic analyses showed that the family Lymnaeidae was more closely related to the family Planorbidae, consistent with previous classification. Nevertheless, due to the position recovered for R. swinhoei, the family Lymnaeidae was not monophyletic. CONCLUSION: This study provides the complete mt genomes of two freshwater snails, which will aid the development of useful molecular markers for epidemiological, ecological and phylogenetic studies. Additionally, the predicted models for mt gene rearrangement might provide novel insights into mt genome evolution in gastropods.

Warming mediates the relationship between plant nutritional properties and herbivore functional responses.

Quantifying the per capita effects of invasive alien species is crucial for assessing their ecological impact. A major challenge to risk assessment of invasive species was to understand the factors that cause per capita effects to vary in different ecological contexts, particularly in a warming world. By conducting functional response experiments, we estimated the per capita effects (attack rate and maximum feeding rate) of an invasive herbivorous snail, Pomacea canaliculata, toward ten host plant species. We tested whether variation in these effects is related to plant nutritional and physical properties (total N and dry matter content (DMC)) and examined how increasing temperature can shift these relationships. We observed stronger per capita effects (i.e., higher attack rate and maximum feeding rate) by the snail on plants with higher total N, but no direct relationship was found with DMC. A significant interaction effect of total N and DMC on the attack rate indicated that DMC probably adjusted the feeding indirectly. Warmer temperatures reduced correlations between snail functional responses and host plant nutritional properties (total N) by increasing maximum feeding rate for plants of low nutrition, but there was no such effect on attack rates. However, given the nonreplacement design used in our study, the nonsignificant effect of temperature on the attack rate should be caveated. Our result suggests that characterizing the per capita effects of herbivores using functional responses can reveal the mechanisms by which climate change may alter herbivore-plant interactions and, thus, the ecological impacts of introduced herbivores.

Spatial variation in adult sex ratio across multiple scales in the invasive golden apple snail, Pomacea canaliculata.

Adult sex ratio (ASR) has critical effects on behavior and life history and has implications for population demography, including the invasiveness of introduced species. ASR exhibits immense variation in nature, yet the scale dependence of this variation is rarely analyzed. In this study, using the generalized multilevel models, we investigated the variation in ASR across multiple nested spatial scales and analyzed the underlying causes for an invasive species, the golden apple snail Pomacea canaliculata. We partitioned the variance in ASR to describe the variations at different scales and then included the explanatory variables at the individual and group levels to analyze the potential causes driving the variation in ASR. We firstly determined there is a significant female-biased ASR for this species when accounting for the spatial and temporal autocorrelations of sampling. We found that, counter to nearly equal distributed variation at plot, habitat and region levels, ASR showed little variation at the town level. Temperature and precipitation at the region level were significantly positively associated with ASR, whereas the individual weight, the density characteristic, and sampling time were not significant factors influencing ASR. Our study suggests that offspring sex ratio of this species may shape the general pattern of ASR in the population level while the environmental variables at the region level translate the unbiased offspring sex ratio to the female-biased ASR. Future research should consider the implications of climate warming on the female-biased ASR of this invasive species and thus on invasion pattern.

The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts.

The Asian arowana (Scleropages formosus), one of the world's most expensive cultivated ornamental fishes, is an endangered species. It represents an ancient lineage of teleosts: the Osteoglossomorpha. Here, we provide a high-quality chromosome-level reference genome of a female golden-variety arowana using a combination of deep shotgun sequencing and high-resolution linkage mapping. In addition, we have also generated two draft genome assemblies for the red and green varieties. Phylogenomic analysis supports a sister group relationship between Osteoglossomorpha (bonytongues) and Elopomorpha (eels and relatives), with the two clades together forming a sister group of Clupeocephala which includes all the remaining teleosts. The arowana genome retains the full complement of eight Hox clusters unlike the African butterfly fish (Pantodon buchholzi), another bonytongue fish, which possess only five Hox clusters. Differential gene expression among three varieties provides insights into the genetic basis of colour variation. A potential heterogametic sex chromosome is identified in the female arowana karyotype, suggesting that the sex is determined by a ZW/ZZ sex chromosomal system. The high-quality reference genome of the golden arowana and the draft assemblies of the red and green varieties are valuable resources for understanding the biology, adaptation and behaviour of Asian arowanas.

Comparative Functional Responses Predict the Invasiveness and Ecological Impacts of Alien Herbivorous Snails.

Understanding determinants of the invasiveness and ecological impacts of alien species is amongst the most sought-after and urgent research questions in ecology. Several studies have shown the value of comparing the functional responses (FRs) of alien and native predators towards native prey, however, the technique is under-explored with herbivorous alien species and as a predictor of invasiveness as distinct from ecological impact. Here, in China, we conducted a mesocosm experiment to compare the FRs among three herbivorous snail species: the golden apple snail, Pomacea canaliculata, a highly invasive and high impact alien listed in "100 of the World's Worst Invasive Alien Species"; Planorbarius corneus, a non-invasive, low impact alien; and the Chinese native snail, Bellamya aeruginosa, when feeding on four locally occurring plant species. Further, by using a numerical response equation, we modelled the population dynamics of the snail consumers. For standard FR parameters, we found that the invasive and damaging alien snail had the highest "attack rates" a, shortest "handling times" h and also the highest estimated maximum feeding rates, 1/hT, whereas the native species had the lowest attack rates, longest handling times and lowest maximum feeding rates. The non-invasive, low impact alien species had consistently intermediate FR parameters. The invasive alien species had higher population growth potential than the native snail species, whilst that of the non-invasive alien species was intermediate. Thus, while the comparative FR approach has been proposed as a reliable method for predicting the ecological impacts of invasive predators, our results further suggest that comparative FRs could extend to predict the invasiveness and ecological impacts of alien herbivores and should be explored in other taxa and trophic groups to determine the general utility of the approach.

An unusual mitochondrial genome structure of the tonguefish, Cynoglossus trigrammus: Control region translocation and a long additional non-coding region inversion.

Flatfishes (Pleuronectiformes) exhibit different types of large-scale gene rearrangements. In the present study, the mitochondrial (mt) genome (18,369bp) of a tonguefish, Cynoglossus trigrammus, was determined using de novo mitochondrion genome sequencing. Compared with other flatfishes, the mt genome of C. trigrammus revealed distinct mitogenome architectures that primarily included two striking findings: 1) insertion of an additional long non-coding region (1647bp) making it the second largest genome length among Pleuronectiformes and 2) the translocation of the control region. The reconstructed phylogenetic tree based on 13 mt protein-coding gene sequences recovered the monophyletic suborder Pleuronectoidei and the family Cynoglossidae. These data provide useful information for a better understanding of the mitogenomic diversities and evolution in fish as well as novel genetic markers for studying population genetics and species identification.

Transcriptome analysis between invasive Pomacea canaliculata and indigenous Cipangopaludina cahayensis reveals genomic divergence and diagnostic microsatellite/SSR markers.

BACKGROUND: Pomacea canaliculata is an important invasive species worldwide. However, little is known about the molecular mechanisms behind species displacement, adaptational abilities, and pesticide resistance, partly because of the lack of genomic information that is available for this species. Here, the transcriptome sequences for the invasive golden apple snail P. canaliculata and the native mudsnail Cipangopaludina cahayensis were obtained by next-generation-sequencing and used to compare genomic divergence and identify molecular markers. RESULTS: More than 46 million high quality sequencing reads were generated from P. canaliculata and C. cahayensis using Illumina paired-end sequencing technology. Our analysis indicated that 11,312 unigenes from P. canaliculata and C. cahayensis showed significant similarities to known proteins families, among which a total of 4,320 specific protein families were identified. KEGG pathway enrichment was analyzed for the unique unigenes with 17 pathways (p-value < 10(-5)) in P. canaliculata relating predominantly to lysosomes and vitamin digestion and absorption, and with 12 identified in C. cahayensis, including cancer and toxoplasmosis pathways, respectively. Our analysis also indicated that the comparatively high number of P450 genes in the P. canaliculata transcriptome may be associated with the pesticide resistance in this species. Additionally, 16,717 simple sequence repeats derived from expressed sequence tags (EST-SSRs) were identified from the 14,722 unigenes in P. canaliculata and 100 of them were examined by PCR, revealing a species-specific molecular marker that could distinguish between the morphologically similar P. canaliculata and C. cahayensis snails. CONCLUSIONS: Here, we present the genomic resources of P. canaliculata and C. cahayensis. Differentially expressed genes in the transcriptome of P. canaliculata compared with C. cahayensis corresponded to critical metabolic pathways, and genes specifically related to environmental stress response were detected. The CYP4 family of P450 cytochromes that may be important factors in pesticide metabolism in P. canaliculata was identified. Overall, these findings will provide valuable genetic data for the further characterization of the molecular mechanisms that support the invasive and adaptive abilities of P. canaliculata.

Molecular characteristics of the HSP70 gene and its differential expression in female and male golden apple snails (Pomacea canaliculata) under temperature stimulation.

Heat-shock protein 70 (HSP70) is one of the most important heat-shock proteins that helps organisms to modulate stress response via over-expression. The HSP70 gene from Pomacea canaliculata was cloned using the RACE approach; the gene is 2,767 bp in length and contains an open reading frame of 1,932 bp, which is encoded by a polypeptide of 643 amino acids. BLAST analysis showed that the predicted amino acid sequence of the P. canaliculata HSP70 gene shared a relatively high similarity with that of other known eukaryotic species that display conserved HSP characteristics. The phylogeny demonstrated a separate clustering of the apple snail HSP70 with other constitutive members from other mollusk species. Quantitative real-time RT-PCR was used to detect the differential expression of HSP70 in both sexes of P. canaliculata at different temperature conditions. These results showed that HSP70 transcript levels decreased slightly under cold shock and increased significantly under heat-shock conditions in both sexes compared to normal temperatures (26 °C). Under cold-shock treatment, the sex effect was not significant. With heat treatment, HSP70 expression could be induced at 36 °C in both females and males, and it peaked at 42 and 39 °C in females and males, respectively. In addition, a clear time-dependent HSP70 expression pattern of the apple snail exposed to the same high temperature (36 °C) was observed at different time points. The maximal induction of HSP70 expression appeared at 12 and 48 h in males and females after heat shock, respectively. The maximal induction in females was significantly higher compared to males under heat stimulus. Taken together, these results strongly suggested that males were more susceptible to heat than females and provided useful molecular information for the ecological adaptability of P. canaliculata against extreme environmental stress.

Mitochondrial DNA as effective molecular markers for the genetic variation and phylogeny of the family Osteoglossidae.

The present study examined the genetic variation of the family Osteoglossidae from different geographical locations based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) and ATPase subunit 6 (ATPase6) genes; we then re-constructed the phylogenetic relationships using the two sequences in combination. The results showed that the partial sequences of mitochondrial ND2 and ATPase6 of the family Osteoglossidae were 813 bp and 669 bp, respectively. A total of 42 species-specific nucleotide positions of the family Osteoglossidae were found to be useful for molecular identification. The sequence variation showed greater differences (8.3%~28.1% for the combined sequences, 8.3%~26.7% for the ND2 gene, and 9.3%~28.7% for the ATPase6 gene) among the different species of Osteoglossidae, and there was a significant association between the genetic difference and geographical location. Phylogenetic analyses using neighbor-joining, Bayesian inference, and maximum parsimony (MP) methods based on the combined sequences of the two genes were able to distinguish the different species and were in agreement with the existing taxonomy based on morphological characters and in association with the geographical distribution among seven species of the family Osteoglossidae.