Effects on growth performance and immunity of Monopterus albus after high temperature stress.

To investigate the impact of the effect of high temperature stimulation on Monopterus albus larvae after a certain period of time, five experimental groups were established at different temperatures. Then, the M. albus under high temperature stress was fed at 30°C for 70 days. After that, the growth index of the M. albus was counted and analyzed. In terms of growth index, high temperature stress had significant effects on FCR, FBW, WGR, and SGR of M. albus (p < 0.05). The SR increased after being stimulated by temperature (p < 0.1). The study revealed that liver cells of M. albus were harmed by elevated temperatures of 36°C and 38°C. In the experimental group, the activities of digestive enzymes changed in the same trend, reaching the highest point in the 32°C group and then decreasing, and the AMS activity in the 38°C group was significantly different from that in the 30°C group (p < 0.05). The activities of antioxidase in liver reached the highest at 34°C, which was significantly different from those at 30°C (p < 0.05). In addition, the expression levels of TLR1, C3, TNF-α, and other genes increased in the experimental group, reaching the highest point at 34°C, and the expression level of the IL-1ß gene reached the highest point at 32°C, which was significantly different from that at 30°C (p < 0.05). However, the expression level of the IRAK3 gene decreased in the experimental group and reached its lowest point at 34°C (p < 0.05). The expression level of the HSP90α gene increased with the highest temperature stimulus and reached its highest point at 38°C (p < 0.05). In the α diversity index of intestinal microorganisms in the experimental group, the observed species, Shannon, and Chao1 indexes in the 34°C group were the highest (p < 0.05), and ß diversity analysis revealed that the intestinal microbial community in the experimental group was separated after high temperature stimulation. At the phylum level, the three dominant flora are Proteus, Firmicutes, and Bacteroides. Bacteroides and Macrococcus abundance increased at the genus level, but Vibrio and Aeromonas abundance decreased. To sum up, appropriate high-temperature stress can enhance the immunity and adaptability of M. albus. These results show that the high temperature stimulation of 32°C-34°C is beneficial to the industrial culture of M. albus.

A delayed and unsynchronized ovary development as revealed by transcriptome of brain and pituitary of Coilia nasus.

Coilia nasus is an anadromous fish that has been successfully domesticated in the last decade due to its high economic value. The fish exhibits a delayed ovary development during the reproductive season, despite breeding and selection for five to six offspring. The molecular mechanism of the delayed ovary development is still unknown, so the obstacles have not been removed in the large-scale breeding program. This study aims to investigate the key genes regulating ovarian development by comparing the transcriptomes of ovarian-stage IV and stage II brain/pituitary of Coilia nasus. Ovarian stages were validated by histological sections. A total of 75,097,641 and 66,735,592 high-quality reads were obtained from brain and pituitary transcriptomes, respectively, and alternatively spliced transcripts associated with gonadal development were detected. Compared to ovarian Ⅱ- brain, 515 differentially expressed genes (DEGs) were upregulated and 535 DEGs were downregulated in ovarian Ⅳ- brain, whereas 470 DEGs were upregulated and 483 DEGs were downregulated in ovarian Ⅳ- pituitary compared to ovarian Ⅱ- pituitary. DEGs involved in hormone synthesis and secretion and in the GnRH signaling pathway were screened. Weighted gene co-expression network analysis identified gene co-expression modules that were positively correlated with ovarian phenotypic traits. The hub genes Smad4 and TRPC4 in the modules were co-expressed with DEGs including Kiss1 receptor and JUNB, suggesting that ovarian development is controlled by a hypothalamic-pituitary-gonadal axis. Our results have provided new insights that advance our understanding of the molecular mechanism of C. nasus reproductive functions and will be useful for future breeding.

Biological Function of Prophage-Related Gene Cluster ΔVpaChn25_RS25055~ΔVpaChn25_0714 of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is the primary foodborne pathogen known to cause gastrointestinal infections in humans. Nevertheless, the molecular mechanisms of V. parahaemolyticus pathogenicity are not fully understood. Prophages carry virulence and antibiotic resistance genes commonly found in Vibrio populations, and they facilitate the spread of virulence and the emergence of pathogenic Vibrio strains. In this study, we characterized three such genes, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055, within the largest prophage gene cluster in V. parahaemolyticus CHN25. The deletion mutants ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 were derived with homologous recombination, and the complementary mutants ΔVpaChn25_0713-com, ΔVpaChn25_0714-com, ΔVpaChn25_RS25055-com, ΔVpaChn25_RS25055-0713-0714-com were also constructed. In the absence of the VpaChn25_RS25055, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055-0713-0714 genes, the mutants showed significant reductions in low-temperature survivability and biofilm formation (p < 0.001). The ΔVpaChn25_0713, ΔVpaChn25_RS25055, and ΔVpaChn25_RS25055-0713-0714 mutants were also significantly defective in swimming motility (p < 0.001). In the Caco-2 model, the above four mutants attenuated the cytotoxic effects of V. parahaemolyticus CHN25 on human intestinal epithelial cells (p < 0.01), especially the ΔVpaChn25_RS25055 and ΔVpaChn25_RS25055-0713-0714 mutants. Transcriptomic analysis showed that 15, 14, 8, and 11 metabolic pathways were changed in the ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 mutants, respectively. We labeled the VpaChn25_RS25055 gene with superfolder green fluorescent protein (sfGFP) and found it localized at both poles of the bacteria cell. In addition, we analyzed the evolutionary origins of the above genes. In summary, the prophage genes VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055 enhance V. parahaemolyticus CHN25's survival in the environment and host. Our work improves the comprehension of the synergy between prophage-associated genes and the evolutionary process of V. parahaemolyticus.

Establishment of a Coilia nasus Spermatogonial Stem Cell Line Capable of Spermatogenesis In Vitro.

The process by which spermatogonial stem cells (SSCs) continuously go through mitosis, meiosis, and differentiation to produce gametes that transmit genetic information is known as spermatogenesis. Recapitulation of spermatogenesis in vitro is hindered by the challenge of collecting spermatogonial stem cells under long-term in vitro culture conditions. Coilia nasus is a commercially valuable anadromous migrant fish found in the Yangtze River in China. In the past few decades, exploitation and a deteriorating ecological environment have nearly caused the extinction of C. nasus's natural resources. In the present study, we established a stable spermatogonial stem cell line (CnSSC) from the gonadal tissue of the endangered species C. nasus. The cell line continued to proliferate and maintain stable cell morphology, a normal diploid karyotype, and gene expression patterns after more than one year of cell culture (>80 passages). Additionally, CnSSC cells could successfully differentiate into sperm cells through a coculture system. Therefore, the establishment of endangered species spermatogonial stem cell lines is a model for studying spermatogenesis in vitro and a feasible way to preserve germplasm resources.

Full-Length Transcriptomes and Sex-Based Differentially Expressed Genes in the Brain and Ganglia of Giant River Prawn Macrobrachium rosenbergii.

Macrobrachium rosenbergii is an important aquaculture prawn that exhibits sexual dimorphism in growth, with males growing much faster than females. However, the mechanisms controlling these complex traits are not well understood. The nervous system plays an important role in regulating life functions. In the present work, we applied PacBio RNA-seq to obtain and characterize the full-length transcriptomes of the brains and thoracic ganglia of female and male prawns, and we performed comparative transcriptome analysis of female and male prawns. A total of 159.1-Gb of subreads were obtained with an average length of 2175 bp and 93.2% completeness. A total of 84,627 high-quality unigenes were generated and annotated with functional databases. A total of 6367 transcript factors and 6287 LncRNAs were predicted. In total, 5287 and 6211 significantly differentially expressed genes (DEGs) were found in the brain and thoracic ganglion, respectively, and confirmed by qRT-PCR. Of the 435 genes associated with protein processing pathways in the endoplasmic reticula, 42 DEGs were detected, and 21/26 DEGs with upregulated expression in the male brain/thoracic ganglion. The DEGs in this pathway are regulated by multiple LncRNAs in polypeptide folding and misfolded protein degradation in the different organs and sexes of the prawn. Our results provide novel theories and insights for studying the nervous system, sexual control, and growth dimorphism.

Approaches to Reduce Rice Blast Disease Using Knowledge from Host Resistance and Pathogen Pathogenicity.

Rice is one of the staple foods for the majority of the global population that depends directly or indirectly on it. The yield of this important crop is constantly challenged by various biotic stresses. Rice blast, caused by Magnaporthe oryzae (M. oryzae), is a devastating rice disease causing severe yield losses annually and threatening rice production globally. The development of a resistant variety is one of the most effective and economical approaches to control rice blast. Researchers in the past few decades have witnessed the characterization of several qualitative resistance (R) and quantitative resistance (qR) genes to blast disease as well as several avirulence (Avr) genes from the pathogen. These provide great help for either breeders to develop a resistant variety or pathologists to monitor the dynamics of pathogenic isolates, and ultimately to control the disease. Here, we summarize the current status of the isolation of R, qR and Avr genes in the rice-M. oryzae interaction system, and review the progresses and problems of these genes utilized in practice for reducing rice blast disease. Research perspectives towards better managing blast disease by developing a broad-spectrum and durable blast resistance variety and new fungicides are also discussed.

Low particle concentrations of nanoplastics impair the gut health of medaka.

The environmental occurrence of nanoplastics (NPs) is now evident but their long-term impacts on organisms are unclear, limiting ecological and health risk assessment. We hypothesized that chronic exposure to low particle concentrations of NPs can result in gut-associated toxicity, and subsequently affect survival of fish. Japanese medaka Oryzias latipes were exposed to polystyrene NPs (diameter 100 nm; 0, 10, 104, and 106 items/L) for 3 months, and histopathology, digestive and antioxidant enzymes, immunity, intestinal permeability, gut microbiota, and mortality were assessed. NP exposures caused intestinal lesions, and increased intestinal permeability of the gut. The trypsin, lipase, and chymotrypsin activities were increased, but the amylase activity was decreased. Oxidative damage was reflected by the decreased superoxide dismutase and alkaline phosphatase and increased malondialdehyde, catalase, and lysozyme. The integrated biomarkers response index values of all NP-exposed medaka were significantly increased compared to the control group. Moreover, NP exposures resulted in a decrease of diversity and changed the intestinal microbiota composition. Our results provide new evidence that long-term NPs exposure impaired the health of fish at extremely low particle concentrations, suggesting the need for long-term toxicological studies resembling environmental particle concentrations when assessing the risk of NPs.

Identification of Elite R-Gene Combinations against Blast Disease in Geng Rice Varieties.

Rice blast, caused by the Magnaporthe oryzae fungus, is one of the most devastating rice diseases worldwide. Developing resistant varieties by pyramiding different blast resistance (R) genes is an effective approach to control the disease. However, due to complex interactions among R genes and crop genetic backgrounds, different R-gene combinations may have varying effects on resistance. Here, we report the identification of two core R-gene combinations that will benefit the improvement of Geng (Japonica) rice blast resistance. We first evaluated 68 Geng rice cultivars at seedling stage by challenging with 58 M. oryzae isolates. To evaluate panicle blast resistance, we inoculated 190 Geng rice cultivars at boosting stage with five groups of mixed conidial suspensions (MCSs), with each containing 5-6 isolates. More than 60% cultivars displayed moderate or lower levels of susceptibility to panicle blast against the five MCSs. Most cultivars contained two to six R genes detected by the functional markers corresponding to 18 known R genes. Through multinomial logistics regression analysis, we found that Pi-zt, Pita, Pi3/5/I, and Pikh loci contributed significantly to seedling blast resistance, and Pita, Pi3/5/i, Pia, and Pit contributed significantly to panicle blast resistance. For gene combinations, Pita+Pi3/5/i and Pita+Pia yielded more stable pyramiding effects on panicle blast resistance against all five MCSs and were designated as core R-gene combinations. Up to 51.6% Geng cultivars in the Jiangsu area contained Pita, but less than 30% harbored either Pia or Pi3/5/i, leading to less cultivars containing Pita+Pia (15.8%) or Pita+Pi3/5/i (5.8%). Only a few varieties simultaneously contained Pia and Pi3/5/i, implying the opportunity to use hybrid breeding procedures to efficiently generate varieties with either Pita+Pia or Pita+Pi3/5/i. This study provides valuable information for breeders to develop Geng rice cultivars with high resistance to blast, especially panicle blast.

Small Peptides Isolated from Enzymatic Hydrolyzate of Pneumatophorus japonicus Bone Promote Sleep by Regulating Circadian Rhythms.

Due to the high addiction and side effects of medicines, people have increasingly inclined to natural and healthy peptides to improve sleep. Herein, we isolated novel peptides with sleep-promoting ability from Pneumatophorus japonicus bone peptides (PBPs) and constructed an insomniac zebrafish model as a demonstration, incorporating behavioral and transcriptomic approaches to reveal the sleep-promoting effect and mechanism of PBPs. Specifically, a sequential targeting isolation approach was developed to refine and identify a peptide with remarkable sleep-promoting activity, namely TG7 (Tyr-Gly-Asn-Pro-Trp-Glu-Lys). TG7 shows comparable effects and a similar action pathway to melatonin in improving sleep. TG7 restores abnormal behavior of insomnia zebrafish to normal levels by upregulating the hnrnpa3 gene. The peptide downregulates per1b gene but upregulates cry1b, cry1ba and per2, improving the circadian rhythm. Furthermore, TG7 upregulates the genes gnb3b, arr3b and opn1mw1 to regulate the visual function. The above results indicate that TG7 improves circadian rhythms and attenuated abnormal alterations in visual function and motility induced by light, allowing for effective sleep promotion. This study isolated sleep-promoting peptides from PBPs, which provides a theoretical basis for the development of subsequent sleep-promoting products based on protein peptides.

Chronic nanoplastic exposure induced oxidative and immune stress in medaka gonad.

Nanoplastic (NP) pollution is a global issue because of its widespread occurrence and potential toxicity. Many studies have investigated the impacts of the short-term toxicity of NPs on organisms. Until now, only a few studies have assessed the toxicological effects of prolonged exposure to NPs at low concentrations in fish. In this study, the effects of NPs (nano-polystyrene microspheres, diameter: 100 nm) on immune and oxidative stress response, histopathology, and survival in medaka were evaluated. The effects of different concentrations (0, 10, 104, and 106 particles/L) of nanoplastics were studied in medaka Oryzias latipes after 3 months of exposure. Lysozyme enzyme activity, oxidative stress-related biomarkers (i.e., superoxide dismutase, catalase, and glutathione peroxidase), and malondialdehyde levels were decreased under NP exposure. The gonadal histology showed that high NP exposure (106 particles/L) inhibited the process of spermatogenesis and oogenesis processes, implying delayed maturation of the gonad. Furthermore, the IBR and PCA analysis revealed the potential biotoxicity of NPs and the total survival rate of medaka was significantly reduced due to the long-term exposure to NPs. Overall, prolonged exposure to low concentrations of NPs is harmful to the health of medaka gonads. In the long run, this may threaten the fish reproduction and population, suggesting the need for long-term toxicological studies to predict the aquatic animal health in nature.

Chromosome-Level Assembly of Male Opsariichthys bidens Genome Provides Insights into the Regulation of the GnRH Signaling Pathway and Genome Evolution.

The hook snout carp Opsariichthys bidens is an important farmed fish in East Asia that shows sexual dimorphism in growth, with males growing faster and larger than females. To understand these complex traits and improve molecular breeding, chromosome-level genome assembly of male O. bidens was performed using Illumina, Nanopore, and Hi-C sequencing. The 992.9 Mb genome sequences with a contig N50 of 5.2 Mb were anchored to 38 chromosomes corresponding to male karyotypes. Of 30,922 functionally annotated genes, 97.5% of BUSCO genes were completely detected. Genome evolution analysis showed that the expanded and contracted gene families in the male O. bidens genome were enriched in 76 KEGG pathways, and 78 expanded genes were involved in the GnRH signaling pathway that regulates the synthesis and secretion of luteinizing hormone and glycoprotein hormones, further acting on male growth by inducing growth hormone. Compared to the released female O. bidens genome, the number of annotated genes in males was much higher (23,992). The male chromosome LG06 exhibited over 97% identity with the female GH14/GH38. Male-specific genes were identified for LG06, where structural variation, including deletions and insertions, occurred at a lower rate, suggesting a centric fusion of acrocentric chromosomes GH14 and GH38. The genome-synteny analysis uncovered significant inter-chromosome conservation between male O. bidens and grass carp, the former originating from ancestral chromosome breakage to increase the chromosome number. Our results provide a valuable genetic resource for studying the regulation of sexual dimorphism, sex-determining mechanisms, and molecular-guided breeding of O. bidens.

Establishment of a Coilia nasus Gonadal Somatic Cell Line Capable of Sperm Induction In Vitro.

Coilia nasus is an important economic anadromous migratory fish of the Yangtze River in China. In recent years, overfishing and the deterioration of the ecological environment almost led to the extinction of the wild resources of C.nasus. Thus, there is an urgent need to protect this endangered fish. Recently, cell lines derived from fish have proven a promising tool for studying important aspects of aquaculture. In this study, a stable C. nasus gonadal somatic cell line (CnCSC) was established and characterized. After over one year of cell culture (>80 passages), this cell line kept stable growth. RT-PCR results revealed that the CnGSC expressed some somatic cell markers such as clu, fshr, hsd3ß, and sox9b instead of germ cell markers like dazl, piwi, and vasa. The strong phagocytic activity of CnGSC suggested that it contained a large number of Sertoli cells. Interestingly, CnGSC could induce medaka spermatogonial cells (SG3) to differentiate into elongated spermatids while co-cultured together. In conclusion, we established a C. nasus gonadal somatic cell line capable of sperm induction in vitro. This research provides scientific evidence for the long-term culture of a gonadal cell line from farmed fish, which would lay the foundation for exploring the regulatory mechanisms between germ cells and somatic cells in fish.

Generation of a Normal Long-Term-Cultured Chinese Hook Snout Carp Spermatogonial Stem Cell Line Capable of Sperm Production In Vitro.

Opsariichthys bidens belongs to the family Cyprinidae and is a small freshwater economic fish widely distributed in China. In recent years, the natural resources of O. bidens have been drastically reduced due to overfishing and the destruction of the water environment. The in vitro culture and long-term preservation of germ stem cells are the key technologies to keep genetic resources from degeneration. However, except for the establishment of the first long-term cultured medaka spermatogonia cell line (SSC) capable of producing sperm in vitro in 2004, no other long-term cultured SSC line has been found in other fish species. In this study, we successfully established another long-term-cultured spermatogonial stem cell line from Opsariichthys bidens (ObSSC). After more than 2 years of culture, ObSSC had a diploid karyotype and stable growth, with the typical gene expression patterns of SSC. Under in vitro culture, ObSSC could be induced to differentiate into sperm and other different types of somatic cells. In vivo, ObSSC could differentiate into different cells of three germ layers upon being transplanted into zebrafish embryos. Our research helps to explore the potential and regulation mechanism of fish SSC differentiation and spermatogenesis in vitro, provides a new way for solving the problem of fish genetic resource degradation and lays a foundation for further research on fish germ cell transplantation.

Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method.

Crucian carp (Carassius auratus) is one of the major freshwater species and is also a common food fish in China. Recently, Carassius auratus herpesvirus (CaHV) could induce fatal viral disease with high mortality of crucian carp, which had caused huge economic losses. In this study, we described a rapid and simple recombinase-aid amplification (RAA) assay coupled with lateral flow dipstick (LFD), which could achieve sensitive diagnosis of tumor necrosis factor receptor (TNFR) of CaHV within 35 min at 40°C. Our RAA-LFD method had a satisfactory detection limit of 100 gene copies per reaction, which was 100-fold more sensitive than traditional PCR. In addition, no cross-reaction was observed with other viral pathogens, including koi herpesvirus (KHV), cyprinid herpesvirus 2 (CyHV-2), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV). Furthermore, the overall cost of the method was cut in half compared to previous studies. In conclusion, RAA-LFD assay is therefore, a promising alternative for point-of-care testing (POCT) of CaHV, which is feasible and of certain value in application of aquatic disease control.

Comparative transcriptome analysis of brain and gonad reveals reproduction-related miRNAs in the giant prawn, Macrobrachium rosenbergii.

Macrobrachium rosenbergii (M. rosenbergii), as a species of common prawn, is a delicacy that is consumed all over the world. By interacting with the target gene 3'-untranslated region (3'-UTR), microRNAs (miRNAs) regulate its expression and ultimately participate in the regulation of reproductive development. However, research focusing on miRNA regulation during gonadal development in M. rosenbergii received very little attention. To explore the association between miRNA and reproduction, we performed RNA sequencing (RNA-seq) on brain and gonad organs in male and female M. rosenbergii. A total of 494 miRNAs were obtained in RNA-seq, including 31 and 59 differentially expressed (DE) miRNAs in the brain and gonads, respectively. Furthermore, 9 DE miRNAs were randomly selected from the brain and gonads, and qRT-PCR was conducted to validate the results of RNA-seq. Interestingly, dpu-miR-133 was found to be substantially expressed in the male brain and testis but poorly expressed in the female brain, ovary, and other organs. Analysis of dpu-miR-133 by Targetscan and MiRanda predicted to target 5-HT1. Furthermore, the dual-luciferase reporter assay manifested that dpu-miR-133 can combine with 5-HT1. Overall, our research work provides basic data for further study on the miRNA-mediated regulation of brain, gonad, and reproductive development of study M. rosenbergii.

Identification and expression analysis of sex biased miRNAs in chinese hook snout carp Opsariichthys bidens.

As an economically important fish, Opsariichthys bidens has obvious sexual dimorphism and strong reproductive capacity, but no epigenetics study can well explain its phenotypic variations. In recent years, many microRNAs involved in the regulation of reproductive development have been explored. In this study, the small RNA libraries of O. bidens on the testis and ovary were constructed and sequenced. A total of 295 known miRNAs were obtained and 100 novel miRNAs were predicted. By comparing testis and ovary libraries, 115 differentially expressed (DE) miRNAs were selected, of which 53 were up-regulated and 62 were down-regulated. A total of 64 GO items (padj < 0.01) and 206 KEGG pathways (padj < 0.01) were enriched in the target gene of miRNA. After that, the expression levels of nine DE miRNAs, including let-7a, miR-146b, miR-18c, miR-202-5p, miR-135c, miR-9-5p, miR-34c-3p, miR-460-5p and miR-338 were verified by qRT-PCR. Furthermore, bidirectional prediction of DE miRNAs and sex-related genes was carried out and the targeting correlation between miR-9-5p and nanos1 was verified by Dual-Luciferase reporter assay. Our findings identified the differentially expressed miRNA and paved the way to new possibilities for the follow-up study on the mechanism of miRNA-mRNA interaction in the gonads of O. bidens.

Integrated mRNA and miRNA Expression Profile Analysis of Female and Male Gonads in Acrossocheilus fasciatus.

MicroRNAs (miRNAs) are regarded as key regulators in gonadal development and sex determination in diverse organisms. However, the functions of miRNAs in gonads of Acrossocheilus fasciatus, an economically important freshwater species in the south of China, are still unclear. Here, high-throughput sequencing was performed to investigate the mRNA and miRNAs on gonads of A. fasciatus. In total, 49,447 unigenes were obtained, including 11,635 differentially expressed genes (DEGs), among which 4147 upregulated genes and 7488 downregulated genes in the testis compared to the ovary, while 300 (237 known, and 63 novel) miRNAs with 36 differentially expressed miRNAs (DEMs) were identified, from which 17 upregulated and 19 downregulated DEMs. GO and KEGG enrichment analysis were performed to analyze the potential biological functions of DEGs and DEMs. Using qRT-PCR, 9 sex-related genes and 9 miRNAs were selected to verify the sequencing data. By dual-luciferase reporter assay, miR-22a-5p and miR-22b-5p interaction with piwil1, and miR-10d-5p interaction with piwil2 were identified. These findings could provide a reference for miRNA-regulated sex control of A. fasciatus and may reveal new insights into aquaculture and breeding concepts.

Development of Rice Variety With Durable and Broad-Spectrum Resistance to Blast Disease Through Marker-Assisted Introduction of Pigm Gene.

Rice blast, caused by Magnaporthe oryzae (M. oryzae), is one of the most destructive diseases threatening rice production worldwide. Development of resistant cultivars using broad-spectrum resistance (R) genes with high breeding value is the most effective and economical approach to control this disease. In this study, the breeding potential of Pigm gene in geng/japonica rice breeding practice in Jiangsu province was comprehensively evaluated. Through backcross and marker-assisted selection (MAS), Pigm was introduced into two geng rice cultivars (Wuyungeng 32/WYG32 and Huageng 8/HG8). In each genetic background, five advanced backcross lines with Pigm (ABLs) and the same genotypes as the respective recurrent parent in the other 13 known R gene loci were developed. Compared with the corresponding recurrent parent, all these ABLs exhibited stronger resistance in seedling inoculation assay using 184 isolates collected from rice growing regions of the lower region of the Yangtze River. With respect to panicle blast resistance, all ABLs reached a high resistance level to blast disease in tests conducted in three consecutive years with the inoculation of seven mixed conidial suspensions collected from different regions of Jiangsu province. In natural field nursery assays, the ABLs showed significantly higher resistance than the recurrent parents. No common change on importantly morphological traits and yield-associated components was found among the ABLs, demonstrating the introduction of Pigm had no tightly linked undesirable effect on rice economically important traits and its associated grain weight reduction effect could be probably offset by others grain weight genes or at least in the background of the aforementioned two varieties. Notably, one rice line with Pigm, designated as Yangnonggeng 3091, had been authorized as a new variety in Jiangsu province in 2021, showing excellent performance on both grain yield and quality, as well as the blast resistance. Together, these results suggest that the Pigm gene has a high breeding value in developing rice varieties with durable and broad-spectrum resistance to blast disease.

Spinyhead Croaker Germ Cells Gene dnd Visualizes Primordial Germ Cells in Medaka.

Spinyhead croaker (Collichthys lucidus) is an economically important fish suffering from population decline caused by overfishing and habitat destruction. Researches on the development of primordial germ cell (PGC) and reproduction biology were an emergency for the long-term conservation of the involved species. Dead end (dnd) gene plays an indispensable role in PGC specification, maintenance, and development. In the current study, we report the cloning and expression patterns of dnd in C. lucidus (Cldnd). RT-PCR analysis revealed that Cldnd was specifically expressed in both sexual gonads. In the ovary, Cldnd RNA was uniformly distributed in the oocytes and abundant in oogonia, and gradually decreased with oogenesis. A similar expression pattern was also detected in testis. Dual fluorescent in situ hybridization of Cldnd and Clvasa demonstrated that they almost had the same distribution except in oocytes at stage I, in which the vasa RNA aggregated into some particles. Furthermore, Cldnd 3' UTR was sufficient to guide the Green Fluorescent Protein (GFP) specifically and stably expressed in the PGCs of medaka. These findings offer insight into that Cldnd is an evolutionarily conserved germline-specific gene and even a potential candidate for PGC manipulation in C. lucidus.

Differential Expression of Duplicate Insulin-like Growth Factor-1 Receptors (igf1rs) in Medaka Gonads.

Insulin-like growth factor-1 receptors (igf1rs) play important roles in regulating development, differentiation, and proliferation in diverse organisms. In the present study, subtypes of medaka igf1r, igf1ra, and igf1rb were isolated and characterized. RT-PCR results showed that igf1ra and igf1rb mRNA were expressed in all tissues and throughout embryogenesis. Using real-time PCR, the differential expression of igf1ra and igf1rb mRNA during folliculogenesis was observed. The results of in situ hybridization (ISH) revealed that both of them were expressed in ovarian follicles at different stages, and igf1rb was also expressed in theca cells and granulosa cells. In the testis, both igf1ra and igf1rb mRNA were highly expressed in sperm, while igf1rb mRNA was also obviously detected in spermatogonia. In addition, igf1ra mRNA was also present in Leydig cells in contrast to the distribution of igf1rb mRNA in Sertoli cells. Collectively, we demonstrated that differential igf1rs RNA expression identifies medaka meiotic germ cells and somatic cells of both sexes. These findings highlight the importance of the igf system in the development of fish gonads.