No abstract

Quantitative PCR (QPCR) methods targeting the 18S rDNA gene (DNA QPCR) and cathepsin L mRNA (RNA QPCR) from Kudoa thyrsites (Gilchrist) were developed and compared with histology for determination of K. thyrsites infection levels in Atlantic salmon Salmo salar L. Both QPCR tests were specific, reproducible and sensitive down to 3 copies. DNA QPCR was able to detect lower K. thyrsites infection levels than those detected by RNA QPCR and histology. The higher sensitivity of the DNA-based test compared with the RNA-based test appeared to be biological in nature and suggested that when infection levels were low, there were fewer copies of cathepsin L mRNA than 18S rDNA genes. However, all 3 diagnostic methods were highly correlated. Regression analyses comparing DNA QPCR and histology data from 2 distinct groups of fish showed that the relationship between these 2 diagnostic methods was reproducible. A logistic regression analysis comparing diagnostic data with a visual assessment of post-mortem flesh quality indicated that histology was the single best predictor of flesh quality, followed by DNA QPCR and then RNA QPCR. KEY WORDS: Kudoa thyrsites · Atlantic salmon · Diagnostics · QPCR · RNA test · DNA test · Histology · Myoliquefaction Resale or republication not permitted without written consent of the publisherDis Aquat Org 75: [239][240][241][242][243][244][245][246][247][248][249] 2007 stage, it is difficult to discern biological and environmental factors affecting parasite abundance in the environment and, ultimately, the risk to farmed salmon. Efforts have been made to understand factors affecting the susceptibility of Atlantic salmon to K. thyrsites infections (Funk et al. 2004), but in the absence of a challenge model this type of work is also difficult. At present, management of the infection relies on site selection, optimising smolt quality and monitoring stock for the presence of myxospores. Therefore, development of an efficacious vaccine is arguably the most promising method with which to minimize the impacts of K. thyrsites on fillet quality. To facilitate vaccine development, improved diagnostics are required for measuring K. thyrsites infection levels.Kudoa thyrsites infection levels are routinely determined through examination of stained histological sections, and this measure has been shown to be a relatively good predictor for post-mortem flesh quality (Dawson-Coates et al. 2003). However, histology lacks the sensitivity required to quantify low-level infections and requires a minimum seawater exposure period of 900 degree days before plasmodia can be detected reliably (Moran et al. 1999b). Therefore, histological methods as a measure of vaccine efficacy preclude evaluation of low infection levels and prevent rapid evaluation of newly developed vaccine candidates.Standard PCR is routinely used to detect the Kudoa thyrsites small subunit ribosomal DNA (18S rDNA) (Hervio et al. 1997) and has detected K. thyrsites in smolts after sea water exposures of only 350 degree days (V. Funk unpu...

Clam gardens are habitat modifications established by coastal Indigenous Peoples of northwest North America to enhance intertidal clam habitat productivity to provide secure and reliable local food resources. These gardens were established long ago and are mostly unmaintained in present times. To determine whether unmaintained clam gardens still provide a more productive and beneficial habitat than unmodified clam beaches, Pacific littleneck clams (Leukoma staminea) were transplanted to unmaintained clam gardens or unmodified reference beaches and then evaluated for growth, survival, and transcriptomic signatures of the gill and digestive gland after 16 weeks. All beaches in the study were characterized for sediment grain-size distribution, carbonate concentration, and organic content to identify sediment characteristics that may differ between clam garden and reference sites, as well as potentially contribute to differences in clam productivity. Clam growth and survival, sediment grain-size distribution, and carbonate and organic content were not significantly affected by unmaintained clam gardens. Wide variations in survival, growth, and sediment characteristics were observed among beaches. Examining across beach location resulted in the identification of significant negative effects of small rocks (2.00 - 4.75 mm), and silt (< 63 μm) on both growth and survival. Sand (250 - 500 μm) had a significant positive influence on both growth and survival while fine sand (125 - 250 μm) had a significant positive effect only on growth. Coarse sand (0.50 - 1.00 mm) had a significant positive effect on survival, while very fine sand (63 - 125 μm), carbonate, and organic content all had significant negative effects. To evaluate transcriptomic effects, a de novo transcriptome for L. staminea was assembled. The final assembly contained 52,000 putative transcripts and those specific to each of the two tissues were identified. This revealed that similar functional categories were enriched in tissue-specific genes, but each tissue had its own transcripts comprising the categories. Transcriptomic analysis revealed differential expression in individuals from clam gardens, and although this effect was small in terms of the numbers of genes, specific response genes were identified consistently in both tissues. In summary, while the unmaintained clam gardens did not impact clam growth and survival over the 16 weeks of the study, it did have an effect at the level of the transcriptome. Furthermore, correlations of transcripts associated with either high or low survival provide new insights into ecological associations of these genes in this non-model organism. In summary, localized environmental factors are likely to have a greater influence on Pacific littleneck clam physiology, growth, and survival than the presence/absence of unmaintained clam gardens.