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John D. Hansen

Publications and source records attributed to John D. Hansen.

At least 19 recordsLinked to original sources
USGS2026

Time course of effects and tissue concentrations of 6PPDQ in three sensitive salmonids with additional data for two centrarchid species

Studies of 6PPD-quinone (6PPDQ) toxicity have identified both sensitive and resistant salmonid species at environmentally relevant concentrations. However, comparisons between species are complicated by varying testing conditions, which can limit the application to derivation of environmental quality guidelines. Furthermore, few studies have reported 6PPDQ tissue concentrations in fish with known exposure concentrations. Using standard methods with flow-through exposures to provide stable 6PPDQ concentrations, we compared time courses of mortality in three sensitive salmonid species (coho salmon Oncorhynchus kisutch , brook trout Salvelinus fontinalis , rainbow trout Oncorhynchus mykiss ), evaluated the potential for latent (delayed) mortality, and measured tissue concentrations in both fish that died during exposure and those that survived an additional 7 days in clean water. Further experiments documented insensitivity in two centrarchids (smallmouth bass Micropterus dolomieu and largemouth bass Micropterus nigricans ). Our study provided evidence for two mortality time courses: one in which nearly all mortality occurs in the first 24 h and one in which mortalities continue to accumulate throughout the exposure, and 96-h and 24-h LC50s are quite different. We did not observe evidence for the delayed onset of mortality due to 6PPDQ exposure. Most fish that survived exposure had undetectable 6PPDQ concentrations after 1 week in clean water, potentially informing biomonitoring strategies for wild populations.

Environmental Science & Technology↗
USGS2026

Toxicity of 6PPD alternatives to salmonid cell lines

Stormwater runoff in urban areas introduces numerous anthropogenic chemicals into surrounding aquatic environments. One such chemical is 6PPD ( N -(1,3-Dimethylbutyl)- N ’-phenyl- p- phenylenediamine), the primary antidegradant responsible for protecting tire rubber from ozone-induced degradation and cracking. When exposed to ozone on the road surface 6PPD is transformed into the toxic transformation product 6PPD-quinone (6PPDQ). This compound is highly lethal to coho salmon ( O ncorhynchus kisutch ), and 24 hour 50% lethal concentrations (LC 50 ; 41-95 ng/L) are frequently detected in urban streams during stormwater runoff events 1-5 . The highest stream concentrations of 6PPDQ and most extensive coho salmon mortality occur in high-traffic urban areas, where tire wear particles accumulate due to the frequent braking and acceleration of cars 6 . In the Seattle area, some coho salmon-bearing streams can lose over 90% of returning females prior to spawning due to 6PPDQ-containing runoff 7 . Prespawn loss of this magnitude could result in the extinction of some urban spawning populations 8 . Importantly, the lethal effects of 6PPDQ have been observed in several other salmonid species, indicating that its impact may threaten fish health for urban aquatic environments globally 9-11 . The ubiquity of 6PPD in automobile tires, high toxicity of the transformation product 6PPDQ, and prevalence of 6PPD and 6PPDQ in environmental matrices has spurred investigations into alternative rubber antiozonants to replace 6PPD that retain tire performance while reducing ecological harm. A technical memorandum provided to the legislature by the Washington State Department of Ecology summarized a list of potential 6PPD alternatives requiring further study, including several structurally similar p-phenylenediamines (PPDs) 12 . However, the structural similarity of other PPDs to 6PPD and their known production of quinone transformation products 13 raises concerns that they may elicit similar toxic effects as 6PPDQ. Currently, investigations into the toxicity of proposed PPD alternatives or their ozonated transformation products in coho salmon are limited, representing a significant data gap in evaluating whether they offer improved environmental safety over 6PPD. This project investigated the toxicity of proposed alternative rubber antiozonants in vitro using immortalized cell lines derived from three salmonid species with known differences in sensitivity to 6PPDQ (coho salmon, Chinook salmon ( O. tshawytscha ), rainbow trout ( O. mykiss )) 1 . These immortalized cell lines replicate toxic responses observed in in vivo studies, while enabling higher-throughput testing and reducing the need for animal use and other resource-intensive procedures. The antiozonants and their transformation products selected for testing were chosen based on multiple criteria, including their inclusion in the Ecology technical memorandum, structural substitutions (e.g., branched/cyclic alkyl vs aryl substitutions), commercial availability, and input from the Washington State Department of Ecology and other industry experts.

Report↗
USGS2026

Roadway runoff induced acute mortality in juvenile coho salmon during spring storm events

Extensive mortalities of adult coho salmon ( Oncorhynchus kisutch ), often called “Urban Runoff Mortality Syndrome” (URMS), have been documented during the fall in creeks where water quality has been degraded by roadway runoff. The primary cause of mortality is 6PPD-quinone (6PPDQ; N -(1,3-dimethylbutyl)- N ′-phenyl- p -phenylenediamine quinone)–an ozone transformation product that forms on all vehicle tires. Laboratory studies have shown that juvenile coho salmon are highly sensitive to 6PPDQ exposure. Unlike adults, juveniles reside in impacted watersheds year-round, including during the spring when 6PPDQ concentrations can frequently exceed lethal thresholds during storms. To assess the potential incidence of URMS in springtime rearing habitats for juvenile coho salmon, we conducted a paired water quality and toxicology study at Miller Creek, a runoff-impacted watershed in Normandy Park, WA, USA. Using a small field facility, three naïve groups of juvenile coho salmon ( N = 720) were exposed to either creek water or groundwater ( N = 120 per treatment per storm), across three spring storms while comparing water quality and mortality end points. In creek water during exposures, peak 6PPDQ concentrations reached 73–110 ng/L, exceeding reported median lethal concentrations (LC 50 ) for coho salmon. Over each 24–73 h storm exposure period, ∼80% of Miller Creek-exposed juvenile salmon died. No mortality occurred among control fish exposed to groundwater. These results indicate previously unidentified mortality risks for juvenile life stages of coho salmon during spring storms, suggesting substantial and year-round water quality impediments to coho salmon health and recovery across roadway runoff-impacted spawning, rearing, and migratory habitats.

Washington↗
USGS2025

Evaluation of 6PPD-quinone lethal toxicity and sublethal effects on disease resistance and swimming fitness in coastal cutthroat trout (Oncorhynchus clarkii clarkii)

6PPD-quinone (6PPDQ), derived from the tire-protectant 6PPD reacting with ozone, is an emerging contaminant of concern owing to its role in coho salmon ( Oncorhynchus kisutch ) deaths via urban runoff mortality syndrome (URMS). Given the impact of 6PPDQ on aquatic life in urban streams, we addressed the acute toxicity of 6PPDQ exposure on coastal cutthroat trout (CCT) ( Oncorhynchus clarkii clarkii ), a species sympatric with coho salmon in natal watersheds. Using static exposures coupled with analytical chemistry, we determined the 24-h LC 50 values for alevin (297.2 ng/L), swim-up fry (39.6 ng/L), 5-month parr (103.3 ng/L), and 13-month juveniles (185.9 ng/L)─values similar to toxicity observed in coho salmon. Additionally, the 96-h LC 50 (77.6 ng/L) was 2.4 times more lethal for juvenile CCT. We assessed potential effects of sublethal 6PPDQ exposure on disease resistance to infectious hematopoietic necrosis (IHN), an endemic viral disease of Pacific salmon, and to swimming performance. Sublethal 6PPDQ (53.6 ng/L) did not affect survival of parr exposed to IHN virus compared to virus alone. Conversely, 6PPDQ exposure as low as 72.2 ng/L significantly reduced 15- and 24-month juvenile swimming performance, and 120.5 ng/L 6PPDQ increased blood hematocrit. Overall, CCT are the second most sensitive species tested to date for 6PPDQ sensitivity which further emphasizes the need for identifying alternatives to 6PPD.

Environmental Science and Technology↗
USGS2024

Structurally selective ozonolysis of p-phenylenediamines and toxicity in coho salmon and rainbow trout

The tire-rubber-derived ozonation product of N -(1,3-dimethylbutyl)- N ′-phenyl- p -phenylenediamine (6PPD), N -(1,3-dimethylbutyl)- N ′-phenyl- p -phenylenediamine-quinone (6PPD-Q), was recently discovered to cause acute mortality in coho salmon ( Oncorhynchus kisutch ). para -Phenylenediamines (PPDs) with variable side chains distinct from 6PPD have been identified as potential replacement antioxidants, but their toxicities remain unclear under environmentally relevant ozone conditions. We herein tested the multiphase gas-surface ozone reactivity of four select PPDs [6PPD, N -isopropyl- N ′-phenyl- p -phenylenediamine (IPPD), N , N ′-diphenyl- p -phenylenediamine (DPPD), and N -phenyl- N ′-cyclohexyl- p -phenylenediamine (CPPD)] and evaluated the toxicity of their reaction mixtures in coho salmon, rainbow trout ( Oncorhynchus mykiss ), and fathead minnow ( Pimephales promelas ). 6PPD and IPPD were found to rapidly react with ozone, while no significant multiphase ozone reactivity was observed for DPPD or CPPD. The viability of coho salmon CSE-119 cells was strongly affected by the ozonolysis products of 6PPD but not by those of the other three PPDs. Acute mortality was only observed in juvenile rainbow trout that were exposed to oxidized 6PPD, suggesting a common mechanism of toxic action in the two salmonid fish species. This study reports the structurally selective ozone reactivity of PPDs and the unique toxicity of 6PPD ozonolysis mixtures, which demonstrates that other PPDs are potential alternative antioxidants.

Environmental Science and Technology↗
USGS2024

Tire-derived contaminants 6PPD and 6PPD-Q: Analysis, sample handling, and reconnaissance of United States stream exposures

The environmental ubiquity of tire and road wear particles (TRWP) underscores the need to understand the occurrence, persistence, and environmental effects of tire-related chemicals in aquatic ecosystems. One such chemical is 6PPD-quinone (6PPD-Q), a transformation product of the tire antioxidant 6PPD. In urban stormwater runoff 6PPD-Q can exceed acute toxicity thresholds for several salmonid species and is being implicated in significant coho salmon losses in the Pacific Northwest. There is a critical need to understand the prevalence of 6PPD-Q across watersheds to identify habitats heavily affected by TRWPs. We conducted a reconnaissance of 6PPD and 6PPD-Q in surface waters across the United States from sites (N = 94) with varying land use (urban, agricultural, and forested) and streamflow to better understand stream exposures. A rapid, low-volume direct-inject, liquid chromatography mass spectrometry method was developed for the quantitation of 6PPD-Q and screening for 6PPD. Laboratory holding times, bottle material, headspace, and filter materials were investigated to inform best practices for 6PPD-Q sampling and analysis. Glass bottles with PTFE-lined caps minimized sorption and borosilicate glass fiber filters provided the highest recovery. 6PPD-Q was stable for at least 5 months in pure laboratory solutions and for 75 days at 5 °C with minimal headspace in the investigated surface water and stormwaters. Results also indicated samples can be frozen to extend holding times. 6PPD was not detected in any of the 526 analyzed samples and there were no detections of 6PPD-Q at agricultural or forested sites. 6PPD-Q was frequently detected in stormwater (57%, N = 90) and from urban impacted sites (45%, N = 276) with concentrations ranging from 0.002 to 0.29 μg/L. The highest concentrations, above the lethal level for coho salmon, occurred during stormwater runoff events. This highlights the importance of capturing episodic runoff events in urban areas near ecologically relevant habitat or nursery grounds for sensitive species.

Chemosphere↗
USGS2024

Structurally selective ozonolysis of p-phenylenediamines and toxicity in coho salmon and rainbow trout

The tire rubber-derived ozonation product of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD), N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q), was recently discovered to cause acute mortality in Coho Salmon (Oncorhynchus kisutch). para-Phenylenediamines (PPDs) with variable side chains distinct from 6PPD have been identified as potential replacement commercial antioxidants, but their structure-related ozone reactivities and toxicities remain unexplored. We herein tested the multiphase gas-surface ozone reactivity of four select PPDs and evaluated the toxicity of their reaction mixtures in Coho Salmon and Rainbow Trout (Oncorhynchus mykiss). 6PPD and N-Isopropyl-N'-phenyl-p-phenylenediamine (IPPD) were found to rapidly react with ozone to form 22 and 16 transformation products, respectively, including PPD-Qs. No significant multiphase ozone reactivity was observed for N,N'-Diphenyl-p-phenylenediamine (DPPD) or N-Cyclohexyl-N'-phenyl-p-phenylenediamine (CPPD) despite their structural similarity to 6PPD. The viability of Coho Salmon CSE-119 cells was strongly affected by the ozonolysis products of 6PPD, but not by those of the other three PPDs. The cytotoxicity of the 6PPD reaction mixture increased with ozonolysis time, with the strongest toxicity being observed after 7 days of oxidation by 100 ppb of ozone. As with Coho Salmon cells, acute mortality was only observed in juvenile Rainbow Trout that were exposed to the oxidized 6PPD reaction mixture, suggesting a common mechanism of toxic action in the two salmonid fish species. Compound- and regio-selective formation of hydroxylated metabolites of 6PPD-Q were detected in Rainbow Trout exposed to the 6PPD reaction mixture, which may be related to its selective toxicity. This study reports the structurally selective ozone reactivity of PPDs, and the unique toxicity of 6PPD ozonolysis mixtures, which demonstrates that other PPDs are potential alternative antioxidants.

ChemRxiv↗
USGS2023

Tire-derived transformation product 6PPD-quinone induces mortality and transcriptionally disrupts vascular permeability pathways in developing coho salmon

Urban stormwater runoff frequently contains the car tire transformation product 6PPD-quinone, which is highly toxic to juvenile and adult coho salmon ( Onchorychus kisutch ). However, it is currently unclear if embryonic stages are impacted. We addressed this by exposing developing coho salmon embryos starting at the eyed stage to three concentrations of 6PPD-quinone twice weekly until hatch. Impacts on survival and growth were assessed. Further, whole-transcriptome sequencing was performed on recently hatched alevin to address the potential mechanism of 6PPD-quinone-induced toxicity. Acute mortality was not elicited in developing coho salmon embryos at environmentally measured concentrations lethal to juveniles and adults, however, growth was inhibited. Immediately after hatching, coho salmon were sensitive to 6PPD-quinone mortality, implicating a large window of juvenile vulnerability prior to smoltification. Molecularly, 6PPD-quinone induced dose-dependent effects that implicated broad dysregulation of genomic pathways governing cell–cell contacts and endothelial permeability. These pathways are consistent with previous observations of macromolecule accumulation in the brains of coho salmon exposed to 6PPD-quinone, implicating blood–brain barrier disruption as a potential pathway for toxicity. Overall, our data suggests that developing coho salmon exposed to 6PPD-quinone are at risk for adverse health events upon hatching while indicating potential mechanism(s) of action for this highly toxic chemical.

Environmental Science & Technology↗
USGS2023

Establishing an in vitro model to assess the toxicity of 6PPD-quinone and other tire wear transformation products

The tire wear transformation product 6PPD-quinone (6PPDQ) has been implicated as the causative factor for broad scale mortality events for coho salmon in the Pacific Northwest. Highly variable sensitivity to 6PPDQ in closely related salmonids complicates efforts to evaluate the broader toxicological impacts to aquatic ecosystems. Our goals were to (1) validate the large range of in vivo species sensitivities reported for coho, Chinook, and sockeye salmon and (2) develop an in vitro platform for assessing 6PPDQ toxicity. In vivo studies confirmed the acute sensitivity of juvenile coho (12 h LC 50 = 80.4 ng/L) and demonstrated that sockeye salmon were not vulnerable to mortality. Chinook salmon were sensitive to 6PPDQ mortality at initial concentrations >25 μg/L, ∼10-fold greater than reported environmental measurements. In vitro , the coho salmon cell line CSE-119 was acutely sensitive to 6PPDQ (metabolic EC 50 = 7.9 μg/L, cytotoxicity EC 50 = 6.1 μg/L). Analogous Chinook (CHSE-214) and sockeye salmon (SSE-5) cell lines were nonresponsive in both assays, and rainbow trout RTG-2 cells began showing metabolic effects at 68 μg/L (EC 5 ). Recreation of species-specific 6PPDQ sensitivity in vitro implicates conserved modes of action in CSE-119 that could be utilized for mechanistic studies of 6PPDQ toxicity and screening of other PPD transformation products.

Environmental Science and Technology Letters↗
USGS2022

Exposure to 17α-ethinylestradiol results in differential susceptibility of largemouth bass (Micropterus salmoides) to bacterial infection

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass ( Micropterus salmoides ) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2 Low , 0.87 ng/L) or high (EE2 High , 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD 40 dose of the Gram-negative bacteria Edwardsiella piscicida . Unexpectedly, this prior exposure to EE2 High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2 Low -exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2 High treatment that could underlie the observed protective effect and metabolic shift in EE2 High -infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

Environmental Science and Technology↗
USGS2022

Evaluation of Francisella orientalis ΔpdpA as a live attenuated vaccine against piscine Francisellosis in Nile tilapia

Francisella orientalis is an important bacterial pathogen of marine and freshwater fish with worldwide distribution. Fish francisellosis is a severe subacute to chronic granulomatous disease, with high mortalities and high infectivity rates in cultured and wild fish. To date, there is no approved vaccine for this disease. In this study, we evaluated the efficacy of a defined F. orientalis pathogenicity determinant protein A ( pdpA ) mutant (Δ pdpA ) as a live attenuated immersion vaccine against subsequent immersion challenge with the wild-type organism. Immunized Nile tilapia Oreochromis niloticus were protected (45% relative percent survival) from the lethal challenges and presented significantly lower mortality than nonvaccinated and challenged treatments. Although serum IgM was significantly higher in immunized fish, similar bacterial loads were detected in vaccinated and nonvaccinated survivors. In conclusion, although the F. orientalis Δ pdpA is attenuated and effectively stimulated an adaptive immune response, the low relative percent survival and high bacterial persistence in survivors of immunized and challenged treatments indicates low suitability of Δ pdpA as a mucosal vaccine for tilapia under conditions used in this study.

Journal of Aquatic Animal Health↗
USGS2021

Neither microcystin, nor nodularin, nor cylindrospermopsin directly interact with human toll-like receptors

Various stressors including temperature, environmental chemicals, and toxins can have profound impacts on immunity to pathogens. Increased eutrophication near rivers and lakes coupled with climate change are predicted to lead to increased algal blooms. Currently, the effects of cyanobacterial toxins on disease resistance in mammals is a largely unexplored area of research. Recent studies have suggested that freshwater cyanotoxins can elicit immunomodulation through interaction with specific components of innate immunity, thus potentially altering disease susceptibility parameters for fish, wildlife, and human health owing to the conserved nature of the vertebrate immune system. In this study, we investigated the effects of three microcystin congeners (LR, LA, and RR), nodularin-R, and cylindrospermopsin for their ability to directly interact with nine different human Toll-like receptors (TLRs)—key pathogen recognition receptors for innate immunity. Toxin concentrations were verified by LC/MS/MS prior to use. Using an established HEK293-hTLR NF-κB reporter assay, we concluded that none of the tested toxins (29–90 nM final concentration) directly interacted with human TLRs in either an agonistic or antagonistic manner. These results suggest that earlier reports of cyanotoxin-induced NF-κB responses likely occur through different surface receptors to mediate inflammation.

Chemosphere↗
USGS2021

Disruption of the Francisella noatunensis orientalis pdpA gene results in virulence attenuation and protection in zebrafish

Several Francisella spp. including F. noatunensis are regarded as important emerging pathogens of wild and farmed fish. However, very few studies have investigated the virulence factors that allow these bacterial species to be pathogenic in fish. The Francisella Pathogenicity Island (FPI) is a well-described, gene-dense region encoding major virulence factors for the genus Francisella. PdpA is a member of the pathogenicity determining protein genes encoded by the FPI that are implicated in the ability of the mammalian pathogen, F. tularensis , to escape and replicate in infected host cells. Using a sacB suicide approach, we generated pdpA knockouts to address the role of PdpA as a virulence factor for F. noatunensis . Because polarity can be an issue in gene-dense regions, we generated two different marker-based mutants in opposing polarity ( Fno Δ pdpA1 and Δ pdpA2 ). Both mutants were attenuated (p<0.0001) in zebrafish challenges and displayed impaired intracellular replication (p<0.05) and cytotoxicity (p<0.05), all of which could be restored to wild-type (WT) levels by complementation for Fno Δ pdpA 1. Importantly, differences were found for bacterial burden and induction of acute phase and pro-inflammatory genes for Fno Δ pdpA 1 and Δ pdpA 2 compared to WT during acute infection. In addition, neither mutant resulted in significant histopathological changes. Finally, immunization with Fno Δ pdpA1 led to protection (p<0.012) against an acute lethal-dose 40 challenge with WT Fno in the zebrafish model of infection. Taken together, this study further demonstrates physiological similarities within the genus Francisella relative to their phylogenetic relationships and the utility of zebrafish for addressing virulence factors for the genus.

Infection and Immunity↗
USGS2020

Genomic architecture and repertoire of the rainbow trout immunoglobulin light chain genes

The genomic loci encoding the four immunoglobulin light chains (IgL1, IgL2, IgL3, and IgL4) in the Swanson trout genome assembly were annotated in order to provide a measurement of the potential IgL repertoire. IgL1 and IgL3 gene segments are co-localized on chromosomes 21, 18, 15, and 7 while IgL2 and IgL4 were found on chromosomes 13 and 17, respectively. In total, 48 constant (C L ), 87 variable (V L ), and 59 joining (J L ) productive genes are described. Pairwise alignment of the V L segments revealed that they belong to nine different families, three of which (kappa IV, V, and VI) are described for the first time in this study. V L and C L sequences on chromosome 15 and 21 and those on chromosomes 7 and 18 clustered together in phylogenetic analysis. PCR was used to examine IgL C L and V L genes in 9 lines of rainbow trout. IgL4 in the Hot Creek and Golden trout lines was missing 42 nucleotides resulting in a loss of 14 amino acids. The sigma IV variable family was completely absent from the Swanson, Arlee, Hot Creek, and wild type lines and silenced in the Skamania line with the addition of 176 bp mini-satellite insert. Similarly, the Whale Rock, Arlee, and wild type lines were all found to encode two sigma II products, a functional 252 bp product and a larger 425 bp product that contained a 172 bp insert. Results from this study indicate that there are genomic differences in IgL repertoire between different lines of trout that could affect humoral immune responses post vaccination and during disease.

Developmental and Comparative Immunology↗
USGS2019

Effect of growth rate on transcriptomic responses to immune stimulation in wild-type, domesticated, and GH-transgenic coho salmon

Background Transcriptomic responses to immune stimulation were investigated in coho salmon (Oncorhynchus kisutch) with distinct growth phenotypes. Wild-type fish were contrasted to strains with accelerated growth arising either from selective breeding (i.e. domestication) or genetic modification. Such distinct routes to accelerated growth may have unique implications for relationships and/or trade-offs between growth and immune function. Results RNA-Seq was performed on liver and head kidney in four ‘growth response groups’ injected with polyinosinic-polycytidylic acid (Poly I:C; viral mimic), peptidoglycan (PGN; bacterial mimic) or PBS (control). These groups were: 1) ‘W’: wild-type, 2) ‘TF’: growth hormone (GH) transgenic salmon with ~ 3-fold higher growth-rate than W, 3) ‘TR’: GH transgenic fish ration restricted to possess a growth-rate equal to W, and 4) ‘D’: domesticated non-transgenic fish showing growth-rate intermediate to W and TF. D and TF showed a higher similarity in transcriptomic response compared to W and TR. Several immune genes showed constitutive expression differences among growth response groups, including perforin 1 and C-C motif chemokine 19-like. Among the affected immune pathways, most were up-regulated by Poly I:C and PGN. In response to PGN, the c-type lectin receptor signalling pathway responded uniquely in TF and TR. In response to stimulation with both immune mimics, TR responded more strongly than other groups. Further, group-specific pathway responses to PGN stimulation included NOD-like receptor signalling in W and platelet activation in TR. TF consistently showed the most attenuated immune response relative to W, and more DEGs were apparent in TR than TF and D relative to W, suggesting that a non-satiating ration coupled with elevated circulating GH levels may cause TR to possess enhanced immune capabilities. Alternatively, TF and D salmon are prevented from acquiring the same level of immune response as TR due to direction of energy to high overall somatic growth. Further study of the effects of ration restriction in growth-modified fishes is warranted. Conclusions These findings improve our understanding of the pleiotropic effects of growth modification on the immunological responses of fish, revealing unique immune pathway responses depending on the mechanism of growth acceleration and nutritional availability.

BMC Genomics↗
USGS2019

Generation of lamprey monoclonal antibodies (Lampribodies) using the phage display system

The variable lymphocyte receptors (VLRs) consist of leucine rich repeats (LRRs) and comprise the humoral antibodies produced by lampreys and hagfishes. The diversity of the molecules is generated by stepwise genomic rearrangements of LRR cassettes dispersed throughout the VLRB locus. Previously, target-specific monovalent VLRB antibodies were isolated from sea lamprey larvae after immunization with model antigens. Further, the cloned VLR cDNAs from activated lamprey leukocytes were transfected into human cell lines or yeast to select best binders. Here, we expand on the overall utility of the VLRB technology by introducing it into a filamentous phage display system. We first tested the efficacy of isolating phage into which known VLRB molecules were cloned after a series of dilutions. These experiments showed that targeted VLRB clones could easily be recovered even after extensive dilutions (1 to 10 9 ). We further utilized the system to isolate target-specific “lampribodies” from phage display libraries from immunized animals and observed an amplification of binders with relative high affinities by competitive binding. The lampribodies can be individually purified and ostensibly utilized for applications for which conventional monoclonal antibodies are employed.

Biomolecules↗
USGS2019

Standardized IMGT nomenclature of salmonidae IGH genes, the paradigm of Atlantic salmon and rainbow trout: From genomics to repertoires

In teleost fish as in mammals, humoral adaptive immunity is based on B lymphocytes expressing highly diverse immunoglobulins (IG). During B cell differentiation, IG loci are subjected to genomic rearrangements of V, D, and J genes, producing a unique antigen receptor expressed on the surface of each lymphocyte. During the course of an immune response to infections or immunizations, B cell clones specific of epitopes from the immunogen are expanded and activated, leading to production of specific antibodies. Among teleost fish, salmonids comprise key species for aquaculture. Rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) are especially important from a commercial point of view and have emerged as critical models for fish immunology. The growing interest to capture accurate and comprehensive antibody responses against common pathogens and vaccines has resulted in recent efforts to sequence the IG repertoire in these species. In this context, a unified and standardized nomenclature of salmonid IG heavy chain (IGH) genes is urgently required, to improve accuracy of annotation of adaptive immune receptor repertoire dataset generated by high-throughput sequencing (AIRRseq) and facilitate comparisons between studies and species. Interestingly, the assembly of salmonids IGH genomic sequences is challenging due to the presence of two large size duplicated IGH loci and high numbers of IG genes and pseudogenes. We used data available for Atlantic salmon to establish an IMGT standardized nomenclature of IGH genes in this species and then applied the IMGT rules to the rainbow trout IGH loci to set up a nomenclature, which takes into account the specificities of Salmonid loci. This unique, consistent nomenclature for Salmonid IGH genes was then used to construct IMGT sequence reference directories allowing accurate annotation of AIRRseq data. The complex issues raised by the genetic diversity of salmon and trout strains are discussed in the context of IG repertoire annotation.

Frontiers in Immunology↗