The complete germline repertoires of the channel catfish,Ictalurus punctatus, T cell receptor (TR... more The complete germline repertoires of the channel catfish,Ictalurus punctatus, T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish ...
Summary. The use of a striped bass MHC class II A gene as a heterologous probe allowed for the is... more Summary. The use of a striped bass MHC class II A gene as a heterologous probe allowed for the isolation and identification of MHC class II A genes from both an outbred and a second generation gynogenetic, and presumably homozygous, channel catfish (Ictalurus punctatus). Four different clones were isolated from a cDNA library generated from a heterozygous catfish, suggestive of the presence of at least two functional loci. Additionally, both genomic and corresponding class II A cDNA clones were isolated for each of the two loci from gynogenetic cell lines. The amino acids sequences encoding the putative MHC class II a. chain of the catfish possess the general features of those of the three known teleost species. However, the catfish lacks N-linked glycosylation sites. Comparison of the Icpu DAAI*OI and Icpu-DBAI*Ol genomic sequence with the corresponding cDNA sequences revealed that the channel catfish MHC class II A gene has 5 exons and 4 phase 1 introns. Introns 1 and 2 showed only short stretches of identity between the two class II A loci. All three introns differ in length between the two loci. The catfish II A gene has a significantly longer intron 2 versus that found in the zebrafish gene. The relationship of these two loci to the A locus identified in zebrafish, or to the two loci implicated to be present in striped bass, carp, and shark, remain to be elucidated. Genomic sequence determination of class II A genes in these and other species would help detennine the evolutionary history of the II A gene in fishes.
Numerous myxozoan cysts (∼ 1 mm) were found in the musculature of blackfin tuna (Thunnus atlantic... more Numerous myxozoan cysts (∼ 1 mm) were found in the musculature of blackfin tuna (Thunnus atlanticus) harvested off the Caribbean island of St. Kitts. Myxospores were consistent with quadrate members of the Kudoidae, measuring 8.8 (8.2-9.4) μm wide, 7.3 (6.6-8.3) μm thick, and 6.2 (5.8-6.9) μm long with 4 uniform drop-like polar capsules measuring 2.7 (2.2-3.2) μm long and 2.0 (1.7-2.2) μm wide. The 18S small-subunit (SSU) and 28S large-subunit (LSU) ribosomal DNA sequences did not result in direct matches to any published sequences. However, the SSU sequences (1,786 base pairs [bp]) obtained from 6 individual cysts were identical and demonstrated high homology to Kudoa thunni (99.0%) from albacore (Thunnus alalunga). Alternatively, 33 unique sequences were obtained for the LSU (∼ 800 bp), demonstrating 0.1 to 5.0% variability between them, although a majority of these sequences (60%) demonstrated high homology (>99%) to K. thunni. Morphologically, the case isolate was smaller than published descriptions of K. thunni; however, rDNA sequence homology, and phylogenetic placement based on concatenated SSU and LSU rDNA sequences suggests this case isolate and K. thunni are conspecific. To our knowledge this is the first report of K. thunni infection in blackfin tuna from the Caribbean.
Genes encoding MHC class I and II molecules have been identified in a number of fish species, inc... more Genes encoding MHC class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in teleost immune responses. This has in part been due to a lack of appropriate MHC class I and II matched and mismatched animals. To identify such animals, MHC segregation and linkage studies in the channel catfish were undertaken. The results of restriction fragment length polymorphism and fluorescent in situ hybridization studies showed that all the MHC class II genes are linked and most if not all MHC class I genes are linked. These studies also demonstrated that in catfish, as in other teleosts, MHC class I and II genes are not linked. Consequently, catfish matched and mismatched for MHC class I and II genes were identified and preliminary functional studies indicate that spontaneous non-specific allogeneic cytotoxic responses are likely mediated by differences in MHC class I, but not class II, region molecules.
General and Comparative Endocrinology, Sep 1, 2011
Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in... more Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in mammals. In channel catfish, the presence of a partial messenger RNA sequence that encodes a leptin-like peptide (LLP) has been identified and investigated. The objectives of the present studies were to clone and characterize full-length catfish LLP gene, examine tissue expression of LLP mRNA, and determine effects of prolonged fasting and exposure to Edwardsiella ictaluri (E. ictaluri), the bacteria that causes enteric septicemia in catfish, on LLP mRNA expression. Full-length catfish LLP gene was sequenced by genome walking and by 5'- and 3'-RACE. Catfish LLP gene contained three exons with the coding region located in exons 2 and 3. The amino acid sequence of the channel catfish LLP shared very low sequence similarities with leptin of other fish species or the mammalian leptin (24-49%). Using real-time polymerase chain reaction, LLP mRNA expression was detected in various tissues including brain, stomach, spleen, heart, liver, and trunk kidney and was especially high in the liver and trunk kidney. Expression of LLP mRNA in liver and brain was similar between fish that were fasted for 30days and those that received feed daily for 30days (P>0.10). Expression of LLP mRNA was increased in liver, spleen, and trunk kidney within 48h post-exposure to E. ictaluri compared to unexposed fish (P<0.05). Based on the results of the current studies, amino acid sequence of catfish LLP is highly dissimilar to mammalian and fish leptin. Unlike in most mammals, catfish LLP expression is independent of energy status. However, the expression of catfish LLP is increased after exposure to pathogenic bacteria, which is similar to mammals. Further investigations are required to clearly define the biological function and regulation of catfish LLP.
In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight diff... more In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. However, only two of these have confirmed life cycles that involve the oligochaete Dero digitata as the definitive host. During a health screening of farm-raised channel catfish, several fish presented with deformed primary lamellae. Lamellae harbored large, nodular, white pseudocysts 1.25 mm in diameter, and upon rupturing, these pseudocysts released Henneguya myxospores, with a typical lanceolate-shaped spore body, measuring 17.1 ± 1.0 μm (mean ± SD; range = 15.0-19.3 μm) in length and 4.8 ± 0.4 μm (3.7-5.6 μm) in width. Pyriform-shaped polar capsules were 5.8 ± 0.3 μm in length (5.1-6.4 μm) and 1.7 ± 0.1 μm (1.4-1.9 μm) in width. The two caudal processes were 40.0 ± 5.1 μm in length (29.5-50.0 μm) with a spore length of 57.2 ± 4.7 (46.8-66.8 μm). The contiguous SSU rRNA gene sequence obtained from myxospores of five excised cysts did not match any Henneguya sp. in GenBank. The greatest sequence homology (91% over 1,900 bp) was with Henneguya pellis, associated with blister-like lesions on the skin of blue catfish Ictalurus furcatus. Based on the unique combination of pseudocyst and myxospore morphology, tissue location, host, and SSU rRNA gene sequence data, we report this isolate to be a previously unreported species, Henneguya bulbosus sp. nov.
There are more than 200 species of Henneguya described from fish. Of these, only three life cycle... more There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 μm (range = 40.7-61.6 μm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 μm (14.4-19.3 μm) in length and 5.0 ± 0.3 μm (4.5-5.5 μm) in width. The two polar capsules were 6.2 ± 0.4 μm (5.8-7.0 μm) long and 5.0 ± 0.3 μm (4.5-5.5 μm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 μm (22.9-40.6 μm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4 % similarity (100 % coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.
The complete germline repertoires of the channel catfish,Ictalurus punctatus, T cell receptor (TR... more The complete germline repertoires of the channel catfish,Ictalurus punctatus, T cell receptor (TR) loci, TRAD, TRB, and TRG were obtained by analyzing genomic data from PacBio sequencing. The catfish TRB locus spans 214 kb, and contains 112 TRBV genes, a single TRBD gene, 31 TRBJ genes and two TRBC genes. In contrast, the TRAD locus is very large, at 1,285 kb. It consists of four TRDD genes, one TRDJ gene followed by the exons for TRDC, 125 TRAJ genes and the exons encoding the TRAC. Downstream of the TRAC, are 140 TRADV genes, and all of them are in the opposite transcriptional orientation. The catfish TRGC locus spans 151 kb and consists of four diverse V-J-C cassettes. Altogether, this locus contains 15 TRGV genes and 10 TRGJ genes. To place our data into context, we also analyzed the zebrafish TR germline gene repertoires. Overall, our findings demonstrated that catfish possesses a more restricted repertoire compared to the zebrafish. For example, the 140 TRADV genes in catfish ...
Summary. The use of a striped bass MHC class II A gene as a heterologous probe allowed for the is... more Summary. The use of a striped bass MHC class II A gene as a heterologous probe allowed for the isolation and identification of MHC class II A genes from both an outbred and a second generation gynogenetic, and presumably homozygous, channel catfish (Ictalurus punctatus). Four different clones were isolated from a cDNA library generated from a heterozygous catfish, suggestive of the presence of at least two functional loci. Additionally, both genomic and corresponding class II A cDNA clones were isolated for each of the two loci from gynogenetic cell lines. The amino acids sequences encoding the putative MHC class II a. chain of the catfish possess the general features of those of the three known teleost species. However, the catfish lacks N-linked glycosylation sites. Comparison of the Icpu DAAI*OI and Icpu-DBAI*Ol genomic sequence with the corresponding cDNA sequences revealed that the channel catfish MHC class II A gene has 5 exons and 4 phase 1 introns. Introns 1 and 2 showed only short stretches of identity between the two class II A loci. All three introns differ in length between the two loci. The catfish II A gene has a significantly longer intron 2 versus that found in the zebrafish gene. The relationship of these two loci to the A locus identified in zebrafish, or to the two loci implicated to be present in striped bass, carp, and shark, remain to be elucidated. Genomic sequence determination of class II A genes in these and other species would help detennine the evolutionary history of the II A gene in fishes.
Numerous myxozoan cysts (∼ 1 mm) were found in the musculature of blackfin tuna (Thunnus atlantic... more Numerous myxozoan cysts (∼ 1 mm) were found in the musculature of blackfin tuna (Thunnus atlanticus) harvested off the Caribbean island of St. Kitts. Myxospores were consistent with quadrate members of the Kudoidae, measuring 8.8 (8.2-9.4) μm wide, 7.3 (6.6-8.3) μm thick, and 6.2 (5.8-6.9) μm long with 4 uniform drop-like polar capsules measuring 2.7 (2.2-3.2) μm long and 2.0 (1.7-2.2) μm wide. The 18S small-subunit (SSU) and 28S large-subunit (LSU) ribosomal DNA sequences did not result in direct matches to any published sequences. However, the SSU sequences (1,786 base pairs [bp]) obtained from 6 individual cysts were identical and demonstrated high homology to Kudoa thunni (99.0%) from albacore (Thunnus alalunga). Alternatively, 33 unique sequences were obtained for the LSU (∼ 800 bp), demonstrating 0.1 to 5.0% variability between them, although a majority of these sequences (60%) demonstrated high homology (>99%) to K. thunni. Morphologically, the case isolate was smaller than published descriptions of K. thunni; however, rDNA sequence homology, and phylogenetic placement based on concatenated SSU and LSU rDNA sequences suggests this case isolate and K. thunni are conspecific. To our knowledge this is the first report of K. thunni infection in blackfin tuna from the Caribbean.
Genes encoding MHC class I and II molecules have been identified in a number of fish species, inc... more Genes encoding MHC class I and II molecules have been identified in a number of fish species, including the channel catfish, but there is still a dearth of knowledge concerning their functional roles in teleost immune responses. This has in part been due to a lack of appropriate MHC class I and II matched and mismatched animals. To identify such animals, MHC segregation and linkage studies in the channel catfish were undertaken. The results of restriction fragment length polymorphism and fluorescent in situ hybridization studies showed that all the MHC class II genes are linked and most if not all MHC class I genes are linked. These studies also demonstrated that in catfish, as in other teleosts, MHC class I and II genes are not linked. Consequently, catfish matched and mismatched for MHC class I and II genes were identified and preliminary functional studies indicate that spontaneous non-specific allogeneic cytotoxic responses are likely mediated by differences in MHC class I, but not class II, region molecules.
General and Comparative Endocrinology, Sep 1, 2011
Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in... more Leptin is a key pleiotropic cytokine involved in regulation of energy homeostasis and immunity in mammals. In channel catfish, the presence of a partial messenger RNA sequence that encodes a leptin-like peptide (LLP) has been identified and investigated. The objectives of the present studies were to clone and characterize full-length catfish LLP gene, examine tissue expression of LLP mRNA, and determine effects of prolonged fasting and exposure to Edwardsiella ictaluri (E. ictaluri), the bacteria that causes enteric septicemia in catfish, on LLP mRNA expression. Full-length catfish LLP gene was sequenced by genome walking and by 5'- and 3'-RACE. Catfish LLP gene contained three exons with the coding region located in exons 2 and 3. The amino acid sequence of the channel catfish LLP shared very low sequence similarities with leptin of other fish species or the mammalian leptin (24-49%). Using real-time polymerase chain reaction, LLP mRNA expression was detected in various tissues including brain, stomach, spleen, heart, liver, and trunk kidney and was especially high in the liver and trunk kidney. Expression of LLP mRNA in liver and brain was similar between fish that were fasted for 30days and those that received feed daily for 30days (P>0.10). Expression of LLP mRNA was increased in liver, spleen, and trunk kidney within 48h post-exposure to E. ictaluri compared to unexposed fish (P<0.05). Based on the results of the current studies, amino acid sequence of catfish LLP is highly dissimilar to mammalian and fish leptin. Unlike in most mammals, catfish LLP expression is independent of energy status. However, the expression of catfish LLP is increased after exposure to pathogenic bacteria, which is similar to mammals. Further investigations are required to clearly define the biological function and regulation of catfish LLP.
In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight diff... more In the southeastern USA, the channel catfish Ictalurus punctatus is a host to at least eight different species of myxozoan parasites belonging to the genus Henneguya, four of which have been characterized molecularly using sequencing of the small subunit ribosomal RNA (SSU rRNA) gene. However, only two of these have confirmed life cycles that involve the oligochaete Dero digitata as the definitive host. During a health screening of farm-raised channel catfish, several fish presented with deformed primary lamellae. Lamellae harbored large, nodular, white pseudocysts 1.25 mm in diameter, and upon rupturing, these pseudocysts released Henneguya myxospores, with a typical lanceolate-shaped spore body, measuring 17.1 ± 1.0 μm (mean ± SD; range = 15.0-19.3 μm) in length and 4.8 ± 0.4 μm (3.7-5.6 μm) in width. Pyriform-shaped polar capsules were 5.8 ± 0.3 μm in length (5.1-6.4 μm) and 1.7 ± 0.1 μm (1.4-1.9 μm) in width. The two caudal processes were 40.0 ± 5.1 μm in length (29.5-50.0 μm) with a spore length of 57.2 ± 4.7 (46.8-66.8 μm). The contiguous SSU rRNA gene sequence obtained from myxospores of five excised cysts did not match any Henneguya sp. in GenBank. The greatest sequence homology (91% over 1,900 bp) was with Henneguya pellis, associated with blister-like lesions on the skin of blue catfish Ictalurus furcatus. Based on the unique combination of pseudocyst and myxospore morphology, tissue location, host, and SSU rRNA gene sequence data, we report this isolate to be a previously unreported species, Henneguya bulbosus sp. nov.
There are more than 200 species of Henneguya described from fish. Of these, only three life cycle... more There are more than 200 species of Henneguya described from fish. Of these, only three life cycles have been determined, identifying the actinospore and myxospore stages from their respective hosts. Two of these life cycles involve the channel catfish (Ictalurus punctatus) and the freshwater oligochaete Dero digitata. Herein, we molecularly confirm the life cycle of a previously undescribed Henneguya sp. by matching 18S ribosomal RNA (rRNA) gene sequence of the myxospore stage from channel catfish with the previously described actinospore stage (Aurantiactinomyxon mississippiensis) from D. digitata. Gill tissue from naturally infected channel catfish contained pseudocysts restricted to the apical end of the primary lamellae. Myxospores were morphologically consistent with Henneguya spp. from ictalurid fishes in North America. The spores measured 48.8 ± 4.8 μm (range = 40.7-61.6 μm) in total spore length. The lanceolate spore body was 17.1 ± 1.0 μm (14.4-19.3 μm) in length and 5.0 ± 0.3 μm (4.5-5.5 μm) in width. The two polar capsules were 6.2 ± 0.4 μm (5.8-7.0 μm) long and 5.0 ± 0.3 μm (4.5-5.5 μm) wide. The polar capsule contained eight to nine coils in the polar filament. The two caudal processes were of equal length, measuring 31.0 ± 4.1 μm (22.9-40.6 μm). The 1980-bp 18S rRNA gene sequence obtained from two excised cysts shared 99.4 % similarity (100 % coverage) to the published sequence of A. mississippiensis, an actinospore previously described from D. digitata. The sequence similarity between the myxospore from channel catfish and actinospore from D. digitata suggests that they are conspecific, representing alternate life stages of Henneguya mississippiensis n. sp.
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