Formation of autotriploid Carassius auratus and its fertility-related genes analysis

Background Formation of triploid organism is useful in genetics and breeding. In this study, autotriploid Carassius auratus (3nRR, 3n = 150) was generated from Carassius auratus red var. (RCC, 2n = 100) (♀) and autotetraploid Carassius auratus (4nRR, 4n = 200) (♂). The female 3nRR produced haploid, diploid and triploid eggs, whereas the male 3nRR was infertile. The aim of the present study was to explore fertility of potential candidate genes of 3nRR. Results Gonadal transcriptome profiling of four groups (3 females RCC (FRCC), 3 males 4nRR (M4nRR), 3 males 3nRR (M3nRR) and 3 females 3nRR (F3nRR)) was performed using RNA-SEq. A total of 78.90 Gb of clean short reads and 24,262 differentially expressed transcripts (DETs), including 20,155 in F3nRR vs. FRCC and 4,107 in M3nRR vs. M4nRR were identified. A total of 106 enriched pathways were identified through KEGG enrichment analysis. Out of the enriched pathways, 44 and 62 signalling pathways were identified in F3nRR vs. FRCC and M3nRR vs. M4nRR, respectively. A total of 80 and 25 potential candidate genes for fertility-related in F3nRR and M3nRR were identified, respectively, through GO, KEGG analyses and the published literature. Moreover, protein-protein interaction (PPI) network construction of these fertility-associated genes were performed. Analysis of the PPI networks showed that 6 hub genes (MYC, SOX2, BMP4, GATA4, PTEN and BMP2) were involved in female fertility of F3nRR, and 2 hub genes (TP53 and FGF2) were involved in male sterility of M3nRR. Conclusions Establishment of autotriploid fish offers an ideal model to study reproductive traits of triploid fish. RNA-Seq data revealed 6 genes, namely, MYC, SOX2, BMP4, GATA4, PTEN and BMP2, involved in the female fertility of the F3nRR. Moreover, 2 genes, namely, TP53 and FGF2, were related to the male sterility of the M3nRR. These findings provide information on reproduction and breeding in triploid fish.

Incidence of skeletal deformities in induced triploid rainbow trout Oncorhynchus mykiss (Walbaum, 1792)

Due to the cytogenetic incompatibility, triploid fish are usually infertile and are not affected by a decline in growth, survival and meat quality, which accompanies the process of sexual maturation in diploid specimens. Thus, artificial triploidization has been proposed for fish production in the case of species with early sexual maturation, such as rainbow trout. However, the use of this technique is limited by increased ratios of skeletal deformities observed in triploid specimens. The main objective of this research was to compare the proportion and variety of body abnormalities in diploid and triploid 14-month-old rainbow trout from commercial stocks, using external body shape examination, radiography and whole-mount skeletal staining. Individuals with externally observed body deformities (scoliosis, humpback, shortened tail and jaw deformities) accounted for 0.45% of the diploid stock and 3.83% of the triploid stock. X-rays and whole-mount skeletal staining of deformed individuals showed spine deformities, including compressions and fusions of vertebrae. Abnormalities observed in diploid and triploid rainbow trout examined during this study were non-lethal, however, they may negatively affect the condition of fish. Fish with skeletal deformities are not aesthetically pleasing, thus an increased ratio of such deformations in fish produced for commercial purposes may result in real economic losses.

Formation of autotriploid Carassius auratus and its fertility-related genes analysis

Background Formation of triploid organism is useful in genetics and breeding. In this study, autotriploid Carassius auratus (3nRR, 3n = 150) was generated from Carassius auratus red var. (RCC, 2n = 100) (♀) and autotetraploid Carassius auratus (4nRR, 4n = 200) (♂). The female 3nRR produced haploid, diploid and triploid eggs, whereas the male 3nRR was infertile. The aim of the present study was to explore fertility of potential candidate genes of 3nRR. Results Gonadal transcriptome profiling of four groups (3 females RCC (FRCC), 3 males 4nRR (M4nRR), 3 males 3nRR (M3nRR) and 3 females 3nRR (F3nRR)) was performed using RNA-SEq. A total of 78.90 Gb of clean short reads and 24,262 differentially expressed genes (DEGs), including 20,155 in F4nRR vs FRCC and 4,107 in M3nRR vs M4nRR were identified. A total of 106 enriched pathways were identified through KEGG enrichment analysis. Out of the enriched pathways, 44 and 62 signalling pathways were identified in F3nRR vs FRCC and M3nRR vs M4nRR, respectively. A total of 80 and 25 potential candidate genes for fertility-related in F3nRR and M3nRR were identified, through GO, KEGG analyses and the published literature. Moreover, protein-protein interaction (PPI) network construction of these fertility-associated genes were performed. Analysis of the PPI networks showed that 6 hub genes (MYC, SOX2, BMP4, GATA4, PTEN and BMP2) were involved in female fertility of F3nRR, and 2 hub genes (TP53 and FGF2) were involved in male sterility of M3nRR. Conclusions Establishment of autotriploid fish offers an ideal model to study reproductive traits of triploid fish. Further, RNA-Seq data revealed 8 hub genes related to fertility in 3nRR. We further identified 6 genes, namely, MYC, SOX2, BMP4, GATA4, PTEN and BMP2, involved in the female fertility of the F3nRR. Moreover, 2 genes, namely, TP53 and FGF2, were related to the male sterility of the M3nRR. These findings provide information on reproduction and breeding in triploid fish.

Effects of ploidy and salmonid alphavirus infection on the skin and gill microbiome of Atlantic salmon (Salmo salar)

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon.

Digestive tract morphology and enzyme activities of juvenile diploid and triploid Atlantic salmon (Salmo salar) fed fishmeal-based diets with or without fish protein hydrolysates

Triploid, sterile Atlantic salmon (Salmo salar) could make a contribution to the development of the farming industry, but uncertainties about the performance and welfare of triploids have limited their adoption by farmers. In this study, we compared the ontogeny of digestive tract morphology and enzyme activities (pepsin, trypsin, chymotrypsin, alkaline phosphatase and aminopeptidase) of diploid and triploid Atlantic salmon. Fish were fed diets based on fishmeal (STD) or a mix of fishmeal and hydrolysed fish proteins (HFM) whilst being reared at low temperature from start-feeding to completion of the parr-smolt transformation. Fish weights for each ploidy and feed combination were used to calculate thermal growth coefficients (TGCs) that spanned this developmental period, and the data were used to examine possible relationships between enzyme activities and growth. At the end of the experiment, faeces were collected and analyzed to determine the apparent digestibility coefficients (ADCs) of the dietary amino acids (AAs). Digestive tract histo-morphology did not differ substantially between ploidies and generally reflected organ maturation and functionality. There were no consistent differences in proteolytic enzyme activities resulting from the inclusion of HFM in the diet, nor was there improved digestibility and AA bioavailability of the HFM feed in either diploid or triploid fish. The triploid salmon had lower ADCs than diploids for most essential and non-essential AAs in both diets (STD and HFM), but without there being any indication of lower intestinal protease activity in triploid fish. When trypsin-to-chymotrypsin activity and trypsin and alkaline phosphatase (ALP) ratios (T:C and T:ALP, respectively) were considered in combination with growth data (TGC) low T:C and T:ALP values coincided with times of reduced fish growth, and vice versa, suggesting that T:C and T:ALP may be used to predict recent growth history and possible growth potential.

Effects of ploidy and salmonid alphavirus infection on the skin and gill microbiome of Atlantic salmon (Salmo salar)

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon.

Cold temperature shock tetraploidization of striped catfish Pangasianodon hypophthalmus (Sauvage, 1878) with different of temperature and age of zygote

Striped catfish (Pangasianodon hypophthalmus) has a relatively slow growth, so the operational cost of production is high and incomparable with the selling price, thus makes the cultivation of striped catfish is inefficient. Genetic quality improvement through polyploidization is to produce sterile triploid fish (3n) that can overcome this problem. The provision of triploid fish is more efficient through tetraploidization. Tetraploidization in striped catfish using cold shock treatment has never been done before. The purpose of this study was to determine the optimum tetra-ploidization condition using cold shock with temperature and different age of zygote in striped catfish. A completely randomized factorial design with two treatments i.e. three level of temperatures (8oC, 12°C, 16oC ) and two different ages of zygote (29 and 31 minutes after fertilisation-maf) and three replications was used. In addition, a control without applying temperature shock treatment was performed, Soaking process was conducted for 30 minutes. Tetraploid identification was done by calculating the maximum total of nucleoli per cell that was confirmed by calculating the total of chromosome. The results showed that the degree of hatching rate and abnormalities has a significantly different effect (P <0.05). Number of nucleoli per cell was 4 and chromosomes was 112 (4n=112) for tetraploid), whereas for diploid fish the maximum number of nucleoli per cell was 2 and the number of chromosomes was 56 (2n=56). The highest tetraploid percentage was obtained at a treatment of 12oC at age of zygote 29 maf. Thus, the optimum condition of tetraploidization in striped catfish is using cold shock treatment of 12oC with the age of zygote of 29 maf.

Ploidy influences on metabolic substrate deposition of rainbow trout

Triploid fish are usually sterile. Thus, the energy and nutrients intended for sexual maturation may be available to enhance flesh quality and physical growth. The present study aimed to investigate differences in the metabolic substrates, lipids and proteins, between storage tissues from diploid and triploid female rainbow trout. Monthly, metabolic substrates were quantified in liver, muscle, and ovaries, which were collected during the first reproductive cycle. In general, it was possible to identify a seasonal and similar deposition of metabolites in different tissues of 2n and 3n females, mainly at early stages of gonadal maturation. However, from the stages 5-6, the ovaries showed great differences between ploidies, with higher concentration of lipids and protein in 2n females. This result reflects the incorporation of vitellogenin in oocytes, which is a process that does not occur in 3n females. It was possible to observe seasonal hepato-somatic index changes in 2n females, with higher values observed in the post-ovulatory stage, and the triploid animals showed lower values compared to 2n, with no seasonal difference. Viscero-somatic index can reflect the mobilization of substrates, with higher values found for 2n females in stage 5-6, which is the period of active mobilization of tissue substrates.

Analysis on the Meiosis-Related Gene (Dmc1, Ph1) Expression in Autotriploid Carassius auratus

Triploid is usually considered to be unable to perform normal meiosis due to the abnormal behavior of the three sets of chromosomes. But autotriploid Carassius auratus in the Dongting water system (3n = 150, abbreviated as 3nCC) can perform normal meiosis. In artificial autotriploid Carassius auratus (3n = 150, abbreviated as 3nRR), female individuals undergo normal meiosis and produce mature gametes, while male individuals cannot. To better understand the effects of triploidization on meiosis in fish, we study the structure, methylation level, and expression level of meiosis-related genes (Dmc1, Ph1) in diploid Carassius auratus (2n = 100, abbreviated as 2nCC), Carassius auratus red var.(2n = 100, abbreviated as RCC), 3nCC and 3nRR. The results show that, compared with their diploid ancestors (2nCC and RCC), Dmc1 and Ph1 genes are hypomethylated in all 3nCC and female 3nRR, while are hypermethylated in male 3nRR. Correspondingly, Dmc1 and Ph1 genes are highly expressed in all 3nCC and female 3nRR, while are lowly expressed in male 3nRR. These results indicate that high expression of meiosis-related genes can contribute to restoration of bivalent pairing during meiosis in autotriploid Carassius auratus. This study provides new insights into the effect of DNA methylation on the fertility in triploid fish.