The Pacific blue shrimp (Litopenaeus stylirostris) is a premium product in the international seafood market. However, intensified farming has increased disease incidence and reduced genetic diversity. In this study, we developed a transcriptome database for L. stylirostris and mined microsatellite markers to analyze their genetic diversity. Using the Illumina HiSeq 4000 platform, we identified 53,263 unigenes from muscle, hepatopancreas, the intestine, and lymphoid tissues. Microsatellite analysis identified 36,415 markers from 18,657 unigenes, predominantly dinucleotide repeats. Functional annotation highlighted key disease resistance pathways and enriched categories. The screening and PCR testing of 42 transcriptome-based and 58 literature-based markers identified 40 with successful amplification. The genotyping of 200 broodstock samples revealed that Na, Ho, He, PIC, and FIS values were 3, 0.54 ± 0.05, 0.43 ± 0.09, 0.41 ± 0.22, and 0.17 ± 0.27, respectively, indicating moderate genetic variability and significant inbreeding. Four universal microsatellite markers (CL1472.Contig13, CL517.Contig2, Unigene5692, and Unigene7147) were identified for precise diversity analysis in Pacific blue, Pacific white (Litopenaeus vannamei), and black tiger shrimps (Penaeus monodon). The transcriptome database supports the development of markers and functional gene analysis for selective breeding programs. Our findings underscore the need for an appropriate genetic management system to mitigate inbreeding depression, reduce disease susceptibility, and preserve genetic diversity in farmed shrimp populations.
Global warming and pollution could lead to the destruction of marine habitats and loss of species. The anomalous behavior of underwater creatures can be used as a biometer for assessing the health status of our ocean. Advances in behavior recognition have been driven by the active application of deep learning methods, yet many of them render superior accuracy at the cost of high computational complexity and slow inference. This paper presents a real-time anomalous behavior recognition approach that incorporates a lightweight deep learning model (Lite3D), object detection, and multitarget tracking. Lite3D is characterized in threefold: (1) image frames contain only regions of interest (ROI) generated by an object detector; (2) no fully connected layers are needed, the prediction head itself is a flatten layer of 1 × [Formula: see text] @ 1× 1, [Formula: see text]= number of categories; (3) all the convolution kernels are 3D, except the first layer degenerated to 2D. Through the tracking, a sequence of ROI-only frames is subjected to 3D convolutions for stacked feature extraction. Compared to other 3D models, Lite3D is 50 times smaller in size and 57 times lighter in terms of trainable parameters and can achieve 99% of F1-score. Lite3D is ideal for mounting on ROV or AUV to perform real-time edge computing.
The tideland snails Pirenellanipponica and Pirenellaasiatica are distributed north of the central Ryukyu Islands and in South Ryukyu, respectively, in Japan. To reveal their distribution and genetic characteristics in Taiwan, we sampled Pirenella snails along the western coast of Taiwan Island and analyzed the nucleotide sequences of their mitochondrial DNAs. Pirenella nipponica and P. asiatica inhabit the northern and southern parts of the western coast of Taiwan, respectively, and coexist only in the central part. Taiwanese and Japanese populations of P. asiatica showed significant genetic differentiation. The former showed higher genetic diversity and a larger effective population size than the latter. However, the Taiwanese population of P. nipponica was not genetically deviated from the local Japanese population on Kyushu Island. Both the Taiwan and Kyushu populations of P. nipponica showed significant genetic differences from local populations in other regions of Japan, namely, Honshu Island (the Japanese mainland) and Central Ryukyu. They also showed higher genetic diversity and a larger effective population size than the others. The Taiwanese populations of both species might be part of a large panmictic population with individuals from the Asian continent and Kyushu Island.
Stock enhancement, used for replenishing depleted wild finfish populations, is an aggressive approach. Stock enhancement projects in Taiwan involve black sea bream (Acanthopagrus schlegelii), a major commercial species. During 2004-2015, even management agencies conducted stock enhancement projects, leading to numerous private releases that have not been recorded. Stock enhancement by a private hatchery without accurate genetic records may lead to a genetic structure change in wild populations. Using allele frequencies at nine microsatellite loci, we studied the genetic effects of stock enhancement in 19 samples collected from populations in the hatcheries and the wild. In 458 individuals from nine hatchery samples, most populations showed weak but significant genetic differences and complex clusters in structure analysis, indicating dramatic stock change within and among hatcheries. The 10 wild populations (n = 773) also had a complex genetic composition and were genetically different among sampling sites and times. However, a simple and clear cluster in structure analysis was found for only one sampling site, which had no release history. Thus, stock enhancement with complex genetic sources helps maintain genetic diversity but dramatically changes the genetic structure within and among wild populations, especially when stock enhancement is successful.
Taiwan tilapia is one of the primary species used in aquaculture practices in Taiwan. However, as a tropical fish, it is sensitive to cold temperatures that can lead to high mortality rates during winter months. Genetic and broodstock management strategies using marker-assisted selection and breeding are the best tools currently available to improve seed varieties for tilapia species. The purpose of this study was to develop molecular markers for cold stress-related genes using digital gene expression analysis of next-generation transcriptome sequencing in Taiwan tilapia (Oreochromis spp.). We constructed and sequenced cDNA libraries from the brain, gill, liver, and muscle tissues of cold-tolerance (CT) and cold-sensitivity (CS) strains. Approximately 35,214,833,100 nucleotides of raw sequencing reads were generated, and these were assembled into 128,147 unigenes possessing a total length of 185,382,926 bp and an average length of 1446 bp. A total of 25,844 unigenes were annotated using five protein databases and Venny analysis, and 38,377 simple sequence repeats (SSRs) and 65,527 single nucleotide polymorphisms (SNPs) were identified. Furthermore, from the 38-cold tolerance-related genes that were identified using differential gene expression analysis in the four tissues, 13 microsatellites and 37 single nucleotide polymorphism markers were identified. The results of the genotype analysis revealed that the selected markers could be used for population genetics. In addition to the diversity assessment, one of the SNP markers was determined to be significantly related to cold-tolerance traits and could be used as a molecular marker to assist in the selection and verification of cold-tolerant populations. The specific genetic markers explored in this study can be used for the identification of genetic polymorphisms and cold tolerance traits in Taiwan tilapia, and they can also be used to further explore the physiological and biochemical molecular regulation pathways of fish that are involved in their tolerance to environmental temperature stress.
Seafood, especially the traditional one in Taiwan, is rarely sourced from a fixed species and routinely from similar species depending on their availability. Hence, the species composition of seafood can be complicated. While a DNA-based approach has been routinely utilized for species identification, a large scale of seafood identification in fish markets and restaurants could be challenging (e.g., elevated cost and time-consuming only for a limited number of species identification). In the present study, we aimed to identify the majority of fish species potentially consumed in fish markets and nearby seafood restaurants using environmental DNA (eDNA) metabarcoding. Four eDNA samplings from a local fish market and nearby seafood restaurants were conducted using Sterivex cartridges. Nineteen universal primers previously validated for fish species identification were utilized to amplify the fragments of mitochondrial DNA (12S, COI, ND5) of species in eDNA samples and sequenced with NovaSeq 6000 sequencing. A total of 153 fish species have been identified based on 417 fish related operational taxonomic units (OTUs) generated from 50,534,995 reads. Principal Coordinate Analysis (PCoA) further showed the differences in fish species between the sampling times and sampling sites. Of these fish species, 22 chondrichthyan fish, 14 Anguilliformes species, and 15 Serranidae species were respectively associated with smoked sharks, braised moray eels, and grouper fish soups. To our best knowledge, this work represents the first study to demonstrate the feasibility of a large scale of seafood identification using eDNA metabarcoding approach. Our findings also imply the species diversity in traditional seafood might be seriously underestimated and crucial for the conservation and management of marine resources.
Laevistrombus canarium, also known as dog conch, is a marine gastropod mollusk widely distributed in the Indo-Pacific region. It is an economically crucial species; however, its population has been declining due to overfishing and overexploitation. In this study, the suitable salinity for juvenile L. canarium was between 20 and 35. Diatoms and biological detritus by using flow-water from the fish pool were the most favorable diets for newly metamorphosed and 10 mm juveniles. In the polyculture experiment, L. canarium was cultured with whiteleg shrimp, tilapia, small abalone, purple sea urchin, and collector urchin. Better growth was found in all co-culture groups except with whiteleg shrimp. We also found that the polyculture system with or without substrates significantly affected the growth of juveniles. Additionally, we observed that water temperature was the most crucial factor for growth and survival; a water temperature of less than 10 °C might cause the death of L. canarium. We have proposed a novel polyculture and water-flow method for mass production of L. canarium and evaluated the feasibility and benefits of polyculture with other species. The findings from this work reveal the potentiality of L. canarium in integrated multitrophic aquaculture (IMTA) and its implication for aquaculture and resource restoration.
The first complete mitochondrial genome of Metasepia tullbergi has been characterized in this study. The circular mitogenome is 16182 bp in length and comprises 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. The organization of these genes is highly consistent with that of other Sepiidae. The overall base composition of mitogenome is 39.20% A, 36.07% T, 8.98% G, and 15.75% C, with 75.27% AT. Phylogenetic analysis further suggests that M. tullbergi is placed within the Sepiidae and is closely related to Sepia latimanus and S. apama.
Cannibalism is a major problem in lobster and crab aquaculture. Reducing the aggressive characteristics of lobsters and crabs can improve survival during the culturing process. In this study, juvenile scalloped spiny lobsters (Panulirus homarus) and crucifix crabs (Charybdis feriatus) were both cultured under different shelter and live prey conditions. Groups with shelter (seaweed and cotton filter) showed a better survival rate than the control group (no shelter; p < 0.05) for both Pa. homarus and Char. feriatus. Co-culturing with live prey (Litopenaeus vannamei) significantly benefited the juveniles of Pa. homarus and visibly increased the survival of juvenile Char. feriatus. Although providing shelter is currently the main method for reducing agonistic behavior, it must be continually altered as the lobsters and crabs grow. Live prey can grow and attract lobsters and crabs to hunt them, and live prey can be supplemented at any time. They can also be used as an additional source of income during the harvest season.
Laevistrombus canarium is a marine gastropod species with high economical value. The complete mitochondrial genome of L. canarium has been characterized in this study. The circular mitogenome is 15626 bp in length and comprises 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA (rRNA) genes. The organization of these genes is consistent with that of other stromboidae species. The overall base composition of mitochondrial genome is 30.87% A, 38.99% T, 15.54% G, and 14.60% C, with 69.86% AT. Phylogenetic analysis further implies that L. canarium is placed within the Stromboidae.
The accuracy and efficiency of marker-assisted selection (MAS) has been proven for economically critical aquaculture species. The potato grouper (Epinephelus tukula), a novel cultured grouper species in Taiwan, shows large potential in aquaculture because of its fast growth rate among other groupers. Because of the lack of genetic information for the potato grouper, the first transcriptome and expressed sequence tag (EST)-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were developed. Initially, the transcriptome was obtained from seven cDNA libraries by using the Illumina platform. De novo transcriptome of the potato grouper yielded 51.34 Gb and 111,490 unigenes. The EST-derived SSR and SNP markers were applied in genetic management, in parentage analysis, and to discover the functional markers of economic traits. The F1 juveniles were identified as siblings from one pair of parents (80 broodstocks). Fast- and slow-growth individuals were analyzed using functional molecular markers and through their association with growth performance. The results revealed that two SNPs were correlated with growth traits. The transcriptome database obtained in this study and its derived SSR and SNP markers may be applied not only for MAS but also to maintain functional gene diversity in the novel cultured grouper.
There are numerous means to improve the tilapia aquaculture industry, and one is to develop disease resistance through selective breeding using molecular markers. In this study, 11 disease-resistance-associated microsatellite markers including 3 markers linked to hamp2, 4 linked to hamp1, 1 linked to pgrn2, 2 linked to pgrn1, and 1 linked to piscidin 4 (TP4) genes were established for tilapia strains farmed in Taiwan after challenge with Streptococcus inae. The correlation analysis of genotypes and survival revealed a total of 55 genotypes related to survival by the chi-square and Z-test. Although fewer markers were found in B and N2 strains compared with A strain, they performed well in terms of disease resistance. It suggested that this may be due to the low potency of some genotypes and the combinatorial arrangement between them. Therefore, a predictive model was built by the genotypes of the parental generation and the mortality rate of different combinations was calculated. The results show the same trend of predicted mortality in the offspring of three new disease-resistant strains as in the challenge experiment. The present findings is a nonkilling method without requiring the selection by challenge with bacteria or viruses and might increase the possibility of utilization of selective breeding using SSR markers in farms.
DNA/genetics , Disease Resistance/genetics , Fish Diseases/genetics , Genetic Markers , Microsatellite Repeats , Selective Breeding , Tilapia/genetics , Animals , Aquaculture , DNA/analysis , Disease Resistance/immunology , Fish Diseases/immunology , Genotype , Taiwan , Tilapia/growth & development , Tilapia/immunology
Spermatozoan ultrastructure and complete mitochondrial genome in the marine bivalve mollusk Meretrix sp. (Taiwan) from Taiwan are described and contrasted with other bivalves, especially within Meretrix. We have examined the features of the mature gonadal spermatozoa of Meretrix sp. (Taiwan) and provided comparisons with the other four Meretrix species (M. petechialis, M. meretrix, M. lyrata, and M. lamarckii). The morphological characteristics of these spermatozoa are diagnostic for each of the species studied here. The most marked interspecific difference was found in the acrosome. Meretrix sp. (Taiwan) is genetically distinct and is a different species from M. petechialis and M. lusoria (Japan) based on complete mitochondrial genome data. Sperm data for Meretrix are limited but show remarkable congruence with the molecular results. We suggest use Meretrix formosa Gwo and Hsu as the scientific name for Taiwanese hard clams, Meretrix sp. (Taiwan). Additional species, particularly the Japanese hard clam (M. lusoria) require examination before this tentative conclusion can be verified.
Bivalvia/genetics , Bivalvia/ultrastructure , Genome, Mitochondrial , Spermatozoa/ultrastructure , Acrosome/ultrastructure , Animals , Cell Nucleus/ultrastructure , Male , Sperm Tail/ultrastructure , Taiwan
Small abalone (Haliotis diversicolor) is a commercially valuable species for both fisheries and aquaculture. The production of annual farmed small abalone in Taiwan, once the highest in the world, has dramatically decreased in the past 15 years, and currently, the industry is close to collapse. Understanding the genetic diversity of small abalone and developing stock identification methods will be useful for genetic breeding, restoring collapsed stocks, managing stocks, and preventing illegal trade. We investigated 307 cultured and wild individuals from Taiwan, Japan, and Bali Island (Indonesia) by using the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis of mtDNA COI gene sequences revealed that the individuals collected from Taiwan, Japan, and Indonesia could be identified, and showed significant genetic divergence. In addition, the Indonesian population (Haliotis diversicolor squamata) was significantly different from the other populations and might need to be considered a separate species. We discovered a single nucleotide polymorphism marker in the mtDNA COI gene that can be used to distinguish the Taiwan population from the Japan population. We also developed a polymerase chain reaction-restriction fragment length polymorphism method for rapid detection. Furthermore, we could identify the cultured stocks, wild population, and hybrid stocks by using 6 microsatellites and amplified fragment length polymorphism. This study contributes useful tools for stock identification and the production of high-disease resistant small abalone strains (Japan × Taiwan or Taiwan × Japan). Efforts should be made to avoid unintentional random genetic mixing of the Taiwan population with the Japan population and subsequent breakdown of population differentiation, which impair local adaptation of the Taiwan wild population. Molecular markers revealed a split between the Taiwan and Japan populations, and the existence of a possible barrier to the free dispersal of small abalone is discussed.
Gastropoda/genetics , Genetic Variation , Animals , DNA, Mitochondrial/genetics , Electron Transport Complex IV/genetics , Gastropoda/classification , Indonesia , Japan , Phylogeny , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Taiwan
Peanut worm (Sipunculus nudus) is a cosmopolitan species mainly distributed in tropical and subtropical coastal waters. Analysis of the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences among S. nudus from GenBank revealed high genetic variation (p-distance, 0.115-0.235; k2p, 0.128-0.297) and paraphyletic relationships. These indicated misidentification and/or cryptic diversity may be present in the genus Sipunculus. To understand the genetic diversity and to manage the recourse of S. nudus, we collected specimens from coastal waters of southern China and Taiwan. In the phylogenetic topology, specimens can be separated into four distinct clades; three of these clades (clade A, B and C) were only represented from this region (southern China and Taiwan), but the clade D grouped with individuals from Central America (Atlantic coast). Furthermore, individuals of clades A and D were collected at the same location, which does not support the hypothesis that this genetic break reflects contemporary geographical isolation. The four distinct clades observed among coastal waters of southern China and Taiwan indicated underestimated diversity. It is noteworthy that the cryptic diversity is vulnerable under high pressure of human activity.
Arachis/parasitology , DNA Barcoding, Taxonomic , Genetic Variation , Polychaeta/classification , Polychaeta/genetics , Animals , China , Cluster Analysis , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/genetics , Electron Transport Complex IV/genetics , Helminths/classification , Helminths/genetics , Molecular Sequence Data , Phylogeography , Sequence Analysis, DNA , Taiwan
Papaya (Carica papaya L.) is established as a cash crop throughout the tropical and subtropical regions due to its easy adaptation to diverse agricultural conditions, high yields, and prompt returns. The sex types of papaya plants are hermaphrodite, male, and female. Among them, hermaphroditic plants are the major type in papaya production, because the fruit has commercial advantages over that of the other sexes. Sex inheritance in papaya is determined by the M and M(h) dominant alleles in males and hermaphrodites, respectively, and a recessive m allele in females. Currently, all hermaphrodite seeds are not available due to the lethality of dominant homozygosity. Therefore, in this study, six male-hermaphrodite-specific markers were developed for a rapid sex identification using multiplex loop-mediated isothermal amplification (mLAMP) to efficiently and precisely select hermaphroditic individuals in the seedling or early growth stage. The LM1-LAMP assay consisted of two sex-LAMP reactions for amplifying two male-specific markers (T12 and Cpsm90) in one reaction, and showed several advantages in terms of a rapid reaction time (<1 h), isothermal conditions (less equipment required), a high efficiency (0.5 ng of DNA required in the reaction mixture), and an economical reaction system (5 µl in volume). The established method can be easily performed in the field by visual inspection and facilitates the selection of all hermaphroditic individuals in papaya production.
Carica/genetics , Nucleic Acid Amplification Techniques/methods , DNA Primers/genetics , DNA, Plant/genetics , Genetic Markers , Seedlings/genetics , Sensitivity and Specificity
Species and sex identification are among the most important parameters for conservation management. However, it is extremely difficult to perform such identification in Formosa landlocked salmon (Oncorhynchus masou formosanus). Both sexual dimorphism in landlocked dwarf form Formosa landlocked salmon and morphological difference among cherry salmon complex are minimal. We developed a simple, rapid and noninvasive method for identifying sex and species of this critically endangered species using a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay showed the advantage of simple detection (evaluated by visual inspection), rapid reaction time (< 1 h), isothermal condition (less equipment required) and high efficiency (only 0.5-5 pg of DNA was required in the reaction mixture). Therefore, the method is more economical and practical than PCR. The LAMP assay can be easily performed in the field and is a valuable tool for detecting sex ratios in wild populations and identifying species in commercial imports. This is the first application of LAMP in identifying species and sex of salmonids as far as we know and clearly shows the potential application of LAMP in molecular ecology and conservation efforts.
Conservation of Natural Resources/methods , Nucleic Acid Amplification Techniques/methods , Oncorhynchus/genetics , Animals , DNA Primers/genetics , Electrophoresis, Agar Gel , Sensitivity and Specificity , Sex Determination Analysis , Species Specificity , Taiwan
