Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ... more Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes no...
The availability of sexually mature fish often dictates the success of its captive breeding. In t... more The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered folliclestimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH β-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17β-estradiol (E 2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.
The giant grouper is presumed to follow the reproductive pattern of most Epinephelus species, cha... more The giant grouper is presumed to follow the reproductive pattern of most Epinephelus species, characterized by protogynous hermaphroditism wherein male maturation is attained through sex reversal of a functional female. This hypothesis, however, has not been verified due to lack of biological data. The present study addresses this gap by investigating the reproductive development of giant groupers from juvenile stage through sexual maturity. Gonad histological analysis of hatchery-bred juvenile giant grouper from Queensland, Australia (0.8-5.2 kg, n=43) have shown earliest occurrence of primary oocytes (i.e. ovarian differentiation) in 47.8 cm and 2.5 kg fish. Monitoring of sexual maturity by gonadal biopsy was performed in a stock of wild-caught giant groupers (2-20 kg) held in sea cages in the Philippines and Vietnam from 2015 to 2017. Onset of female sexual maturity was at 96.9±1.6 cm and 23.5±1.5 kg in the Philippines, and 103.0±4.1 cm and 33.5±2.5 kg in Vietnam. In both locations, development of primary males was observed wherein fish produced milt (or spermiated) without passing through a functional female phase. The ratio of primary males to females in both locations was about 1:2. Size at maturity of primary males is 86.5±4.8 cm and 17.1±2.1 kg in the Philippines, and 97.3±1.3 cm and 34.3±0.9 kg in Vietnam. To aid in the monitoring of female maturation, we developed a non-invasive method based on immunoassay of vitellogenin in skin mucus and this was shown to be effective in detecting female maturation 9±2 months prior to first observation of oocytes through gonadal biopsy. Our findings suggest that giant grouper is a diandric protogynous hermaphrodite. This study provides novel information on the reproductive biology of giant grouper, an economically important and vulnerable species.
The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphr... more The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphroditic grouper (Epinephelus fuscoguttatus) was investigated. Recombinant giant grouper (E. lanceolatus) FSH (rggFSH) was produced in yeast. Its receptor-binding capacity and steroidogenic potency were confirmed in vitro. Weekly injections of rggFSH to juvenile tiger grouper for 8 weeks (100 μg/kg body weight, BW) resulted in significantly larger and more advanced oocytes (cortical alveolar stage vs primary growth stage in control). Sustained treatment with rggFSH (20 to 38 weeks at 200 μg/kg BW) resulted in significant reduction in gonad size, degeneration of oocytes, and proliferation of spermatogonial cells, indicative of female to male sex change. Gene expression analysis showed that, while initiating female to male sex change, the rggFSH significantly suppressed the steroidogenic genes cyp11b, cyp19a1a, and foxl2 which restrained the endogenous production of sex steroid hormones and thus prevented the differentiation of spermatogonial cells. Expression profile of sex markers dmrt1, amh, figla, and bmp15 suggests that the observed sex change was restricted at the initiation stage. Based on these results, we propose that the process of female to male sex change in the protogynous grouper is initiated by FSH, rather than sex steroids, and likely involves steroid-independent pathway. The cortical alveolar stage in oocyte development is the critical point after which FSH-induced sex change is possible in grouper.
Regulation of inflammation is a critical process for maintaining physiological homeostasis. The l... more Regulation of inflammation is a critical process for maintaining physiological homeostasis. The l-carrageenan (l-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of l-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% l-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the l-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group, 62 proteins got a significant (p < 0.05) upregulation in the l-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the l-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of various classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.
The first artificially fertilized spotted wolffish (Anarhichas minor) eggs hatched in Norway in t... more The first artificially fertilized spotted wolffish (Anarhichas minor) eggs hatched in Norway in the mid-1990s as this species was considered by Norwegian authorities to be a top candidate species for cold-water aquaculture in the North Atlantic regions. Previous research conducted in Norway (since 1992) and Canada (since 2000), focused on identifying key biological parameters for spotted wolffish cultivation which led, respectively, to the rapid establishment of a full commercial production line in northern Norway, while Québec (Canada) is witnessing its first privately driven initiative to establish commercial production of spotted wolffish on its territory. The control of reproduction can be viewed as a major requirement to achieve the development of performant strains using genetic selection tools and/or all-year-round production to bring about maximal productivity and synchronization among a given captive population. Although the basic reproduction aspects are more understood an...
Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to a... more Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the best-understood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture ar...
Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to a... more Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the bestunderstood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture are mainly restricted to the Myoviridae and Podoviridae, and the Siphoviridae, respectively. However, the current functional use of phages against bacterial pathogens of cultured fish is still in its infancy. Based on the available data, in this review, we summarize the current knowledge about phage, identify gaps, and provide insights into the possible bacterial control strategies they might represent for managing aquaculture-related bacterial diseases.
Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ... more Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes no...
The availability of sexually mature fish often dictates the success of its captive breeding. In t... more The availability of sexually mature fish often dictates the success of its captive breeding. In this study, we induced reproductive development in juvenile protogynous tiger grouper through oral administration of a plasmid (p) containing an engineered folliclestimulating hormone (FSH). An expression construct (pcDNA3.1) was designed to express a single-chain FSH consisting of giant grouper FSH β-subunit and glycoprotein subunit-α (CGα), linked by the carboxy-terminal peptide (CTP) sequence from the human chorionic gonadotropin (hCG). Single oral delivery of pFSH encapsulated in liposome and chitosan to tiger grouper yielded a significant increase in plasma FSH protein level after 4 days. Weekly pFSH feeding of juvenile tiger groupers for 8 weeks stimulated ovarian development as indicated by a significant increase in oocyte diameter and progression of oocytes to cortical alveolar stage. As the pFSH treatment progressed from 20 to 38 weeks, female to male sex change was initiated, characterized by oocyte regression, proliferation of spermatogonial cells, and occurrence of spermatogenic cysts. It was also associated with significantly lower mRNA expression of steroidogenic genes (cyp11b, cyp19a1a, and foxl2) and basal plasma levels of sex steroid hormones 17β-estradiol (E 2), testosterone (T), and 11-ketotestosterone (11KT). Results suggest that pFSH stimulates ovarian development up to cortical alveolar stage and then initiates sex change in tiger grouper. These findings significantly contribute to our knowledge on the role of FSH in the development of protogynous hermaphroditic fish. This study is the first to demonstrate induction of reproductive development in fish through oral delivery of plasmid gonadotropin.
The giant grouper is presumed to follow the reproductive pattern of most Epinephelus species, cha... more The giant grouper is presumed to follow the reproductive pattern of most Epinephelus species, characterized by protogynous hermaphroditism wherein male maturation is attained through sex reversal of a functional female. This hypothesis, however, has not been verified due to lack of biological data. The present study addresses this gap by investigating the reproductive development of giant groupers from juvenile stage through sexual maturity. Gonad histological analysis of hatchery-bred juvenile giant grouper from Queensland, Australia (0.8-5.2 kg, n=43) have shown earliest occurrence of primary oocytes (i.e. ovarian differentiation) in 47.8 cm and 2.5 kg fish. Monitoring of sexual maturity by gonadal biopsy was performed in a stock of wild-caught giant groupers (2-20 kg) held in sea cages in the Philippines and Vietnam from 2015 to 2017. Onset of female sexual maturity was at 96.9±1.6 cm and 23.5±1.5 kg in the Philippines, and 103.0±4.1 cm and 33.5±2.5 kg in Vietnam. In both locations, development of primary males was observed wherein fish produced milt (or spermiated) without passing through a functional female phase. The ratio of primary males to females in both locations was about 1:2. Size at maturity of primary males is 86.5±4.8 cm and 17.1±2.1 kg in the Philippines, and 97.3±1.3 cm and 34.3±0.9 kg in Vietnam. To aid in the monitoring of female maturation, we developed a non-invasive method based on immunoassay of vitellogenin in skin mucus and this was shown to be effective in detecting female maturation 9±2 months prior to first observation of oocytes through gonadal biopsy. Our findings suggest that giant grouper is a diandric protogynous hermaphrodite. This study provides novel information on the reproductive biology of giant grouper, an economically important and vulnerable species.
The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphr... more The role of follicle-stimulating hormone (FSH) in the gonadal development of protogynous hermaphroditic grouper (Epinephelus fuscoguttatus) was investigated. Recombinant giant grouper (E. lanceolatus) FSH (rggFSH) was produced in yeast. Its receptor-binding capacity and steroidogenic potency were confirmed in vitro. Weekly injections of rggFSH to juvenile tiger grouper for 8 weeks (100 μg/kg body weight, BW) resulted in significantly larger and more advanced oocytes (cortical alveolar stage vs primary growth stage in control). Sustained treatment with rggFSH (20 to 38 weeks at 200 μg/kg BW) resulted in significant reduction in gonad size, degeneration of oocytes, and proliferation of spermatogonial cells, indicative of female to male sex change. Gene expression analysis showed that, while initiating female to male sex change, the rggFSH significantly suppressed the steroidogenic genes cyp11b, cyp19a1a, and foxl2 which restrained the endogenous production of sex steroid hormones and thus prevented the differentiation of spermatogonial cells. Expression profile of sex markers dmrt1, amh, figla, and bmp15 suggests that the observed sex change was restricted at the initiation stage. Based on these results, we propose that the process of female to male sex change in the protogynous grouper is initiated by FSH, rather than sex steroids, and likely involves steroid-independent pathway. The cortical alveolar stage in oocyte development is the critical point after which FSH-induced sex change is possible in grouper.
Regulation of inflammation is a critical process for maintaining physiological homeostasis. The l... more Regulation of inflammation is a critical process for maintaining physiological homeostasis. The l-carrageenan (l-CGN) is a mucopolysaccharide extracted from the cell wall of red algae (Chondrus crispus) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of l-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) (Danio rerio). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% l-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the l-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group, 62 proteins got a significant (p < 0.05) upregulation in the l-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the l-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of various classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.
The first artificially fertilized spotted wolffish (Anarhichas minor) eggs hatched in Norway in t... more The first artificially fertilized spotted wolffish (Anarhichas minor) eggs hatched in Norway in the mid-1990s as this species was considered by Norwegian authorities to be a top candidate species for cold-water aquaculture in the North Atlantic regions. Previous research conducted in Norway (since 1992) and Canada (since 2000), focused on identifying key biological parameters for spotted wolffish cultivation which led, respectively, to the rapid establishment of a full commercial production line in northern Norway, while Québec (Canada) is witnessing its first privately driven initiative to establish commercial production of spotted wolffish on its territory. The control of reproduction can be viewed as a major requirement to achieve the development of performant strains using genetic selection tools and/or all-year-round production to bring about maximal productivity and synchronization among a given captive population. Although the basic reproduction aspects are more understood an...
Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to a... more Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the best-understood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture ar...
Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to a... more Therapeutic bacteriophages, commonly called as phages, are a promising potential alternative to antibiotics in the management of bacterial infections of a wide range of organisms including cultured fish. Their natural immunogenicity often induces the modulation of a variated collection of immune responses within several types of immunocytes while promoting specific mechanisms of bacterial clearance. However, to achieve standardized treatments at the practical level and avoid possible side effects in cultivated fish, several improvements in the understanding of their biology and the associated genomes are required. Interestingly, a particular feature with therapeutic potential among all phages is the production of lytic enzymes. The use of such enzymes against human and livestock pathogens has already provided in vitro and in vivo promissory results. So far, the bestunderstood phages utilized to fight against either Gram-negative or Gram-positive bacterial species in fish culture are mainly restricted to the Myoviridae and Podoviridae, and the Siphoviridae, respectively. However, the current functional use of phages against bacterial pathogens of cultured fish is still in its infancy. Based on the available data, in this review, we summarize the current knowledge about phage, identify gaps, and provide insights into the possible bacterial control strategies they might represent for managing aquaculture-related bacterial diseases.
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(neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% l-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the l-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group, 62 proteins got a significant (p < 0.05) upregulation in the l-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the l-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D
reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of various classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.
(neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% l-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the l-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group, 62 proteins got a significant (p < 0.05) upregulation in the l-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the l-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D
reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of various classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases.