K. Fraser Clark

Analysis of gene expression in Homarus americanus larvae exposed to sublethal concentrations of endosulfan during metamorphosis.

Agricultural pesticide runoff has been suspected as the cause of numerous fish kills in rivers throughout Prince Edward Island but the impact on the surrounding marine environment is unknown. Endosulfan, an organochlorine pesticide, is a potent neurotoxin and molt inhibitor used to combat the Colorado potato beetle however it has the potential to affect non-target organisms including the American lobster (Homarus americanus). Metamorphosis is a critical stage of development and the effects of contaminant exposure during this time are largely unknown in lobster. A 14day endosulfan exposure was performed to identify the effects on survival, development and gene expression in lobster larvae during metamorphosis; all of which were predicted to be negatively impacted. The higher endosulfan concentrations resulted in greater mortality and a significant increase in the number of days required to reach metamorphosis in surviving animals. A custom made H. americanus microarray was used for monitoring the changes in expression of 14,592 genes at the termination of the exposure. Genes with >1.5 fold change and identified as being significant at p<0.05 using one-way ANOVA were selected for further analysis. A total of 707 genes were identified as being significantly differentiated, however with only ~40% annotation of the array, the majority of these genes were unknown. Annotated genes of interest were involved in many processes: development, metabolism, immunity and oxidative stress response and gene regulation. Nine genes of interest (histone H1, farnesoic acid O-methyltransferase, cuticle protein, glutathione S-transferase, thioredoxin, NADH dehydrogenase, ecdysone nuclear receptor Fushi tarazu F1 (FTZ-F1), ferritin and ecdysone inducible protein E75 (EIP-E75)) were selected for RT-qPCR validation of the microarray results. The RT-qPCR method was more sensitive than the microarray yet detected similar expression patterns. The two highest endosulfan concentrations resulted in increased mortalities, developmental delays in reaching metamorphosis and significant changes in gene expression. This research provides a foundation for using microarray gene expression profiles as screening tools for exploring the impact of environmental contaminants on lobster.

Differential expression of American lobster (Homarus americanus) immune related genes during infection of Aerococcus viridans var. homari, the causative agent of Gaffkemia.

This is the first transcriptomic study focusing on immunity in the commercially valuable American lobster (Homarus americanus). We have conducted an in vivo infection trial using the Gram-positive bacterium Aerococcus viridans var. homari to determine how H. americanus responds to this naturally occurring lethal-pathogen. A novel H. americanus microarray was used to measure the transcriptomic changes occurring in over 14,000 genes in the lobster hepatopancreas. Hundreds of new immune genes and isoforms were identified and measured for the first time in this species, and our findings highlight 148 genes of interest involved in H. americanus pathogen response. We verified our microarray results using RT-qPCR on three anti-lipopolysaccharide (ALFHa-1, ALFHa-2, ALFHa-4), a thioredoxin, acute phase serum amyloid protein A, hexokinase and two trypsin genes. RT-qPCR and microarray findings show close agreement and highlight the significant increase in gene expression in many lobster immune genes during A. viridans infection. Differential expression of the ALFHa isoforms may indicate that the H. americanus immune response can be tailored to the class of pathogen causing disease.

Molecular immune response of the American lobster (Homarus americanus) to the White Spot Syndrome Virus.

The adult American lobster (Homarus americanus) is susceptible to few naturally occurring pathogens, and no viral pathogen is known to exist. Despite this, relatively little is known about the H. americanus immune system and nothing is known about its potential viral immune response. Hundreds of rural communities in Atlantic Canada rely on the lobster fishery for their economic sustainability and could be devastated by large-scale pathogen-mediated mortality events. The White Spot Syndrome Virus is the most economically devastating viral pathogen to global shrimp aquaculture production and has been proposed to be capable of infecting all decapod crustaceans including the European Lobster. An in vivo WSSV injection challenge was conducted in H. americanus and WSSV was found to be capable of infecting and replicating within lobsters held at 20°C. The in vivo WSSV challenge also generated the first viral disease model of H. americanus and allowed for the high-throughput examination of transcriptomic changes that occur during viral infection. Microarray analysis found 136 differentially expressed genes and the expression of a subset of these genes was verified using RT-qPCR. Anti-lipopolysaccharide isoforms and acute phase serum amyloid protein A expression did not change during WSSV infection, contrary to previous findings during bacterial and parasitic infection of H. americanus. This, along with the differential gene expression of thioredoxin and trypsin isoforms, provides compelling evidence that H. americanus is capable of mounting an immune response specific to infection by different pathogen classes.

A transcriptomic analysis of American lobster (Homarus americanus) immune response during infection with the bumper car parasite Anophryoides haemophila

Anophryoides haemophila is an important protistan parasite of American lobster, Homarus americanus, as it has been found to infect lobsters in the wild as well as causing major losses of lobsters maintained in commercial holding facilities. Expression of over 14,500 H. americanus hepatopancreatic genes were monitored during an A. haemophila infection challenge in order to elucidate molecular mechanisms involved in the lobster immune response. One hundred and forty-five genes were found to be differentially expressed during infection. For many genes, this study is the first to link their expression to an immune response to a known lobster pathogen. Several of the genes have previously been linked to crustacean or invertebrate immune response including: several anti-lipopolysaccharide factor isoforms (ALFHa), acute phase serum amyloid protein A (SAA), a serine protease inhibitor, a toll-like receptor, several haemocyanin subunits, phagocyte signaling-impaired protein, vitelline membrane outer layer protein-1, trypsin, and a C-type lectin receptor. Microarray results were verified using RT-qPCR and agreement was good between the two methods. The expression of six ALFHa isoforms was monitored via microarray where ALFHa-1, ALFHa-2, ALFHa-4 and ALFHa-6 were differentially expressed while ALFHa-3 and ALFHa7 were not. RT-qPCR analysis confirmed that ALFHa-1, ALFHA-2 and ALFHa-4 expression increased during infection with a peak at 5-7weeks for ALFHa-1 and 10weeks for ALFHa-2 and ALFHa-4. This suggests that different ALFHa isoforms are temporally expressed during A. haemophila infection. Importantly, these results provide evidence that different ALFHa isoforms have more significant roles in responding to A. haemophila infection. Significant increases in SAA gene expression were also found, corroborating previous findings of increased SAA expression during Aerococcus viridans infections; highlighting the importance of SAA as a marker of H. americanus immune activation and potential indicator of H. americanus health.

Sex matters in Massive Parallel Sequencing: evidence for biases in genetic parameter estimation and investigation of sex determination systems

Using massively parallel sequencing data from two species with different life history traits, American lobster (Homarus americanus) and Arctic Char (Salvelinus alpinus), we highlight how an unbalanced sex ratio in the samples and a few sex-linked markers may lead to false interpretations of population structure and thus to potentially erroneous management recommendations. Here, multivariate analyses revealed two genetic clusters separating samples by sex instead of by expected spatial variation: inshore and offshore locations in lobster, or east and west locations in Arctic Char. To further investigate this, we created several subsamples artificially varying the sex ratio in the inshore/offshore and east/west groups and then demonstrated that significant genetic differentiation could be observed despite panmixia in lobster, and that FST values were overestimated in Arctic Char. This pattern was due to 12 and 94 sex-linked markers driving differentiation for lobster and Arctic Char, respectively. Removing sex-linked markers led to nonsignificant genetic structure in lobster and a more accurate estimation of FST in Arctic Char. The locations of these markers and putative identities of genes containing or nearby the markers were determined using available transcriptomic and genomic data, and this provided new information related to sex determination in both species. Given that only 9.6% of all marine/diadromous population genomic studies to date have reported sex information, we urge researchers to collect and consider individual sex information. Sex information is therefore relevant for avoiding unexpected biases due to sex-linked markers as well as for improving our knowledge of sex determination systems in nonmodel species.

Analysis of expressed sequence tags (ESTs) and gene expression changes under different growth conditions for the ciliate Anophryoides haemophila, the causative agent of bumper car disease in the American lobster (Homarus americanus)

The scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate.

Global gene expression profiling of Homarus americanus (Crustacea) larval stages during development and metamorphosis

Homarus americanus has a life history that is similar to other arthropods, including a pelagic larval phase and a benthic adult phase. The larval phase is divided into three morphologically distinct stages, followed by metamorphosis to the post-larval phase. H. americanus larval development has been studied previously, although the molecular mechanisms that regulate the consequent changes are not fully elucidated. This study is the first to use an oligonucleotide microarray to investigate global gene expression during H. americanus larval development. Stage-specific gene expression profiles of larvae and postlarvae from two-year classes were assessed. We found the expression levels of 1851 genes to be significantly different among larval stages. Functional annotations indicated that various differentially expressed genes were involved with immune function, energy regulation, and development. Ten target genes of interest were selected for expression verification using RT-qPCR. Two Phosphoenolpyruvatecarboxykinases, Argonaute 2, Ecdysone-inducible protein 75, and Procollagen-lysine 2-oxoglutarate 5-dioxygenase 3, had significantly different expression (p < 0.05) among stages. These genes are involved in translation regulation, gene expression, morphological development and energy metabolism. This study provides a foundation for future investigations regarding signaling, morphological remodeling, energy metabolism, and the immune system as they pertain to larval development.

Aerococcus viridans var. homari: The presence of capsule and the relationship to virulence in American lobster (Homarus americanus).

The relationship between virulence and encapsulation of Aerococcus viridans var. homari was evaluated by growing virulent (Rabins) and avirulent (ATCC 10400) strains under varying culture conditions, and during challenge trials. Changes in capsule thickness were monitored using a modified lysine-ruthenium red (LRR) fixation method and transmission electron microscopy. The virulent Rabins strain possessed a prominent capsule of 0.252 μm±0.061 μm that was diminished by in vitro growth conditions to 0.206 μm±0.076 μm. The ATCC 10400 strain capsule thickness decreased from 0.157 μm±0.043 μm to 0.117 μm±0.043 μm after 10 in vitro passages. The virulent Rabins strain capsule was significantly thicker than the avirulent ATCC 10400 strain under all growth conditions. Rabins strain, regardless of pre-challenge growth conditions or dose (high dose 10(7) or low dose 10(2)), was able to kill lobsters in 7 days at 15°C. ATCC 10400 strain, regardless of pre-challenge growth conditions, killed lobster only at high doses (10(7)) with varying median time to death of ∼15 days, while at low doses (10(2)) all lobsters survived and no bacteria were present after 42 days. This work demonstrates the importance of the thickness of the A. viridans capsule to virulence in the American lobster.