Extensive age-dependent loss of antibody diversity in naturally short-lived turquoise killifish.

Aging individuals exhibit a pervasive decline in adaptive immune function, with important implications for health and lifespan. Previous studies have found a pervasive loss of immune-repertoire diversity in human peripheral blood during aging; however, little is known about repertoire aging in other immune compartments, or in species other than humans. Here, we perform the first study of immune-repertoire aging in an emerging model of vertebrate aging, the African turquoise killifish (Nothobranchius furzeri). Despite their extremely short lifespans, these killifish exhibit complex and individualized heavy-chain repertoires, with a generative process capable of producing millions of distinct productive sequences. Whole-body killifish repertoires decline rapidly in within-individual diversity with age, while between-individual variability increases. Large, expanded B-cell clones exhibit far greater diversity loss with age than small clones, suggesting important differences in how age affects different B-cell populations. The immune repertoires of isolated intestinal samples exhibit especially dramatic age-related diversity loss, related to an elevated prevalence of expanded clones. Lower intestinal repertoire diversity was also associated with transcriptomic signatures of reduced B-cell activity, supporting a functional role for diversity changes in killifish immunosenescence. Our results highlight important differences in systemic vs. organ-specific aging dynamics in the adaptive immune system.

The impact of salinity and dissolved oxygen regimes on transcriptomic immune responses to oil in early life stage Fundulus grandis.

Understanding the effects of oil exposure on early life stage fish species is critical to fully assessing the environmental impacts of oil spills. Oil released from the 2010 Deepwater Horizon spill reached habitats where estuarine fish routinely spawn. In addition, estuaries are highly dynamic environments, therefore, fish in these areas are routinely exposed to varying salinity and dissolved oxygen (DO) levels, each of which are known to modulate transcriptional responses. Fish exposed to oil often display altered immune competence, and several studies have shown that Deepwater Horizon oil in particular causes modulation of various immune functions. However, few studies have directly examined how environmental parameters may affect oil-induced immunomodulation, particularly in early life stage fishes when the immune system is still developing. To this end, we examined transcriptional patterns of immune genes and pathways in Fundulus grandis larvae to various oil (0, 15 µg/L), salinity (3, 30 ppt), and DO (2.5, 6 mg/L) regimes in a fully factorial design. Our results suggest that immune pathways are generally activated in all treatment groups with the exception of the Low Salinity/No Oil/Hypoxia treatment where immune pathways are largely suppressed, and the High Salinity/No Oil/Hypoxia treatment where pathways are unchanged. The High Salinity/Oil/Hypoxia treatment had the largest number of enriched immune pathways (44 as defined by IPA and 43 as defined by ConsensusPathDB), indicating that oil under certain environmental conditions has the potential to further modulate immune-related genes, pathways, and responses in fish.

Development, characterization, and technical applications of a fish lysozyme-specific monoclonal antibody (mAb M24-2).

Lysozyme is one of several humoral and cellular factors associated with front line, innate immunity in all vertebrates. Historically, circulating lysozyme has been quantified in teleosts by measuring enzymatic activity against heat-killed Mycococcus lysodieticus using whole serum or plasma at a low pH. However, the amount of serum or plasma required for standard lysozyme activity exceeds that which can be easily acquired from small fish, thus making lysozyme a difficult endpoint to measure in limited sample volumes. Moreover, while circulating lysozyme is considered to be an indicator of proinflammatory phagocyte activity, the cellular source of this protein is not easily detected in fish. While several antibodies against lysozyme are commercially available for use in higher vertebrates, neither reacts with lysozyme in fish. In this study, a monoclonal antibody (mAb) for detecting and quantifying lysozyme was developed from mummichog, Fundulus heteroclitus, myeloid cells that also recognizes hen egg lysozyme (HEL), then tested for cross-reactivity in different species of teleosts. A single protein of ≈14-15 kDa mass was identified by the mAb in fish cell lysates and plasma samples, as well as denatured HEL. Total circulating lysozyme protein was compared to lysozyme activity using standard ELISA procedures and was found to correlate with enzymatic activity. Using mAb M24-2, intracellular lysozyme protein was detected in formalin-fixed and permeabilized lymphoid cells adhered to glass cover slips. Moreover, mAb M24-2 localizes lysozyme to myeloid cells. Finally, it was demonstrated that mAb M24-2 is suitable for immunohistochemistry in that lysozyme could be detected in plastic-embedded lymphoid tissues.

Bioindicators of immune function in creosote-adapted estuarine killifish, Fundulus heteroclitus.

Several populations of the estuarine killifish, Fundulus heteroclitus, also known as the mummichog, exhibit characteristics of adaptation to priority pollutants. One such population of mummichog inhabits the Elizabeth River in Virginia at the Atlantic Wood site (AW), a U.S. Environmental Protection Agency (EPA) Superfund site heavily contaminated with creosote containing a mixture of polyaromatic hydrocarbons (PAHs). Although PAHs are known to be immunotoxic in experimental animals, resident AW mummichogs seem to thrive. Mummichogs from the AW site and a reference site were subsequently examined over a 2-yr period for total immunoglobin (IgM), as well as circulating antibody levels against 5 ubiquitous marine bacteria. Expression profiles of circulating and lymphoid lysozyme and lymphoid cyclooxygenase-2 (COX-2) were also examined. Compared to relatively high total IgM and specific antibody responses in reference fish, AW mummichogs had lower circulating IgM and lower specific antibody levels against all bacteria examined, however they had higher levels of circulating lysozyme. Lymphoid cells in the AW mummichogs also expressed higher levels of lysozyme, as well as COX-2, which may indicate a state of macrophage activation. Elevated COX-2 levels may be associated with enhanced metabolism of PAHs through cooxidation-peroxidase pathways. Additional studies attempted to immunize AW mummichogs reared in uncontaminated water to compare their antibody responses to that of reference fish. AW mummichogs did not survive 40 d post culture, while reference fish thrived. Our findings suggest that the chemical environment at the AW site may be vicariously enhancing components of innate immunity, probably through oxidative stress pathways, in resident mummichogs, while actively suppressing humoral immune responses.

Development and characterization of polyclonal antibodies against the aryl hydrocarbon receptor protein family (AHR1, AHR2, and AHR repressor) of Atlantic killifish Fundulus heteroclitus.

The aryl hydrocarbon receptor (AHR) and AHR repressor (AHRR) proteins regulate gene expression in response to some halogenated aromatic hydrocarbons and polycyclic aromatic hydrocarbons. The Atlantic killifish is a valuable model of the AHR signaling pathway, but antibodies are not available to fully characterize AHR and AHRR proteins. Using bacterially expressed AHRs, we developed specific and sensitive polyclonal antisera against the killifish AHR1, AHR2, and AHRR. In immunoblots, these antibodies recognized full-length killifish AHR and AHRR proteins synthesized in rabbit reticulocyte lysate, proteins expressed in mammalian cells transfected with killifish AHR and AHRR constructs, and AHR proteins in cytosol preparations from killifish tissues. Killifish AHR1 and AHR2 proteins were detected in brain, gill, kidney, heart, liver, and spleen. Antisera specifically precipitated their respective target proteins in immunoprecipitation experiments with in vitro-expressed proteins. Killifish ARNT2 co-precipitated with AHR1 and AHR2. These sensitive, specific, and versatile antibodies will be valuable to researchers investigating AHR signaling and other physiological processes involving AHR and AHRR proteins.

Persistence of Mhc heterozygosity in homozygous clonal killifish, Rivulus marmoratus: implications for the origin of hermaphroditism.

The mangrove killifish Rivulus marmoratus, a neotropical fish in the order Cyprinodontiformes, is the only known obligatorily selfing, synchronous hermaphroditic vertebrate. To shed light on its population structure and the origin of hermaphroditism, major histocompatibility complex (Mhc) class I genes of the killifish from seven different localities in Florida, Belize, and the Bahamas were cloned and sequenced. Thirteen loci and their alleles were identified and classified into eight groups. The loci apparently arose approximately 20 million years ago (MYA) by gene duplications from a single common progenitor in the ancestors of R. marmoratus and its closest relatives. Distinct loci were found to be restricted to different populations and different individuals in the same population. Up to 44% of the fish were heterozygotes at Mhc loci, as compared to near homozygosity at non-Mhc loci. Large genetic distances between some of the Mhc alleles revealed the presence of ancestral allelic lineages. Computer simulation designed to explain these findings indicated that selfing is incomplete in R. marmoratus populations, that Mhc allelic lineages must have diverged before the onset of selfing, and that the hermaphroditism arose in a population containing multiple ancestral Mhc lineages. A model is proposed in which hermaphroditism arose stage-wise by mutations, each of which spread through the entire population and was fixed independently in the emerging clones.