Characterization of gene expression endpoints during postembryonic development of the northern green frog (Rana clamitans melanota).

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.

Evidence of disruption in estrogen-associated signaling in the liver transcriptome of in-migrating sockeye salmon of British Columbia, Canada.

The health of sockeye (Oncorhynchus nerka) salmon stocks is of increasing concern; reflecting both a sentinel of human-impacted aquatic environments and as a key fishery for British Columbia, Canada. The spawning migration of Pacific sockeye salmon represents a critical life stage where significant demands are made on animal biology and important BC fisheries are linked to this migration in the Skeena and Fraser River watersheds. These watersheds present very different environments; the former being sparsely populated with little industrial impact, while the latter flows through highly-populated areas. The present study used quantitative real-time PCR analysis of adult sockeye salmon from four 2008 stocks [Fulton River and Pinkut Creek (Skeena) and Weaver Creek and Harrison River (Fraser)] to evaluate ten hepatic gene transcripts associated with reproduction, stress, energy metabolism, and exposure to environmental contaminants. Dynamic changes in mRNA abundance were observed in Fulton River stock animals from the Skeena River mouth to the spawning ground which reflect the physiological demands of in-river migration and reproductive maturation. Inter-stock comparisons of migrants at spawning grounds demonstrated a marked difference in the sex-specific gene hepatic gene expression profiles. Our original hypothesis was that a greater diversity in mRNA profiles is associated with watersheds with higher human impact. However, our observations contradict this posit. Skeena males and females displayed poor definition in their molecular profiles between sexes while the Fraser River fish had very distinctive sex differences that were consistent with the previous year's migration. The genetic sex distribution and ratio of milt versus roe production did not differ between the Skeena and Fraser River spawning site fish. However, a significant percentage of Skeena animals displayed marked discordance of these characteristics with gender-specific hepatic mRNA profiles implying that an alteration in estrogen-mediated signaling has occurred. Continued geospatial and longitudinal assessments will help determine to what extent the dynamic molecular biology of late life-stage sockeye salmon reflects natural variation or modulation by anthropogenic causative agents.

Triclosan exposure alters postembryonic development in a Pacific tree frog (Pseudacris regilla) Amphibian Metamorphosis Assay (TREEMA).

The Amphibian Metamorphosis Assay (AMA), developed for Xenopus laevis, is designed to identify chemicals that disrupt thyroid hormone (TH)-mediated biological processes. We adapted the AMA for use on an ecologically-relevant North American species, the Pacific tree frog (Pseudacris regilla), and applied molecular endpoints to evaluate the effects of the antibacterial agent, triclosan (TCS). Premetamorphic (Gosner stage 26-28) tadpoles were immersed for 21 days in solvent control, 1.5 µg/L thyroxine (T(4)), 0.3, 3 and 30 µg/L (nominal) TCS, or combined T(4)/TCS treatments. Exposure effects were scored by morphometric (developmental stage, wet weight, and body, snout-vent and hindlimb lengths) and molecular (mRNA abundance using quantitative real time polymerase chain reaction) criteria. T(4) treatment alone accelerated development concomitant with altered levels of TH receptors α and ß, proliferating cell nuclear antigen, and gelatinase B mRNAs in the brain and tail. We observed TCS-induced perturbations in all of the molecular and morphological endpoints indicating that TCS exposure disrupts coordination of postembryonic tadpole development. Clear alterations in molecular endpoints were evident at day 2 whereas the earliest morphological effects appeared at day 4 and were most evident at day 21. Although TCS alone (3 and 30 µg/L) was protective against tadpole mortality, this protection was lost in the presence of T(4). The Pacific tree frog is the most sensitive species examined to date displaying disruption of TH-mediated development by a common antimicrobial agent.

Biological effects of the anti-parasitic chemotherapeutant emamectin benzoate on a non-target crustacean, the spot prawn (Pandalus platyceros Brandt, 1851) under laboratory conditions.

The potential impact of commercial salmon aquaculture along the coast of British Columbia on the health of non-target marine wildlife is of growing concern. In the current initiative, the biological effects on gene expression within spot prawn (Pandalus platyceros) exposed to the sea lice controlling agent, emamectin benzoate (EB; 0.1-4.8 mg/kg sediment), were investigated. A mean sediment/water partitioning coefficient (K(p)) was determined to be 21.81 and significant levels of EB were detected in the tail muscle tissue in all exposed animals. Animals selected for the experiment did not have eggs and were of similar weight. Significant mortality was observed within 8 days of EB treatment at concentrations between 0.1 and 0.8 mg/kg and there was no effect of EB on molting. Twelve spot prawn cDNA sequences were isolated from the tail muscle either by directed cloning or subtractive hybridization of control versus EB exposed tissues. Three of the transcripts most affected by EB exposure matched sequences encoding the 60S ribosomal protein L22, spliceosome RNA helicase WM6/UAP56, and the intracellular signal mediator histidine triad nucleotide binding protein 1 suggesting that translation, transcription regulation, and apoptosis pathways were impacted. The mRNA encoding the molting enzyme, ß-N-acetylglucosaminidase, was not affected by EB treatment. However, the expression of this transcript was extremely variable making it unsuitable for effects assessment. The results suggest that short-term exposure to EB can impact biological processes within this non-target crustacean.

Identification of in vivo P-glycoprotein mRNA decay intermediates in normal liver but not in liver tumors.

Post-transcriptional regulation at the level of mRNA stability is one important mechanism for over-expression of P-glycoprotein (Pgp) genes observed in cultured cells and in animals. A previous study has shown that mRNA half-lives for Pgp genes in normal liver were less than 2 h, in contrast to greater than 12 h measured in a transplantable liver tumor line. This lower turnover rate of Pgp mRNA may, in large part, contribute to the abundance of Pgp mRNA in liver tumors. The current study sought to investigate the underlying mechanism for the lower turnover rate of Pgp2 mRNA previously determined in liver tumors. As a first approach, we set out to understand the Pgp2 mRNA decay in both normal liver and liver tumors by first identifying and characterizing Pgp2 mRNA degradation intermediates. In this study, we showed that the sensitive ligation-mediated polymerase chain reaction (LM-PCR) method can be used to detect a homogenous pool of in vitro transcribed RNA down to 0.4 ng. By employing gene-specific primers in the LM-PCR method, we successfully identified four Pgp2 mRNA decay intermediates in normal liver. All four decay intermediates detected correspond to the 5' coding region of Pgp2 mRNA, and surprisingly no decay intermediates which correspond to 3' untranslated region, 3' coding region or middle coding region were found using LM-PCR. The identified decay intermediates are unique to the normal liver as they were absent or present at very low level in all three liver tumor samples analyzed. This observation supports our previous findings that the Pgp mRNA turnover rate is lower in liver tumors than in normal liver. These findings have implications for our understanding of the regulation of Pgp mRNA turnover in normal and malignant tissues.

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method.

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.