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Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

ABSTRACT: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt.Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes.Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry.This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

SUBMITTER: Gendron RL

PROVIDER: S-EPMC3198499 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

Gendron Robert L RL Armstrong Elizabeth E Paradis Hélène H Haines Lacey L Desjardins Mariève M Short Connie E CE Clow Kathy A KA Driedzic William R WR

Molecular vision 20111005

<h4>Purpose</h4>The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental ...[more]

PMID: 22025894

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Project description:BackgroundAnadromous rainbow smelt (Osmerus mordax) have experienced a large range reduction in recent decades and the status of remnant spawning populations is poorly known in Maine, where these fish have significant ecological, cultural, and commercial relevance. Defining the remnant range of anadromous smelt is more difficult than for many declining fish species because adults are only ephemerally present while spawning in small coastal streams at night during spring runoff periods when traditional assessments can be unreliable or even hazardous. We hypothesized that eDNA might facilitate improved survey efforts to define smelt spawning habitat, but that detection could also face challenges from adult eDNA quickly flushing out of these small stream systems. We combined daytime eDNA sampling with nighttime fyke netting to ascertain a potential window of eDNA detection before conducting eDNA surveys in four streams of varying abundance. Hierarchical occupancy modeling was in turn employed to estimate eDNA encounter probabilities relative to numbers of sampling events (date), samples within events, and qPCR replicates within samples.ResultsResults from the combined eDNA and fyke net study indicated eDNA was detectable over an extended period, culminating approximately 8-13 days following peak spawning, suggesting developing smelt larvae might be the primary source of eDNA. Subsequently, smelt eDNA was readily detected in eDNA surveys of four streams, particularly following remediation of PCR inhibitors. Hierarchical occupancy modeling confirmed our surveys had high empirical detection for most sites, and that future surveys employing at least three sampling events, three samples per event, and six qPCR replicates can afford greater than 90% combined detection capability in low abundance systems.ConclusionsThese results demonstrate that relatively modest eDNA sampling effort has high capacity to detect this ephemerally present species of concern at low to moderate abundances. As such, smelt eDNA detection could improve range mapping by providing longer survey windows, safer sampling conditions, and lower field effort in low density systems, than afforded by existing visual and netting approaches.

Project description:The rainbow smelt (Osmerus mordax, Mitchill, 1814) is an anadromous teleost that overwinters in the estuaries and inshore waters along the North American Atlantic coastline. In the winter months, smelt avoid freezing in subzero temperatures by the production of antifreeze proteins and high levels of glycerol. Glycerol production (glyceroneogenesis) occurs in the liver via a branch point in glycolysis and gluconeogenesis and is directly activated by low temperature. In these studies, hepatocytes were isolated from the liver of individual warm fish and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature over a 72 h time course. Functional genomic techniques were used to identify and validate hepatic transcripts that were differentially expressed between the warm and cold cells. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries enriched for cold-responsive liver transcripts were constructed at the 72 h incubation time. Microarray analyses using the consortium for Genomic Research on All Salmonids Project (cGRASP) 16K (salmonid) cDNA array were performed at the 24, 48 and 72 h incubation times. For quantitative reverse transcription – polymerase chain reaction (QPCR) studies, we focused specifically on the non-colligative [type II antifreeze protein (AFPII)] and colligative (glycerol accumulation) freeze prevention strategies. AFPII (SSH identified) and 21 transcripts (SSH and/or microarray identified or selected by the authors based on a conceptual link to glycerol production) involved in the metabolism of glycolytic (glycogen, glucose) and gluconeogenic (amino acids) sources of glycerol and of lipids with a glycerol backbone (triglyceride, phosphoplipid) were analyzed using QPCR. Rainbow smelt were collected by seine netting from Mount Arlington Heights, Placentia Bay, Newfoundland in late October 2007 and transported to the Ocean Sciences Centre, Memorial University of Newfoundland. The fish were held in a 3000 L indoor free-flowing seawater tank maintained at 8°C to 10°C and followed a natural photoperiod with fluorescent lights set by an outdoor photocell. Hepatocyte isolations were performed for individual male fish (i.e. no pooling of cells from different fish) from Nov. 27th to Dec.17th, 2007. Hepatocytes were not pooled as individual smelt produce different amounts of glycerol in response to cold temperature challenge. Each liver was perfused with medium containing collagenase and yielded approximately 300 million cells per individual. Initial or “pre-incubation” samples were collected and the remaining cell suspension was divided into 20 ml glass scintillation vials (6 x106 cells per vial) containing 2 ml of incubation medium and incubated at either a warm (8ºC; non-glycerol accumulating) or cold (0.4ºC; glycerol accumulating) temperature. Duplicate vials from each temperature were sampled at 24, 48 and 72 h incubation times for RNA isolation. RNA isolated from the hepatocytes of the 9 fish with the highest levels of glycerol production at 0.4°C were used to generate 2 mRNA pools (a “cold” pool and a “warm” pool) for each incubation time (24, 48 and 72 h). To summarize, the “cold” pools contained cells from 9 individual fish incubated at 0.4°C for either 24, 48 or 72 h and which exhibited the highest increase in glycerol levels at 72 h relative to pre-incubation levels, and the “warm” pools contained cells from these same 9 individual fish but incubated at 8°C for either 24, 48 or 72 h and therefore exhibited no increase in glycerol levels at 72 h relative to pre-incubation levels. Comparisons were made for the cold compared to warm pools at the 24, 48 and 72 h time points. For each time point, technical quadruplicate slides including two dye swaps were run per comparison. Incubation time: 24 h: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Incubation time: 48 h: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Incubation time: 72 h: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4 Technical replicate: Cold_24h_1, Cold_24h_2, Cold_24h_3, Cold_24h_4 Technical replicate: Cold_48h_1, Cold_48h_2, Cold_48h_3, Cold_48h_4 Technical replicate: Cold_72h_1, Cold_72h_2, Cold_72h_3, Cold_72h_4

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Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

Publications

Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

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