Comparative tissue expression of American lobster (Homarus americanus) immune genes during bacterial and scuticociliate challenge.

The American lobster (Homarus americanus) fishery is the most economically significant fishery in Canada; although comparatively little is known about the lobsters' response to pathogenic challenge. This is the first study to investigate the expression of immune genes in tissues outside of the lobster hepatopancreas in response to challenges by the Gram-positive bacteria, Aerococcus viridans var. homari or the scuticociliate parasite, Anophryoides haemophila. The hepatopancreas has been regarded as the major humoral immune organ in crustaceans, but the contribution of other organs and tissues to the molecular immune response has largely been overlooked. This study used RT-qPCR to monitor the gene expression of several immune genes including three anti-lipopolysaccharide isoforms (ALF) Homame ALF-B1, Homame ALF-C1 and ALFHa-1, acute phase serum amyloid protein A (SAA), as well as thioredoxin and hexokinase, in antennal gland and gill tissues. Our findings indicate that the gene expression of the SAA and all ALF isoforms in the antennal gland and gill tissues increased in response to pathogenic challenge. However, there was differential expression of individual ALF isoforms that were dependent on both the tissue, and the pathogen used in the challenge. The gene expression changes of several immune genes were found to be higher in the antennal gland than have been previously reported for the hepatopancreas. This study demonstrates that increased immune gene expression from the gill and antennal gland over the course of pathogen induced disease contributes to the immune response of H. americanus.

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.

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.

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.