Transcriptomics and gut microbiome analysis of the edible herb Bidens pilosa as a functional feed additive to promote growth and metabolism in tilapia (Oreochromis spp.).

To reduce the use of antibiotics and chemicals in aquaculture, an edible herb, Bidens pilosa, has been selected as a multifunctional feed additive. Although there has been considerable research into the effects of B. pilosa on poultry, the wider effects of B. pilosa, particularly on the growth and gut microbiota of fish, remain largely unexplored. We aimed to investigate the interactive effects between the host on growth and the gut microbiota using transcriptomics and the gut microbiota in B. pilosa-fed tilapia. In this study, we added 0.5% and 1% B. pilosa to the diet and observed that the growth performance of tilapia significantly increased over 8 weeks of feeding. Comparative transcriptome analysis was performed on RNA sequence profiles obtained from liver and muscle tissues. Functional enrichment analysis revealed that B. pilosa regulates several pathways and genes involved in amino acid metabolism, lipid metabolism, carbohydrate metabolism, endocrine system, signal transduction, and metabolism of other amino acids. The expression of the selected growth-associated genes was validated by qRT-PCR. The qRT-PCR results indicated that B. pilosa may enhance growth performance by activating the expression of the liver igf1 and muscle igf1rb genes and inhibiting the expression of the muscle negative regulator mstnb. Both the enhancement of liver endocrine IGF1/IGF1Rb signaling and the suppression of muscle autocrine/paracrine MSTN signaling induced the expression of myogenic regulatory factors (MRFs), myod1, myog and mrf4 in muscle to promote muscle growth in tilapia. The predicted function of the gut microbiota showed several significantly different pathways that overlapped with the KEGG enrichment results of differentially expressed genes in the liver transcriptomes. This finding suggested that the gut microbiota may influence liver metabolism through the gut-liver axis in B. pilosa-fed tilapia. In conclusion, dietary B. pilosa can regulate endocrine IGF1 signaling and autocrine/paracrine MSTN signaling to activate the expression of MRFs to promote muscle growth and alter the composition of gut bacteria, which can then affect liver amino acid metabolism, carbohydrate metabolism, endocrine system, lipid metabolism, metabolism of other amino acids, and signal transduction in the host, ultimately enhancing growth performance. Our results suggest that B. pilosa has the potential to be a functional additive that can be used as an alternative to reduce antibiotic use as a growth promoter in aquaculture.

Apoptosis in gonadal somatic cells of scleractinian corals: implications of structural adjustments for gamete production and release.

Apoptosis is an evolutionarily conserved process of programmed cell death. Here, we show structural changes in the gonads caused by apoptosis during gametogenesis in the scleractinian coral, Euphyllia ancora. Anatomical and histological analyses revealed that from the non-spawning to the spawning season, testes and ovaries increased in size due to active proliferation, differentiation and development of germ cells. Additionally, the thickness and cell density of the gonadal somatic layer decreased significantly as the spawning season approached. Further analyses demonstrated that the changes in the gonadal somatic layer were caused by apoptosis in a subpopulation of gonadal somatic cells. The occurrence of apoptosis in the gonadal somatic layer was also confirmed in other scleractinian corals. Our findings suggest that decreases in thickness and cell density of the gonadal somatic layer are structural adjustments facilitating oocyte and spermary (male germ cell cluster) enlargement and subsequent gamete release from the gonads. In animal reproduction, apoptosis in germ cells is an important process that controls the number and quality of gametes. However, apoptosis in gonadal somatic cells has rarely been reported among metazoans. Thus, our data provide evidence for a unique use of apoptosis in animal reproduction.

Immunodetection of acetylated alpha-tubulin in stony corals: Evidence for the existence of flagella in coral male germ cells.

The molecular and cellular characteristics of male germ cell development remain largely unknown in corals. This study focused on the expression pattern of acetylated α-tubulin (Ac-α-Tu), which is involved in male germ cell development in various animals across taxa, to gain a better understanding of male germ cell development in the stony coral Euphyllia ancora. Immunohistochemical analysis of the different stages of male germ cells showed the presence of filamentous Ac-α-Tu in the early to late stages of male germ cells-such as spermatogonia, spermatocytes, and spermatids-as well as in the flagella of mature sperm. Immunocytochemical and transmission electron microscope analyses demonstrated that early-stage male germ cells possess long flagella containing Ac-α-Tu. The presence of filamentous Ac-α-Tu was also immunohistochemically demonstrated in the male germ cells from 14 other coral species, implying that possession of flagella with Ac-α-Tu is a common characteristic of male germ cells in stony corals. Therefore, as a distinctive cellular characteristic of male germ cells, Ac-α-Tu could be used as a male germ cell marker in stony corals; indeed, immunolabeling for Ac-α-Tu may be a useful method to aid in the identification and morphological observation of male germ cells in various corals in basic and applied biology (e.g., aquaculture) as well as in ecological studies.

Genetic and pathogenic difference between Streptococcus agalactiae serotype Ia fish and human isolates.

BACKGROUND: Streptococcus agalactiae (GBS) is a common pathogen to infect newborn, woman, the elderly, and immuno-compromised human and fish. 37 fish isolates and 554 human isolates of the GBS in 2007-2012 were investigated in serotypes, antibiotic susceptibility, genetic difference and pathogenicity to tilapia. RESULTS: PCR serotyping determined serotype Ia for all fish GBS isolates and only in 3.2 % (3-4.2 %) human isolates. For fish isolates, all consisted a plasmid less than 6 kb and belonged to ST7 type, which includes mainly pulsotypes I and Ia, with a difference in a deletion at the largest DNA fragment. These fish isolates were susceptible to all antimicrobials tested in 2007 and increased in non-susceptibility to penicillin, and resistance to clindamycin and ceftriaxone in 2011. Differing in pulsotype and lacking plasmid from fish isolates, human serotype Ia isolates were separated into eight pulsotypes II-IX. Main clone ST23 included pulsotypes II and IIa (50 %) and ST483 consisted of pulsotype III. Human serotype Ia isolates were all susceptible to ceftriaxone and penicillin and few were resistant to erythromycin, azithromycin, clindamycin, levofloxacin and moxifloxacine with the resistant rate of 20 % or less. Using tilapia to analyze the pathogenesis, fish isolates could cause more severe symptoms, including hemorrhage of the pectoral fin, hemorrhage of the gill, and viscous black and common scites, and mortality (>95 % for pulsotype I) than the human isolates (<30 %); however, the fish pulostype Ia isolate 912 with deletion caused less symptoms and the lowest mortality (<50 %) than pulsotype I isolates. CONCLUSION: Genetic, pathogenic, and antimicrobial differences demonstrate diverse origin of human and fish serotype Ia isolates. The pulsotype Ia of fish serotype Ia isolates may be used as vaccine strains to prevent the GBS infection in fish.

Complete genome sequence of a potential new species Vibrio sp. NTOU-M3 isolated from hard clam, Meretrix taiwanica, in Taiwan.

Vibrio sp. NTOU-M3 is a potential new bacterium isolated from hard clam (Meretrix taiwanica) in the estuarine region of Taiwan. The complete sequences obtained using Oxford Nanopore Technologies and Illumina sequencing consist of a 3,272,438-bp large circular chromosome and a 1,584,497-bp small circular chromosome.

Transcriptomic Insights and the Development of Microsatellite Markers to Assess Genetic Diversity in the Broodstock Management of Litopenaeus stylirostris.

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.

FastAnnotator--an efficient transcript annotation web tool.

BACKGROUND: Recent developments in high-throughput sequencing (HTS) technologies have made it feasible to sequence the complete transcriptomes of non-model organisms or metatranscriptomes from environmental samples. The challenge after generating hundreds of millions of sequences is to annotate these transcripts and classify the transcripts based on their putative functions. Because many biological scientists lack the knowledge to install Linux-based software packages or maintain databases used for transcript annotation, we developed an automatic annotation tool with an easy-to-use interface. METHODS: To elucidate the potential functions of gene transcripts, we integrated well-established annotation tools: Blast2GO, PRIAM and RPS BLAST in a web-based service, FastAnnotator, which can assign Gene Ontology (GO) terms, Enzyme Commission numbers (EC numbers) and functional domains to query sequences. RESULTS: Using six transcriptome sequence datasets as examples, we demonstrated the ability of FastAnnotator to assign functional annotations. FastAnnotator annotated 88.1% and 81.3% of the transcripts from the well-studied organisms Caenorhabditis elegans and Streptococcus parasanguinis, respectively. Furthermore, FastAnnotator annotated 62.9%, 20.4%, 53.1% and 42.0% of the sequences from the transcriptomes of sweet potato, clam, amoeba, and Trichomonas vaginalis, respectively, which lack reference genomes. We demonstrated that FastAnnotator can complete the annotation process in a reasonable amount of time and is suitable for the annotation of transcriptomes from model organisms or organisms for which annotated reference genomes are not avaiable. CONCLUSIONS: The sequencing process no longer represents the bottleneck in the study of genomics, and automatic annotation tools have become invaluable as the annotation procedure has become the limiting step. We present FastAnnotator, which was an automated annotation web tool designed to efficiently annotate sequences with their gene functions, enzyme functions or domains. FastAnnotator is useful in transcriptome studies and especially for those focusing on non-model organisms or metatranscriptomes. FastAnnotator does not require local installation and is freely available at http://fastannotator.cgu.edu.tw.

Development of Disease-Resistance-Associated Microsatellite DNA Markers for Selective Breeding of Tilapia (Oreochromis spp.) Farmed in Taiwan.

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.

Mathematical modeling suggests high potential for the deployment of floating photovoltaic on fish ponds.

Rising energy needs and pressure to reduce greenhouse gas emissions have led to a significant increase in solar power projects worldwide. Recently, the development of floating photovoltaic (FPV) systems offers promising opportunities for land scarce areas. We present a dynamic model that simulates the main biochemical processes in a milkfish (Chanos chanos) pond subject to FPV cover. We validated the model against experimental data collected from ponds with and without cover during two production seasons (winter and summer) and used it to perform a Monte-Carlo analysis of the ecological effects of different extents of cover. Our results show that the installation of FPV on fish ponds may have a moderate negative impact on fish production, due to a reduction in dissolved oxygen levels. However, losses in fish production are more than compensated by gains in terms of energy (capacity of around 1.13 MW/ha). We estimated that, with approximately 40,000 ha of aquaculture ponds in Taiwan, the deployment of FPV on fish ponds in Taiwan could accommodate an installed capacity more twice as high as the government's objective of 20 GW solar power by 2025. We argue that the rules and regulations pertaining to the integration of FPV on fish ponds should be updated to allow realizing the full potential of this new green technology.

Hard clam extracts induce atypical apoptosis in human gastric cancer cells.

Hard clams (HCs) are a nutritionally high-quality and popular seafood, and are established to be a potent antitumor food. The aim of the present study was to determine whether HC extracts induce apoptosis in the human gastric cancer cell line, AGS. In contrast with previously reported methods of extraction, crude extracts of HC were obtained by freezing and thawing and by a method free of hot water or organic solvents. The composition, quality and properties of the HC extracts were demonstrated to be stable since the extracts that were evaluated by capillary electrophoresis and HPLC analysis at different timepoints were similar. HC extracts also have an inhibitory effect against the survival of AGS cells. Treatment with HC extracts induced a marked sub-G1 DNA peak and reduced the expression of the anti-apoptotic genes BIRC5 and KPNA2. However, hallmarks of classical apoptosis such as DNA fragmentation and apoptotic body formation were not observed, indicating atypical apoptosis. Furthermore, it was revealed that HC extracts interrupted cell cycle progression in AGS cells through altered expression of six cell cycle-associated genes: CDC20, KPNA2, BIRC5, ANAPC2, CDKN1A and RB1. The present findings suggest that HC may contribute to a novel future anticancer agent.