Molecular basis of parental contributions to the behavioural tolerance of elevated pCO2 in a coral reef fish.

Knowledge of adaptive potential is crucial to predicting the impacts of ocean acidification (OA) on marine organisms. In the spiny damselfish, Acanthochromis polyacanthus, individual variation in behavioural tolerance to elevated pCO2 has been observed and is associated with offspring gene expression patterns in the brain. However, the maternal and paternal contributions of this variation are unknown. To investigate parental influence of behavioural pCO2 tolerance, we crossed pCO2-tolerant fathers with pCO2-sensitive mothers and vice versa, reared their offspring at control and elevated pCO2 levels, and compared the juveniles' brain transcriptional programme. We identified a large influence of parental phenotype on expression patterns of offspring, irrespective of environmental conditions. Circadian rhythm genes, associated with a tolerant parental phenotype, were uniquely expressed in tolerant mother offspring, while tolerant fathers had a greater role in expression of genes associated with histone binding. Expression changes in genes associated with neural plasticity were identified in both offspring types: the maternal line had a greater effect on genes related to neuron growth while paternal influence impacted the expression of synaptic development genes. Our results confirm cellular mechanisms involved in responses to varying lengths of OA exposure, while highlighting the parental phenotype's influence on offspring molecular phenotype.

Elevated CO2 affects anxiety but not a range of other behaviours in juvenile yellowtail kingfish.

Elevated seawater CO2 can cause a range of behavioural impairments in marine fishes. However, most studies to date have been conducted on small benthic species and very little is known about how higher oceanic CO2 levels could affect the behaviour of large pelagic species. Here, we tested the effects of elevated CO2, and where possible the interacting effects of high temperature, on a range of ecologically important behaviours (anxiety, routine activity, behavioural lateralization and visual acuity) in juvenile yellowtail kingfish, Seriola lalandi. Kingfish were reared from the egg stage to 25 days post-hatch in a full factorial design of ambient and elevated CO2 (~500 and ~1000 µatm pCO2) and temperature (21 °C and 25 °C). The effects of elevated CO2 were trait-specific with anxiety the only behaviour significantly affected. Juvenile S. lalandi reared at elevated CO2 spent more time in the dark zone during a standard black-white test, which is indicative of increased anxiety. Exposure to high temperature had no significant effect on any of the behaviours tested. Overall, our results suggest that juvenile S. lalandi are largely behaviourally tolerant to future ocean acidification and warming. Given the ecological and economic importance of large pelagic fish species more studies investigating the effect of future climate change are urgently needed.

Exposure to Crude Oil from the Deepwater Horizon Oil Spill Impairs Oil Avoidance Behavior without Affecting Olfactory Physiology in Juvenile Mahi-Mahi (Coryphaena hippurus).

The understanding of the detection threshold and behavioral response of fishes in response to crude oil is critical to predicting the effects of oil spills on wild fish populations. The Deepwater Horizon oil spill released approximately 4.9 million barrels of crude oil into the northern Gulf of Mexico in 2010, overlapping spatially and temporally with the habitat of many pelagic fish species. Yet, it is unknown whether highly migratory species, such as mahi-mahi (Coryphaena hippurus), might detect and avoid oil contaminated waters. We tested the ability of control and oil-exposed juvenile mahi-mahi (15-45 mm) to avoid two dilutions of crude oil in a two-channel flume. Control fish avoided the higher concentration (27.1 µg/L Σ50PAH), while oil-exposed (24 h, 18.0 µg/L Σ50PAH) conspecifics did not. Electro-olfactogram (EOG) data demonstrated that both control and oil-exposed (24 h, 14.5 µg/L Σ50PAH) juvenile mahi-mahi (27-85 mm) could detect crude oil as an olfactory cue and that oil-exposure did not affect the EOG amplitude or duration in response to oil or other cues. These results show that a brief oil exposure impairs the ability of mahi-mahi to avoid oil and suggests that this alteration likely results from injury to higher order central nervous system processing rather than impaired olfactory physiology.

Damsels in Distress: Oil Exposure Modifies Behavior and Olfaction in Bicolor Damselfish (Stegastes partitus).

In fishes, olfactory cues evoke behavioral responses that are crucial to survival; however, the receptors, olfactory sensory neurons, are directly exposed to the environment and are susceptible to damage from aquatic contaminants. In 2010, 4.9 million barrels of crude oil were released into the northern Gulf of Mexico from the Deepwater Horizon disaster, exposing marine organisms to this environmental contaminant. We examined the ability of bicolor damselfish (Stegastes partitus), exposed to the water accommodated fraction (WAF) of crude oil, to respond to chemical alarm cue (CAC) using a two-channel flume. Control bicolor damselfish avoided CAC in the flume choice test, whereas WAF-exposed conspecifics did not. This lack of avoidance persisted following 8 days of control water conditions. We then examined the physiological response to CAC, brine shrimp rinse, bile salt, and amino acid cues using the electro-olfactogram (EOG) technique and found that WAF-exposed bicolor damselfish were less likely to detect CAC as an olfactory cue but showed no difference in EOG amplitude or duration compared to controls. These data indicate that a sublethal WAF exposure directly modifies detection and avoidance of CAC beyond the exposure period and may suggest reduced predator avoidance behavior in oil-exposed fish in the wild.

Lymphocytic choriomeningitis virus Clone 13 infection causes either persistence or acute death dependent on IFN-1, cytotoxic T lymphocytes (CTLs), and host genetics.

Understanding of T cell exhaustion and successful therapy to restore T cell function was first described using Clone (Cl) 13 variant selected from the lymphocytic choriomeningitis virus (LCMV) Armstrong (ARM) 53b parental strain. T cell exhaustion plays a pivotal role in both persistent infections and cancers of mice and humans. C57BL/6, BALB, SWR/J, A/J, 129, C3H, and all but one collaborative cross (CC) mouse strain following Cl 13 infection have immunosuppressed T cell responses, high PD-1, and viral titers leading to persistent infection and normal life spans. In contrast, the profile of FVB/N, NZB, PL/J, SL/J, and CC NZO mice challenged with Cl 13 is a robust T cell response, high titers of virus, PD-1, and Lag3 markers on T cells. These mice all die 7 to 9 d after Cl 13 infection. Death is due to enhanced pulmonary endothelial vascular permeability, pulmonary edema, collapse of alveolar air spaces, and respiratory failure. Pathogenesis involves abundant levels of Cl 13 receptor alpha-dystroglycan on endothelial cells, with high viral replication in such cells leading to immunopathologic injury. Death is aborted by blockade of interferon-1 (IFN-1) signaling or deletion of CD8 T cells.

An interplay between plasticity and parental phenotype determines impacts of ocean acidification on a reef fish.

The impacts of ocean acidification will depend on the ability of marine organisms to tolerate, acclimate and eventually adapt to changes in ocean chemistry. Here, we use a unique transgenerational experiment to determine the molecular response of a coral reef fish to short-term, developmental and transgenerational exposure to elevated CO2, and to test how these responses are influenced by variations in tolerance to elevated CO2 exhibited by the parents. Within-generation responses in gene expression to end-of-century predicted CO2 levels indicate that a self-amplifying cycle in GABAergic neurotransmission is triggered, explaining previously reported neurological and behavioural impairments. Furthermore, epigenetic regulator genes exhibited a within-generation specific response, but with some divergence due to parental phenotype. Importantly, we find that altered gene expression for the majority of within-generation responses returns to baseline levels following parental exposure to elevated CO2 conditions. Our results show that both parental variation in tolerance and cross-generation exposure to elevated CO2 are crucial factors in determining the response of reef fish to changing ocean chemistry.

Heritability of behavioural tolerance to high CO2 in a coral reef fish is masked by nonadaptive phenotypic plasticity.

Previous studies have demonstrated limited potential for acclimation of adversely affected olfactory behaviours in reef fishes under elevated CO 2, indicating that genetic adaptation will be required to maintain behavioural performance in the future. Adaptation depends on the presence of heritable phenotypic variation in the trait, which may differ between populations and environments. We used parent-offspring regressions to estimate the heritability (h2) of variation in behavioural tolerance to high CO 2 (754 µatm) in both field-collected and laboratory-reared families of Acanthochromis polyacanthus. Tolerance to elevated CO 2 was measured by determining the behavioural response of individuals to chemical alarm cues. Both populations exhibited high heritability of olfactory behaviour phenotype (father-mid-offspring h2 = 0.56 & 0.65, respectively) when offspring were acutely exposed to high CO 2 for 4 days. However, there was no heritability in the behavioural phenotype when juveniles were chronically exposed to high CO 2 for 6 weeks in the laboratory-reared families. Parental exposure to high CO 2 during the breeding season did not alter this relationship between heritability and length of juvenile exposure to high CO 2. These results demonstrate that variation in behavioural tolerance to high CO 2 is heritable, but adaptive potential may be constrained by a loss of phenotypic variation when juveniles permanently experience a high-CO 2 environment, as will occur with rising CO 2 levels in the ocean.

Effects of elevated CO2 on predator avoidance behaviour by reef fishes is not altered by experimental test water.

Pioneering studies into the effects of elevated CO2 on the behaviour of reef fishes often tested high-CO2 reared fish using control water in the test arena. While subsequent studies using rearing treatment water (control or high CO2) in the test arena have confirmed the effects of high CO2 on a range of reef fish behaviours, a further investigation into the use of different test water in the experimental arena is warranted. Here, we used a fully factorial design to test the effect of rearing treatment water (control or high CO2) and experimental test water (control or high CO2) on antipredator responses of larval reef fishes. We tested antipredator behaviour in larval clownfish Amphiprion percula and ambon damselfish Pomacentrus amboinensis, two species that have been used in previous high CO2 experiments. Specifically, we tested if: (1) using control or high CO2 water in a two channel flume influenced the response of larval clownfish to predator odour; and (2) using control or high CO2 water in the test arena influenced the escape response of larval damselfish to a startle stimulus. Finally, (3) because the effects of high CO2 on fish behaviour appear to be caused by altered function of the GABA-A neurotransmitter we tested if antipredator behaviours were restored in clownfish treated with a GABA antagonist (gabazine) in high CO2 water. Larval clownfish reared from hatching in control water (496 µatm) strongly avoided predator cue whereas larval clownfish reared from hatching in high CO2 (1,022 µatm) were attracted to the predator cue, as has been reported in previous studies. There was no effect on fish responses of using either control or high CO2 water in the flume. Larval damselfish reared for four days in high CO2 (1,051 µatm) exhibited a slower response to a startle stimulus and slower escape speed compared with fish reared in control conditions (464 µatm). There was no effect of test water on escape responses. Treatment of high-CO2 reared clownfish with 4 mg l-1 gabazine in high CO2 seawater restored the normal response to predator odour, as has been previously reported with fish tested in control water. Our results show that using control water in the experimental trials did not influence the results of previous studies on antipredator behaviour of reef fishes and also supports the results of novel experiments conducted in natural reef habitat at ambient CO2 levels.

Type I interferon is a therapeutic target for virus-induced lethal vascular damage.

The outcome of a viral infection reflects the balance between virus virulence and host susceptibility. The clone 13 (Cl13) variant of lymphocytic choriomeningitis virus--a prototype of Old World arenaviruses closely related to Lassa fever virus--elicits in C57BL/6 and BALB/c mice abundant negative immunoregulatory molecules, associated with T-cell exhaustion, negligible T-cell-mediated injury, and high virus titers that persist. Conversely, here we report that in NZB mice, despite the efficient induction of immunoregulatory molecules and high viremia, Cl13 generated a robust cytotoxic T-cell response, resulting in thrombocytopenia, pulmonary endothelial cell loss, vascular leakage, and death within 6-8 d. These pathogenic events required type I IFN (IFN-I) signaling on nonhematopoietic cells and were completely abrogated by IFN-I receptor blockade. Thus, IFN-I may play a prominent role in hemorrhagic fevers and other acute virus infections associated with severe vascular pathology, and targeting IFN-I or downstream effector molecules may be an effective therapeutic approach.

Pathogenesis of Lassa fever virus infection: I. Susceptibility of mice to recombinant Lassa Gp/LCMV chimeric virus.

Lassa virus (LASV) is a BSL-4 restricted agent. To allow study of infection by LASV under BSL-2 conditions, we generated a recombinant virus in which the LASV glycoprotein (Gp) was placed on the backbone of lymphocytic choriomeningitis virus (LCMV) Cl13 nucleoprotein, Z and polymerase genes (rLCMV Cl13/LASV Gp). The recombinant virus displayed high tropism for dendritic cells following in vitro or in vivo infection. Inoculation of immunocompetent adults resulted in an acute infection, generation of virus-specific CD8(+) T cells and clearance of the infection. Inoculation of newborn mice with rLCMV Cl13/LASV Gp resulted in a life-long persistent infection. Interestingly, adoptive transfer of rLCMV Cl13/LASV Gp immune memory cells into such persistently infected mice failed to purge virus but, in contrast, cleared virus from mice persistently infected with wt LCMV Cl13.

Is the TAM receptor Axl a receptor for lymphocytic choriomeningitis virus?

A recent publication indicated that overexpression of Axl, a cellular receptor that negatively regulates Toll-like receptor signaling, enhanced the entry of viruses pseudotyped with the glycoprotein of lymphocytic choriomeningitis virus (LCMV) in vitro. In testing the biological relevance of these observations, we found differences in neither viral kinetics between LCMV infections of Axl(-/-) and wild-type mice nor T-cell responses prior to spontaneous viral clearance. Thus, Axl is not required for productive LCMV infection of mice.

Molecular anatomy and number of antigen specific CD8 T cells required to cause type 1 diabetes.

We quantified CD8 T cells needed to cause type 1 diabetes and studied the anatomy of the CD8 T cell/beta (ß) cell interaction at the immunologic synapse. We used a transgenic model, in situ tetramer staining to distinguish antigen specific CD8 T cells from total T cells infiltrating islets and a variety of viral mutants selected for functional deletion(s) of various CD8 T cell epitopes. Twenty percent of CD8 T cells in the spleen were specific for all immunodominant and subdominant viral glycoprotein (GP) epitopes. CTLs to the immunodominant LCMV GP33-41 epitope accounted for 63% of the total (12.5% of tetramers). In situ hybridization analysis demonstrated only 1 to 2% of total infiltrating CD8 T cells were specific for GP33 CD8 T cell epitope, yet diabetes occurred in 94% of mice. The immunologic synapse between GP33 CD8 CTL and ß cell contained LFA-1 and perforin. Silencing both immunodominant epitopes (GP33, GP276-286) in the infecting virus led to a four-fold reduction in viral specific CD8 CTL responses, negligible lymphocyte infiltration into islets and absence of diabetes.

Toll-like receptor 7 is required for effective adaptive immune responses that prevent persistent virus infection.

TLR7 is an innate signaling receptor that recognizes single-stranded viral RNA and is activated by viruses that cause persistent infections. We show that TLR7 signaling dictates either clearance or establishment of life-long chronic infection by lymphocytic choriomeningitis virus (LCMV) Cl 13 but does not affect clearance of the acute LCMV Armstrong 53b strain. TLR7(-/-) mice infected with LCMV Cl 13 remained viremic throughout life from defects in the adaptive antiviral immune response-notably, diminished T cell function, exacerbated T cell exhaustion, decreased plasma cell maturation, and negligible antiviral antibody production. Adoptive transfer of TLR7(+/+) LCMV immune memory cells that enhanced clearance of persistent LCMV Cl 13 infection in TLR7(+/+) mice failed to purge LCMV Cl 13 infection in TLR7(-/-) mice, demonstrating that a TLR7-deficient environment renders antiviral responses ineffective. Therefore, methods that promote TLR7 signaling are promising treatment strategies for chronic viral infections.

CD8 T cell defect of TNF-α and IL-2 in DNAM-1 deficient mice delays clearance in vivo of a persistent virus infection.

DNAM-1 gene-deficient (-/-) mice take significantly longer to clear an acute and persistent LCMV infection in vivo than DNAM-1 +/+ mice. During acute LCMV priming, at the single cell level, DNAM-1 -/- mice made significantly less cytoplasmic CD8 TNF-α and IL-2 but not IFN-γ than their DNAM-1 +/+ counterparts. Restimulated immune memory CD8 T cells from DNAM-1 -/- and DNAM-1 +/+ mice were equivalent in cytolytic activity against LCMV-infected target cells but DNAM-1 -/- CD8 T cells had significant reductions in TNF-α and IL-2 that were associated on adoptive transfer with the inability to terminate the persistent viral infection.

Point mutation in the glycoprotein of lymphocytic choriomeningitis virus is necessary for receptor binding, dendritic cell infection, and long-term persistence.

Arenaviruses are a major cause of hemorrhagic fevers endemic to Sub-Saharan Africa and South America, and thus a major public health and medical concern. The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is widely used as a model system for studying persistent and acute infections, as well as for gaining an understanding of mammalian immune function. When originally characterized three decades ago, the LCMV isolate, Armstrong, which causes an acute infection in adult mice, was found to differ from the LCMV Clone 13 strain that causes a persistent infection by two amino acid changes, one within the virus surface glycoprotein (GP1: F260L) and the other within the virus L polymerase (K1076Q). Mutation F260L was considered solely responsible for the exceptionally strong binding affinity of Clone 13 (L at GP1 260) to its cellular receptor, α-dystroglycan, which among cells of the immune system is preferentially expressed on dendritic cells, and consequently, alters dendritic cell function leading to viral persistence. Recently, we noted a previously overlooked nucleotide difference between these two strains that results in an additional amino acid change in GP1, N176D. To investigate the potential contribution of this newly identified mutation to the Clone 13 phenotype, we used reverse-genetics approaches to generate recombinant LCM viruses with each of these individual mutations. Phenotypic characterization of these rLCMV showed that mutation F260L, but not N176D, in the GP1 of LCMV is essential for mediating the long-term persistence of Clone 13 infections. This work emphasizes the importance of subtle differences in viral strains that determine disease outcomes.

RNA editing enzyme adenosine deaminase is a restriction factor for controlling measles virus replication that also is required for embryogenesis.

Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses.

Treatment with a sphingosine analog does not alter the outcome of a persistent virus infection.

There is no known antiviral drug treatment that routinely terminates persistent virus infections. A recent provocative report indicated that low dosage of the sphingosine analog FTY720 caused lymphopenia in mice persistently infected with lymphocytic choriomeningitis virus (LCMV)-clone 13 (Cl 13) and induced viral clearance within 30 days post-treatment (Premenko-Lanier et al., 2008). However, we find that low dosage of FTY720 fails to purge LCMV-Cl 13 infection and does not induce lymphopenia in LCMV-Cl 13-infected mice. In fact, infection with non-persistent LCMV-Arm53b or with persistent LCMV-Cl 13 induces an equivalent lymphopenia, demonstrating that the quantity of circulating cells has little bearing on viral persistence. In addition, treatment with FTY720 or the sphingosine-1-phosphate receptor 1 (S1P1)-specific agonist, AUY954, does not alleviate T cell exhaustion and exacerbates disruption of the CD8(+) T cells response following LCMV-Cl 13 infection. Therefore, treatment with a sphingosine analog does not ameliorate persistent LCMV-Cl 13 infection.

In vivo conversion of BM plasmacytoid DC into CD11b+ conventional DC during virus infection.

DC are a highly heterogeneous population that plays a critical role in host defense. We previously demonstrated that virus infection induces BM plasmacytoid DC (pDC) differentiation into CD11b(+) conventional DC (cDC) upon in vitro culture with Fms-like tyrosine kinase 3 ligand (Flt3L). Here we use immunoglobulin D-J rearrangements and pDC adoptive transfer to provide definitive proof supporting BM pDC conversion into CD11b(+) cDC during in vivo viral infection. We show that in vivo BM pDC conversion into CD11b(+) cDC relates to enhanced ability to prime virus-specific T cells. Furthermore, we demonstrate that in vivo pDC conversion does not rely on viral infection of BM pDC, but instead is mediated by type I IFN signaling. Finally, by exploiting recently identified pDC-specific Ab, we provide further characterizations of the BM pDC fraction that exhibits this broader developmental plasticity. Collectively, these data indicate that BM pDC actively contribute to the CD11b(+) cDC pool during in vivo viral infection and delineates molecular, functional, and phenotypic features of this novel developmental pathway.