Costimulatory receptors in the channel catfish: CD28 family members and their ligands.

The CD28-B7 interaction is required to deliver a second signal necessary for T-cell activation. Additional membrane receptors of the CD28 and B7 families are also involved in immune checkpoints that positively or negatively regulate leukocyte activation, in particular T lymphocytes. BTLA is an inhibitory receptor that belongs to a third receptor family. Fish orthologs exist only for some of these genes, and the potential interactions between the corresponding ligands remain mostly unclear. In this work, we focused on the channel catfish (Ictalurus punctatus), a long-standing model for fish immunology, to analyze these co-stimulatory and co-inhibitory receptors. We identified one copy of cd28, ctla4, cd80/86, b7h1/dc, b7h3, b7h4, b7h5, two btla, and four b7h7 genes. Catfish CD28 contains the highly conserved mammalian cytoplasmic motif for PI3K and GRB2 recruitment, however this motif is absent in cyprinids. Fish CTLA4 share a C-terminal putative GRB2-binding site but lacks the mammalian PI3K/GRB2-binding motif. While critical V-domain residues for human CD80 or CD86 binding to CD28/CTLA4 show low conservation in fish CD80/86, C-domain residues are highly conserved, underscoring their significance. Catfish B7H1/DC had a long intracytoplasmic domain with a P-loop-NTPase domain that is absent in mammalian sequences, while the lack of NLS motif in fish B7H4 suggests this protein may not regulate cell growth when expressed intracellularly. Finally, there is a notable expansion of fish B7H7s, which likely play diverse roles in leukocyte regulation. Overall, our work contributes to a better understanding of fish leukocyte co-stimulatory and co-inhibitory receptors.

Interferons and interferon receptors in the channel catfish, Ictalurus punctatus.

In this work, we describe the complete repertoire of channel catfish, Ictalurus punctatus, IFNs and IFN receptor genes. Based on multiple genomic and transcriptomic resources we identified 16 type I IFN genes, which represent the six type I IFN subgroups previously defined in salmonids (a-f.) No representatives of subgroup h previously only found in percomorphs were identified. An expansion in copy numbers of subgroup d IFN genes was of particular interest, as this has not been reported in other fish species to date. Furthermore, we confirmed the presence of two type II ifn genes encoding orthologs of IFNγ and the teleost-specific IFNγRel. Six homologs of IFN type I receptor genes were found in an array that shows conserved synteny with human chromosome 21. Three homologs of type II IFN receptor genes were also identified. These type I and type II receptor sequences are compatible with the dual type I IFN receptors, and the potentially more complex type II IFN receptors described in teleosts. Our data provide a comprehensive resource for future studies of channel catfish innate antiviral immunity.

Analysis of specific mRNA gene expression profiles as markers of egg and embryo quality for hybrid catfish aquaculture.

Despite best efforts to optimize reproduction, egg incubation, and larval performance in captivity, inconsistencies in hatchery fish production are still created by high variations in egg quality from individual females. In some hatchery species, egg quality and generation of viable embryos are correlated to abundances of specific mRNAs. Channel catfish females show considerable extremes in egg quality, causing inconsistencies in channel catfish, Ictalurus punctatus, female × blue catfish, Ictalurus furcatus, male hybrid fry production. The objectives of this study were to examine relative transcripts linked to egg and embryo quality and determine expression between low-hatch and high-hatch egg batches through early development (0, 24, 48, and 96 h post-fertilization; HPF). RNA was extracted from eggs/embryos of nine females (n = 4 high-quality, n = 5 low-quality) and Real-Time PCR was used to quantify relative gene expression. The transcripts assessed in this study perform critical cellular functions, including tubulin ß (tubb), cathepsin D (ctsd), cathepsin Z (ctsz), cathepsin B (ctsb), cyclin B (ccnb1), exportin-1 (xpo1), ring finger protein 213 (rnf213), glucocorticoid receptor-1 (GR-1), and heat shock protein 70 (hsp70). Relative gene expression of all transcripts except GR-1 and hsp70 were up-regulated in the high-hatch group and peaked at 48 HPF (neurulation stage), indicating the importance of these gene products at this threshold to normally progress until hatch. Due to lack of expression during earlier stages, maternally derived mRNAs for these genes do not seem to impact early embryonic development. Using mRNA markers as a selection mechanism for hatchery broodstock may lead to more high-hatch egg batches by reducing problems associated with poor egg quality.

Characterization of a third ghrelin receptor, GHS-R3a, in channel catfish reveals novel expression patterns and a high affinity for homologous ligand.

A novel third channel catfish growth hormone secretagogue (ghrelin) receptor, GHS-R3a, gene was characterized. Identification and analysis of the genomic organization of channel catfish GHS-R3a revealed differences in exon/intron structure relative to the previously published GHS-R1a and GHS-R2a sequences. Amino acid sequence alignment of catfish GHS-R3a with -R1a and -R2a revealed 48 and 52% sequence identity, respectively. Phylogenetic analysis predicted a new clade of GHS-R3a receptors found only in fish, with representation in the teleost infradivisions Osteoglossomorpha, Clupeomorpha, and Euteleostei. In functional analyses, homologous catfish ghrelin increased intracellular Ca2+ concentration in human embryonic kidney (HEK) 293 cells stably expressing catfish GHS-R3a. On the contrary, intracellular Ca2+ concentration was unaffected by treatment with the synthetic growth hormone secretagogues GHRP-6 and hexarelin. Realtime PCR results indicated high expression of GHS-R3a in the brain and gonads, demonstrating tissue specificity among the catfish GHS-Rs. The effects of fasting and refeeding on all three ghrelin receptors were evaluated in catfish brain, pituitary, stomach, and Brockmann bodies. Most notably, GHS-R3a was the only receptor observed to significantly increase (2.9-6.3-fold) in brain, pituitary, and stomach within 4 days of fasting (P < .05). Stomach GHS-R1a also increased (P < .05) after 4 days; however, GHS-R2a was only elevated in brain and pituitary after refeeding for 1 week. Expression of all three ghrelin receptors were elevated (P < .05) in the Brockmann bodies after 2 weeks of fasting and returned to prefasting levels following refeeding. Together with the previously published characterization of GHS-R1a and -R2a, these results establish three ghrelin receptors, each altered by energy state, in channel catfish and add to the growing body of information on GHS-R evolution and function.

A Leukocyte Immune-Type Receptor Subset Is a Marker of Antiviral Cytotoxic Cells in Channel Catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, leukocyte immune type receptors (LITRs) represent a multigene family that encodes Ig superfamily proteins that mediate activating or inhibitory signaling. In this study, we demonstrate the use of mAb CC41 to monitor viral cytotoxic responses in catfish and determine that CC41 binds to a subset of LITRs on the surface of catfish clonal CTLs. Homozygous gynogenetic catfish were immunized with channel catfish virus (CCV)-infected MHC-matched clonal T cells (G14D-CCV), and PBL were collected at various times after immunization for flow cytometric analyses. The percentage of CC41(+) cells was significantly increased 5 d after primary immunization with G14D-CCV and at 3 d after a booster immunization as compared with control fish only injected with G14D. Moreover, CC41(+) cells magnetically isolated from the PBL specifically killed CCV-infected targets as measured by (51)Cr release assays and expressed messages for CD3γδ, perforin, and at least one of the CD4-like receptors as analyzed by RNA flow cytometry. When MLC effector cells derived from a G14D-CCV-immunized fish were preincubated with CC41 mAb, killing of G14D-CCV targets was reduced by ∼40%, suggesting that at least some LITRs have a role in target cell recognition and/or cytotoxicity. The availability of a LITR-specific mAb has allowed, to our knowledge for the first time, functional characterization of LITRs in an autologous system. In addition, the identification of an LITR subset as a cytotoxic cell marker will allow for more effective monitoring of catfish immune responses to pathogens.

Characterization of two channel catfish, Ictalurus punctatus, glucocorticoid receptors and expression following an acute stressor.

Two channel catfish glucocorticoid receptor genes, ipGR1 (NR3C1_1) and ipGR2 (NR3C1_2) were partially characterized. Identification and analysis of the genomic organization of two channel catfish glucocorticoid (GC) receptors (GRs) revealed differences in the lengths of exons 1 and 2 and the addition of an extra 27-bp exon inserted after exon 2 in the GR1 gene, yielding a 9-aa insert in the receptor protein. Sequence of the 9-aa insert in ipGR1 (WRARQNTHG) is unique compared to other teleost fish GRs. Amino acid sequence alignment of the two channel catfish GRs, revealed 55% sequence identity between them, with a high degree of sequence conservation (82%) in the DNA binding and ligand binding domains. Real-time PCR indicated that ipGR1 and ipGR2 were expressed in all tissues evaluated. Channel catfish GR1 was predominantly expressed in the gills, nearly 25-fold higher than in the liver. GR1 expression was higher than GR2 expression in gills, intestine, head kidney and heart (P<0.05). Channel catfish hepatic GR1 mRNA expression was significantly (P<0.05) increased from pre-stress expression 30min following removal of the acute stressor. After 30min of stress and during the 2h recovery period, ipGR1 mRNA expression was higher relative to ipGR2 expression. Hepatic ipGR2 expression was not affected (P>0.05) by the acute stress event. The present study adds to the growing body of information on GR evolution and function and further demonstrates the unique regulation of the GC/GR system in teleost fish.

Biomphalaria straminea (Mollusca: Planorbidae) as an intermediate host of Drepanocephalus spp. (Trematoda: Echinostomatidae) in Brazil: a morphological and molecular study.

Species of trematodes belonging to the genus Drepanocephalus are intestinal parasites of piscivorous birds, primarily cormorants (Phalachrocorax spp.), and are widely reported in the Americas. During a 4-year malacological study conducted on an urban lake in Brazil, 27-collar-spined echinostome cercariae were found in 1665/15,459 (10.7 %) specimens of Biomphalaria straminea collected. The cercariae were identified as Drepanocephalus spp. by sequencing the 18S (SSU) rDNA, ITS1/5.8S rDNA/ITS2 (ITS), 28S (LSU) rDNA region, cytochrome oxidase subunit 1 (CO1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) markers. In experimental life cycle studies, metacercariae developed in laboratory-reared guppies (Poecilia reticulata); however, attempts to infect birds and rodents were unsuccessful. Two closely related morphotypes of cercariae were characterized. One species, identified by molecular markers as a genetic variant of Drepanocephalus auritus (99.9 % similarity at SSU, ITS, LSU; 97.2 % at CO1; 95.8 % at ND1), differs slightly from an archived North American isolate of this species also sequenced as part of this study. A second species, putatively identified as Drepanocephalus sp., has smaller cercariae and demonstrates significant differences from D. auritus at the CO1 (11.0 %) and ND1 (13.6 %) markers. Aspects related to the morphological taxonomic identification of 27-collar-spined echinostome metacercariae are briefly discussed. This is the first report of the involvement of molluscs of the genus Biomphalaria in the transmission of Drepanocephalus and the first report of D. auritus in South America.

Myxobolus ictiobus n. sp. and Myxobolus minutus n. sp. (Cnidaria: Myxobolidae) from the gills of the smallmouth buffalo Ictiobus bubalus Rafinesque (Cypriniformes: Catostomidae).

The smallmouth buffalo Ictiobus bubalus Rafinesque (Catostomidae) is native to North American waterways and occasionally grown in pond aquaculture. Species of Myxobolus Bütschli, 1882 have been reported from the gills, integument, and intestinal tract of buffalo fish, although there is ambiguity in some host records. In the summer of 2013, thirteen adult smallmouth buffalo were seined from a 0.1-acre (0.04-hectare) experimental research pond at the Thad Cochran National Warmwater Aquaculture Center in Stoneville, Mississippi, USA, and examined for the presence of parasitic infection. Two previously unknown species of Myxobolus were observed parasitising the gills. Plasmodia of the two species differed from each other in both size and shape. Morphologically the two species were distinct from one another and from other Myxobolus spp. previously reported from buffalo fish. Myxospores of Myxobolus ictiobus n. sp. were spherical and measured 12.7-14.5 (13.9 ± 0.4) µm in length and 10.7-13.6 (12.5 ± 0.7) µm in width with a thickness of 10.3-14.8 (12.6 ± 2.3) µm. Polar capsules measured 5.6-7.4 (6.6 ± 0.4) µm in length and 3.7-4.9 (4.5 ± 0.8) µm in width and each contained a coiled polar filament with 5-6 turns. Myxospores of Myxobolus minutus n. sp. were circular in shape and measured 7.4-9.6 (8.6 ± 0.7) µm in length and 7.5-9.9 (8.8 ± 0.7) µm in width with a thickness of 6.5-7.3 (6.7 ± 0.3) µm. Polar capsules measured 3.6-4.9 (4.3 ± 0.3) µm in length and 2.8-3.8 (3.3 ± 0.3) µm and each contained a coiled polar filament with 5-6 turns. Supplemental 18S rRNA gene sequencing identified unique sequences for each isolate. Phylogenetic analysis of 18S rRNA sequences demonstrated a strong clustering of both isolates with other species of Myxobolus from cypriniform fish.

Small subunit ribosomal RNA sequence links the myxospore stage of Henneguya mississippiensis n. sp. from channel catfish Ictalurus punctatus to an actinospore released by the benthic oligochaete Dero digitata.

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.

Identification and characterization of TCRγ and TCRδ chains in channel catfish, Ictalurus punctatus.

Channel catfish, Ictalurus punctatus, T cell receptors (TCR) γ and δ were identified by mining of expressed sequence tag databases, and full-length sequences were obtained by 5'-RACE and RT-PCR protocols. cDNAs for each of these TCR chains encode typical variable (V), diversity (D), joining (J), and constant (C) regions. Three TCRγ V families, seven TCRγ J sequences, and three TCRγ C sequences were identified from sequencing of cDNA. Primer walking on bacterial artificial chromosomes (BACs) confirmed that the TRG locus contained seven TRGJ segments and indicated that the locus consists of (Vγ3-Jγ6-Cγ2)-(Vγ1n-Jγ7-Cγ3)-(Vγ2-Jγ5-Jγ4-Jγ3-Jγ2-Jγ1-Cγ1). In comparison for TCRδ, two V families, four TCRδ D sequences, one TCRδ J sequence, and one TCRδ C sequence were identified by cDNA sequencing. Importantly, the finding that some catfish TCRδ cDNAs contain TCR Vα-D-Jδ rearrangements and some TCRα cDNAs contain Vδ-Jα rearrangements strongly implies that the catfish TRA and TRD loci are linked. Finally, primer walking on BACs and Southern blotting suggest that catfish have four TRDD gene segments and a single TRDJ and TRDC gene. As in most vertebrates, all three reading frames of each of the catfish TRDD segments can be used in functional rearrangements, and more than one TRDD segment can be used in a single rearrangement. As expected, catfish TCRδ CDR3 regions are longer and more diverse than TCRγ CDR3 regions, and as a group they utilize more nucleotide additions and contain more nucleotide deletions than catfish TCRγ rearrangements.

Edwardsiella piscicida identified in the Southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR.

A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with >99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but <87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.

18S rRNA gene sequencing identifies a novel species of Henneguya parasitizing the gills of the channel catfish (Ictaluridae).

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.

Comprehensive survey and genomic characterization of Toll-like receptors (TLRs) in channel catfish, Ictalurus punctatus: identification of novel fish TLRs.

A comprehensive survey of channel catfish Toll-like receptors (TLRs) was undertaken following a genomic PCR approach based on degenerate primers. Twenty different TLRs were identified in channel catfish. Channel catfish TLR sequences were characterized by phylogenetic analysis based on their conserved Toll/interleukin-1 receptor domain and by in-depth analysis of leucine-rich repeat (LRR) motifs of the ligand binding extracellular domain (ECD). The catfish have representatives of all the TLR types defined in vertebrates with the exception of TLR6, TLR10, TLR11, TLR12, TLR13, TLR15, TLR23, and TLR24. Additionally, two new types were discovered: TLR25 and TLR26. TLR25 is also present in cyprinids, cichlids, plecoglossids, and adrianichthyids, suggesting its presence early in fish evolution. To date, TLR26 was found only in channel catfish. Like TLR18-23, TLR25 and TLR26 were not found in any other vertebrate classes and appear to be fish specific. Data mining using the catfish TLR sequences revealed that in addition to ictalurids and cyprinids, TLR4 is also present in salmonids. TLR19 and TLR20 were both found in ictalurids, cyprinids, and salmonids, demonstrating a wider range than previously known. The LRR structure within ECDs appeared generally well conserved. TLR7 demonstrated a very high identity to human TLR7 strongly suggesting that ligand specificity maybe conserved. Finally, expression profiling confirmed that most TLRs are widely expressed in a diversity of tissues and revealed marked differences of expression level.

A subset of leukocyte immune-type receptors (LITRs) regulates phagocytosis in channel catfish (Ictalurus punctatus) leukocytes.

Channel catfish, Ictalurus punctatus, leukocyte immune-type receptors (LITRs) constitute a large family of paired, immunoregulatory receptors unique to teleosts. A role for LITRs in phagocytosis has been proposed based on studies in mammalian cell lines; however, LITR-mediated phagocytosis has not been examined in the catfish model. In this study, we use two anti-LITR monoclonal antibodies, CC41 and 125.2, to contrast the effects of crosslinking subsets of inhibitory and activating LITRs. Briefly, LITRs expressed by catfish γδ T cells, αß T cells, and macrophage cell lines were crosslinked using mAb-conjugated fluorescent microbeads, and bead uptake was evaluated by flow cytometry and confirmed by confocal microscopy. A clear difference in the uptake of 125.2- and CC41-conjugated beads was observed. Crosslinking LITRs with mAb 125.2 resulted in efficient bead internalization, while mAb CC41 crosslinking of inhibitory LITRs resulted predominantly in a capturing phenotype. Pretreating catfish macrophages with mAb CC41 resulted in a marked decrease in LITR-mediated phagocytosis of 125.2-conjugated beads. Overall, these findings provide insight into fish immunobiology and validate LITRs as regulators of phagocytosis in catfish macrophages and γδ T cells.

Channel catfish CD8α and CD8ß co-receptors: characterization, expression and polymorphism.

In this study we report the identification and characterization of channel catfish, Ictalurus punctatus CD8α and CD8ß genes. Both genes encode predicted proteins containing a leader, a immunoglobulin superfamily V domain, a stalk/hinge region, a transmembrane region and a positively charged cytoplasmic tail (CYT) containing the conserved teleost C-X-H motif. Catfish CD8α and CD8ß are encoded as single copy genes and as in other vertebrates exhibit a conserved head to tail synteny; the CD8ß gene is found 14.1kb upstream of the CD8α gene. Both CD8α and CD8ß transcripts showed a low degree of polymorphism. Finally, as determined by q-PCR both CD8α and CD8ß are expressed in various catfish lymphoid tissues with the highest expression observed in thymus from 2 month old catfish-fry. In the future these results will provide the basis for evaluating the role of CD8(+) CTL and other CD8-bearing cells in response to immunization or infection in the catfish.

A Comprehensive Annotation of the Channel Catfish (Ictalurus punctatus) T Cell Receptor Alpha/Delta, Beta, and Gamma Loci.

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 form eight subgroups based on members sharing 75% nucleotide identity. However, the 149 TRAD genes in zebrafish form 53 subgroups. This difference in subgroup numbers between catfish and zebrafish is best explained by expansions of catfish TRADV subgroups, which likely occurred through multiple, relatively recent gene duplications. Similarly, 112 catfish TRBV genes form 30 subgroups, while the 51 zebrafish TRBV genes are placed into 36 subgroups. Notably, several catfish and zebrafish TRB subgroups share ancestor nodes. In addition, the complete catfish TR gene annotation was used to compile a TR gene segment database, which was applied in clonotype analysis of an available gynogenetic channel catfish transcriptome. Combined, the TR annotation and clonotype analysis suggested that the expressed TRA, TRB, and TRD repertoires were generated by different mechanisms. The diversity of the TRB repertoire depends on the number of TRBV subgroups and TRBJ genes, while TRA diversity relies on the many different TRAJ genes, which appear to be only minimally trimmed. In contrast, TRD diversity relies on nucleotide additions and the utilization of up to four TRDD segments.

Cloning and characterization of antiviral cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

To determine the role of piscine anti-viral cytotoxic cells, we analyzed the response of channel catfish to Ictalurid herpesvirus 1, commonly designated channel catfish virus (CCV). Peripheral blood leukocytes (PBL) from catfish immunized with MHC-matched, CCV-infected G14D cells (G14D-CCV) showed marked lysis of G14D-CCV but little to no lysis of uninfected allogenic (3B11) or syngeneic (G14D) cells. Expansion of effectors by in vitro culture in the presence of irradiated G14D-CCV cells generated cultures with enhanced cytotoxicity and often broader target range. Cytotoxic effectors expressed rearranged TCR genes, perforin, granzyme, and IFN-γ. Four clonal cytotoxic lines were developed and unique TCR gene rearrangements including γδ were detected. Furthermore, catfish CTL clones were either CD4+/CD8- or CD4-/CD8-. Two CTL lines showed markedly enhanced killing of G14D-CCV targets, while the other two lines displayed a broader target range. Collectively, catfish virus-specific CTL display unique features that illustrate the diversity of the ectothermic vertebrate immune response.

Catfish lymphocytes expressing CC41-reactive leukocyte immune-type receptors (LITRs) proliferate in response to Edwardsiella ictaluri infection in vitro.

Monoclonal antibodies (mAbs) CC34 and CC41 recognize overlapping subsets of leukocyte immune-type receptors (LITRs). The mAb CC34 was raised against the clonal TS32.15 cytotoxic T cell line and the mAb CC41 was raised against the clonal NK cell line TS10.1. In this study, an in vitro model was developed to monitor CC34- and CC41-reactive cells in response to Edwardsiella ictaluri infection. Briefly, head kidney leukocytes and peripheral blood lymphocytes (PBL) were isolated from individual catfish and labeled with CellTrace Violet and CellTrace FarRed dye, respectively. Head kidney-derived macrophages were infected with E. ictaluri and then cocultured with autologous PBL. The combined cell cultures were then analyzed using flow cytometry. A significant increase in CC41 staining was observed in the PBL population at 2, 5 and 7 days after culture, which suggest that LITRs are involved in cell-mediated immunity to E. ictaluri.

Sequence, genomic organization and expression of two channel catfish, Ictalurus punctatus, ghrelin receptors.

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration on target tissue mRNA expression. Analysis of sequence similarities indicated two genes putatively encoding GHS-R1 and GHS-R2, respectively, which have been known to be present in zebrafish. Organization and tissue expression of the GHS-R1 gene was similar to that reported for other species, and likewise yielded two detectable mRNA products as a result of alternative splicing. Expression of both full-length, GHS-R1a, and splice variant, GHS-R1b, mRNA was highest in the pituitary. Gene organization of GHS-R2 was similar to GHS-R1, but no splice variant was identified. Expression of GHS-R2a mRNA was highest in the Brockmann bodies. GHS-R1a mRNA was detected in unfertilized eggs and throughout embryogenesis, whereas GHR-R2a mRNA was not expressed in unfertilized eggs or early developing embryos and was the highest at the time of hatching. Catfish intraperitoneally injected with catfish ghrelin-Gly had greater mRNA expression of GHS-R1a in pituitaries at 2 h and Brockmann bodies at 4 h, and of GHS-R2a in Brockmann bodies at 6 h post injection. Amidated catfish ghrelin (ghrelin-amide) had no observable effect on expression of either pituitary receptor; however, GHS-R1a and GHS-R2a mRNA expression levels were increased 4 h post injection of ghrelin-amide in Brockmann bodies. This is the first characterization of GHS-R2a and suggests regulatory and functional differences between the two catfish receptors.

Insights into the dynamics of memory, effector and apoptotic cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus.

In this study, we used the channel catfish model clonal TS32.15 alloantigen-specific cytotoxic T cell (CTL) line to examine the dynamics of memory CTL expansion and senescence in teleosts. Although TS32.15 has been routinely cultured to study catfish CTL responses and killing mechanisms, little is known about the dynamics of the CTLs in these cultures. Here we show that this cell line consists of small non-cytotoxic T cells and larger granular effector T cells and that their ratios vary with time after stimulation. Small CTLs, when exposed to their irradiated targets, replicate and differentiate to morphologically distinct cytotoxic effectors, which do not replicate. After lysing target cells, or with prolonged absence of stimulation, the effector cells transition to a non-cytolytic senescent stage or become apoptotic. In addition, we demonstrate that natural IgM in catfish serum binds lipids, including PIP2, on early apoptotic CTLs, and that these IgM+ CTL can be cleared by catfish head kidney-derived macrophages.