Spencer J. Greenwood

The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): Illuminating the Functional Diversity of Eukaryotic Life in the Oceans through Transcriptome Sequencing.

Current sampling of genomic sequence data from eukaryotes is relatively poor, biased, and inadequate to address important questions about their biology, evolution, and ecology; this Community Page describes a resource of 700 transcriptomes from marine microbial eukaryotes to help understand their role in the worlds oceans.

Anti-lipopolysaccharide factors in the American lobster Homarus americanus: Molecular characterization and transcriptional response to Vibrio fluvialis challenge

Two partial mRNA sequences predicted to encode anti-lipopolysaccharide factors (ALFs) were identified among expressed sequence tags generated from the American lobster Homarus americanus and complete cDNA sequences were obtained from library clones. Comparison of the translated amino acid sequences to those publicly available confirmed similarity to arthropod anti-lipopolysaccharide factors. Both protein sequences, designated ALFHa-1 and ALFHa-2, contained an N-terminal signal peptide and two half-cysteines participating in a disulfide bridge, features conserved in other ALFs. Predicted secondary structures were similar to that described for the ALF from the horseshoe crab Limulus polyphemus. As part of an exploratory study of immunity in H. americanus, lobsters were injected with the bacterium Vibrio fluvialis and gill, hematopoietic, and hepatopancreas tissues were sampled for analysis of gene expression of ALFHa-1 and ALFHa-2 by quantitative PCR. The relative abundance of ALFHa-2 mRNA was not significantly affected by Vibrio injection in any of the three tissues tested. In contrast, ALFHa-1 mRNA levels in gills were increased by the treatment some 17-fold. Our results support a molecularly specific regulation of antimicrobial proteins in response to bacterial infection in H. americanus.

Seascape genomics provides evidence for thermal adaptation and current-mediated population structure in American lobster (Homarus americanus).

Investigating how environmental features shape the genetic structure of populations is crucial for understanding how they are potentially adapted to their habitats, as well as for sound management. In this study, we assessed the relative importance of spatial distribution, ocean currents and sea surface temperature (SST) on patterns of putatively neutral and adaptive genetic variation among American lobster from 19 locations using population differentiation (PD) approaches combined with environmental association (EA) analyses. First, PD approaches (using bayescan, arlequin and outflank) found 28 outlier SNPs putatively under divergent selection and 9770 neutral SNPs in common. Redundancy analysis revealed that spatial distribution, ocean current‐mediated larval connectivity and SST explained 31.7% of the neutral genetic differentiation, with ocean currents driving the majority of this relationship (21.0%). After removing the influence of spatial distribution, no SST were significant for putatively neutral genetic variation whereas minimum annual SST still had a significant impact and explained 8.1% of the putatively adaptive genetic variation. Second, EA analyses (using Pearson correlation tests, bayescenv and lfmm) jointly identified seven SNPs as candidates for thermal adaptation. Covariation at these SNPs was assessed with a spatial multivariate analysis that highlighted a significant temperature association, after accounting for the influence of spatial distribution. Among the 505 candidate SNPs detected by at least one of the three approaches, we discovered three polymorphisms located in genes previously shown to play a role in thermal adaptation. Our results have implications for the management of the American lobster and provide a foundation on which to predict how this species will cope with climate change.

Differential gene expression of γ-actin, Toll-like receptor 2 (TLR-2) and interleukin-1 receptor-associated kinase 4 (IRAK-4) in Mya arenaria haemocytes induced by in vivo infections with two Vibrio splendidus strains.

Immune function gene expression in Mya arenaria haemocytes was evaluated following in vivo infection with Vibrio splendidus LGP32-GFP and 7SHRW. Elongation factor 1alpha (EF-1alpha) with 2 (EF-2), after challenge with LGP32-GFP, and EF-1alpha with the ribosomal protein S-18, after challenge with 7SHRW, were found to be the most stable housekeeping genes. Using these internal controls and comparing the regulation induced by both strains, up-regulation of gamma-actin, down-regulation of TLR-2 and up-regulation of IRAK-4 was significantly higher after challenge with LGP32-GFP (p<0.001, p=0.001 and p<0.05, respectively). These results suggest specific responses at a molecular level modulated by the bacterial strains. LGP32-GFP induced marked responses which coincide with a similar trend previously found on phenotypic responses under our experimental model.

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada.

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II. All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.

Giardia and Cryptosporidium on Dairy Farms and the Role these Farms May Play in Contaminating Water Sources in Prince Edward Island, Canada

BACKGROUND Cattle represent a reservoir for Giardia and Cryptosporidium and may contaminate water sources. OBJECTIVES To determine the distribution of Cryptosporidium and Giardia on dairy farms and in water bodies near the farms. FARMS AND WATER SOURCES: Twenty dairy farms and 20 wells and 13 surface water samples associated with dairy farms. METHODS Proportions of samples positive for Cryptosporidium or Giardia were determined by a direct immunofluorescence assay. Fecal and water samples were taken at different times. RESULTS Thirty-two (95% CI: 29-35%) and 14% (95% CI: 12-17%) of fecal samples, and 100 (95% CI: 96-100) and 55% (95% CI: 32-77%) of herds, were positive for Giardia and Cryptosporidium, respectively. Giardia duodenalis assemblage E was detected in high proportions (90%) of fecal samples. Cryptosporidium bovis predominated (51%) in all cattle. C. andersoni predominated in adult cattle (53%), whereas the predominant species in animals < 2 months and 2-6 months was C. bovis, respectively. Only calves < 2 months of age were positive for C. parvum. In 46% (95% CI: 19-75%) and 85% (95% CI: 55-98%) of surface water, concentrations of Giardia cysts and Cryptosporidium oocysts were higher in downstream, than in upstream, locations of farms, whereas only 1 groundwater sample was positive for Cryptosporidium. CONCLUSIONS This sample of dairy cattle was predominantly infected with nonzoonotic species and genotypes of Cryptosporidium, Giardia, or both. More studies are needed to determine if the presence of Giardia or Cryptosporidium in surface water was associated with shedding in animals from nearby farms.

Zoonotic potential of Giardia duodenalis and Cryptosporidium spp. and prevalence of intestinal parasites in young dogs from different populations on Prince Edward Island, Canada

The prevalence of Giardia duodenalis, Cryptosporidium spp. and other intestinal parasites was determined in dogs <1 year old from Prince Edward Island, Canada. Fecal samples were collected from the local animal shelter (n=62), private veterinary clinics (n=78) and a pet store (n=69). Intestinal parasites isolated included G. duodenalis, Cryptosporidium spp., Toxocara canis, Isospora spp. and Uncinaria stenocephala. To estimate the zoonotic risk associated with these infections, genotypes of G. duodenalis and Cryptosporidium spp. were determined using 16S rRNA and Hsp70 gene sequencing, respectively. Dogs from the pet store had the highest prevalence of intestinal parasites (78%, 95% CI: 68-88%), followed by the private veterinary clinics (49%, 95% CI: 37-60%), and the local animal shelter (34%, 95% CI: 22-46%). The majority G. duodenalis belonged to host-adapted assemblages D (47%, 95% CI: 31-64%) and C (26%, 95% CI: 13-43%), respectively. Zoonotic assemblages A and B were isolated alone or in mixed infections from 16% (95% CI: 6-31%) of G. duodenalis-positive dogs. All Cryptosporidium spp. were the host-adapted C. canis. While host-adapted, non-zoonotic G. duodenalis genotypes were more common, the presence of G. duodenalis assemblages A and B, T. canis, and U. stenocephala suggests that these dogs may present a zoonotic risk. The zoonotic risk from Cryptosporidium-infected dogs was minimal.

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: changes in cell structure, numbers and adherence.

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p<0.01) than with 7SHRW. The cell adherence was markedly diminished (p<0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p<0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.

Foodborne illness associated with Cryptosporidium and Giardia from livestock.

Waterborne outbreaks caused by Cryptosporidium and Giardia are well documented, while the public health implications for foodborne illness from these parasites have not been adequately considered. Cryptosporidium and Giardia are common in domestic livestock, where young animals can have a high prevalence of infection, shedding large numbers of oocysts and cysts. Molecular epidemiological studies have advanced our knowledge on the distribution of Cryptosporidium and Giardia species and genotypes in specific livestock. This has enabled better source tracking of contaminated foods. Livestock generate large volumes of fecal waste, which can contaminate the environment with (oo)cysts. Evidence suggests that livestock, particularly cattle, play a significant role in food contamination, leading to outbreaks of cryptosporidiosis. However, foodborne giardiasis seems to originate primarily from anthroponotic sources. Foodborne cryptosporidiosis and giardiasis are underreported because of the limited knowledge of the zoonotic potential and public health implications. Methods more sensitive and cheaper are needed to detect the often-low numbers of (oo)cysts in contaminated food and water. As the environmental burden of Cryptosporidium oocysts and Giardia cysts from livestock waste increases with the projected increase in animal agriculture, public health is further compromised. Contamination of food by livestock feces containing Cryptosporidium oocysts and Giardia cysts could occur via routes that span the entire food production continuum. Intervention strategies aimed at preventing food contamination with Cryptosporidium and Giardia will require an integrated approach based on knowledge of the potential points of entry for these parasites into the food chain. This review examines the potential for foodborne illness from Cryptosporidium and Giardia from livestock sources and discusses possible mechanisms for prevention and control.

Identification and cardiotropic actions of brain/gut-derived tachykinin-related peptides (TRPs) from the American lobster Homarus americanus

Two tachykinin-related peptides (TRPs) are known in decapods, APSGFLGMRamide and TPSGFLGMRamide. The former peptide appears to be ubiquitously conserved in members of this taxon, while the latter has been suggested to be a genus (Cancer)- or infraorder (Brachyura)-specific isoform. Here, we characterized a cDNA from the American lobster Homarus americanus (infraorder Astacidea) that encodes both TRPs: six copies of APSGFLGMRamide and one of TPSGFLGMRamide. Mass spectral analyses of the H. americanus supraoesophageal ganglion (brain) and commissural ganglia confirmed the presence of both peptides in these neural tissues; both isoforms were also detected in the midgut. Physiological experiments showed that both APSGFLGMRamide and TPSGFLGMRamide are cardioactive in H. americanus, eliciting identical increases in both heart contraction frequency and amplitude. Collectively, our data represent the first genetic confirmation of TRPs in H. americanus and of TPSGFLGMRamide in any species, demonstrate that TPSGFLGMRamide is not restricted to brachyurans, and show that both this peptide and APSGFLGMRamide are brain-gut isoforms, the first peptides thus far confirmed to possess this dual tissue distribution in H. americanus. Our data also suggest a possible role for TRPs in modulating the output of the lobster heart.