title for learn-about section of A SNAIL'S ODYSSEY
  Life in the intertidal zone

Heat-shock proteins (HSPs) are well known in intertidal invertebrates. These special entities are produced in response to high temperature and other stresses such as osmotic, low oxygen (hypoxia), and certain pollutants. Their job is to bind to damage proteins, thus preventing their aggregation into toxic clusters and/or being degraded by cellular enzymes (proteases), and to help refold them 3-dimensionally into their previous functional shapes.  They are more abundant in animals subjected to high levels of protein damage and their presence is generally indicative of greater tolerance to heat, or other, stresses.

NOTE  heat-shock proteins represent a class of molecules known as “chaperones”, because they care for or stabilise thermally denatured proteins.  HSPs are considered in other locations in the ODYSSEY, including LEARN ABOUT ABALONES: HEAT-SHOCK PROTEINS and LEARN ABOUT LIMPETS: LIFE IN THE INTERTIDAL ZONE: TEMPERATURE STRESS

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  Heat-shock proteins

Topics dealing with life in the intertidal zone include heat-shock proteins considered here, and TEMPERATURE EFFECTS, GAS EXCHANGE, WAVE EFFECTS, SALINITY EFFECTS, TRANSLOCATION STUDIES, and WATER-CHEMISTRY EFFECTS, considered in other sections.

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Research study 1

graph showing expression of heat-shock protein in mussels Mytilus spp. at different incubation temperaturesphotograph of mussels Mytilus trossulus and M. galloprovincialis courtesy Linda Schroeder, Northwest Shell ClubBased on latitudinal temperature differences on west-coast shores we would predict that Hsp expression would be higher in a subtropical mussel species than in a temperate/boreal mussel species. Thus, southern species would be more physiologically able to deal with thermal damage to its proteins.  Indeed, studies on heat-stress responses in Mytilus galloprovincialis, introduced from the Mediterranean and now distributed from central California to Baja California, and in M. trossulus, an indigenous species distributed from Alaska to central California, reveal greater synthesis of Hsp70 in the former, especially at incubation temperatures of 28-30oC (see graph).  The authors additionally show higher residual levels of ubiquitin conjugates and the expression of an additional stress protein in M. galloprovincialis, but not in M. trossulus, which also suggest “better physiological preparedness” for heat stress in the southern species.  The authors conclude that M. trossulus is a more cold-adapted species than M. galloprovincialis.  Hofmann & Somero 1996 Mar Biol 126: 65; see also Hofmann & Somero 1995 J Exp Biol 198: 1509. Photograph courtesy Linda Schroeder, Pacific Northwest Shell Club, Seattle, Washington PNWSC.

NOTE  these are low-molelcular mass proteins that bind to damaged proteins and target them for destruction by cellular proteases.  Higher levels of ubiquitin conjugates are indicative of heat stress and of the presence of irreversibly damaged proteins. For an account of ubiquitin expression in heat-stressed mussels Mytilus trossulus during emersion see Hoffmann & Somero 1996 Molecular Mar Biol Biotech 5: 175.

Research study 2

graph showing heat-shock protein induction in gills of mussels Mytilus californianus at different seasons and different tidal heights on the coast of central OregonHSPs  are synthesised preferentially when an animal is under stress; hence, may preempt synthesis of other proteins critical for the normal functioning of the organism.  Over longer term, then, the energy costs necessary for their synthesis may reduce the energy available for the animal to grow and reproduce.  In mussels, one would expect to see different induction temperatures for HSPs in at different tidal heights.  This is examined in a study on Mytilus californianus at Strawberry Hill on the central Oregon coast, with results showing that differences relating to intertidal heights are generally not significant (see graphs).  However, the authors do find a clear acclimatisation effect on induction temperatures.  In March, HSPs are 4 times higher at experimental temperatures of 28oC than at 20oC, and induction begins at 23oC.  As the season progresses, however, the magnitude of induction response decreases until, by August, there is no obvious induction profile at all.  The August mussels have become adapted to warm seasonal temperatures. Roberts et al. 1997 Biol Bull 192: 309.

NOTE  HSP induction is measured in the laboratory in excised gills of the test animals

Research study 3

graphs showing temperature variation in a tidepool at Pacific Grove, California measured seasonallygraph showing temperature variations seasonally on a horizontal microsite at Pacific Grove, Californiagraph showing temperature variation seasonally at a northward, vertical-facing site in a mussel bed Mytilus californianus at Pacific Grove, CaliforniaThe thermal landscape in a bed of mussels Mytilus californianus is highly variable and depends, not just upon time of year and intertidal height, but also upon features such as animal size, aspect to sun and wind, slope, and extent of residual water.  Thus, any description of thermal stress for an organism must be carefully and minutely documented.  As an example of how even closely located sites at Pacific Grove, California can vary greatly in their temperature profiles, note the temperature differences recorded at 2 "microsites" in a mussel bed and in a nearby tidepool over a 1-yr period.  The first is a horizontal microsite (see graph upper Left); the second, a vertical, northward-facing microsite located just 20cm away (see graph lower Left). Temperatures are on average 3-4oC higher at the horizontal site as compared with the vertical, north-facing site.  A nearby tidepool has a different temperature profile (see graph upper Right).

histograms showing heat-shock protein induction in mussels Mytilus californianus in different intertidal locations seasonally at Pacific Grove, CaliforniaLevels of inducible heat-shock proteins in the gills of the mussels are significantly higher in individuals from the horizontal site than in ones from the north-facing site (see histogram lower Right).  In comparison, seasonal effects are generally absent in the tidepool mussels. The authors note that temperature may not have acted alone to induce the high levels recorded for May. At this time, desiccation and hypoxia may also have contributed to the stress and to induction of the heat-shock proteins.  Helmuth & Hofmann 2001 Biol Bull 201: 374.

  most of the mussel-bed data in this study are obtained from thermistor-loggers embedded in “model” mussels in order to standardise size and thermal-response characteristi. A "model" mussel is an empty shell + silicone + embedded data-logger thermistor.  Ordinary thermistors implanted into mussels are used for the tidepool record 


Research study 4

The temperature at which the repair proteins appear, that is, when the heat-shock genes express them, is known as the threshold-induction temperature.  Not surprisingly, this threshold varies with the past temperature history of an animal.  For example, studies on mussels Mytilus trossulus iat Friday Harbor Laboratories, Washington show that mussels in February have a much lower threshold than mussels in August (23oC vs. 28oC), indicating an adjustment of gene expression in response to seasonal environmental change.  Buckley et al.  2001 J Exper Biol 204: 3571.

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Although the significance to a mussel in having different threshold-induction temperatures is probably quite obvious, let's review some ideas here. CLICK HERE for explanations.

The mussel saves energy by not producing repair proteins at temperatures too low for damage to have occurred. 

Is the difference of 5oC in seasonal response in induction of heat-shock proteins shown in the foregoing Research Study reflective of a 5oC seasonal difference in ambient seawater temperature? 

Mussels communicate in some way the changing seasonal temperatures to the DNA in the nuclei of their cells. 

Research study 5

histogram showing expression of heat-shock proteins in mussels Mytilus californianus in different seasons and at different intertidal heights in Oregonphotograph of shore zonation on a rocky shore in Oregongraph showing effect of intertidal height on induction temperature for heat-shock proteins in mussels Mytilus californianus in OregonWhat is known about the effect of intertidal height on expression of heat-shock proteins? Wouldn’t mussels living at higher intertidal levels and therefore experiencing greater summer heat stress than low level ones have higher levels of the repair proteins?  Studies in Oregon on Mytilus californianus show that this is indeed the case, with high-level mussels having almost 4 times more heat-shock proteins than low-level ones in May, at the beginning of seasonal temperature stress, and also in August, towards the end of seasonal temperature stress (see histogram upper Right).  Moreover, in August, the low-level mussels produce the repair proteins at much lower temperatures than the high-level ones indicating a physiological habituation to heat stress (see graph lower Right).  Halpin et al. 2004 Mar Ecol Progr Ser 276: 137; see also review in Halpin et al. 2002 Integ Comp Biol 42: 815



Zonation of mussels (blue-
white patches in mid-upper
part of photograph) in Oregon

Research study 6

Distributions of marine invertebrates, such as mussels Mytilus californianus, generally have highest densities in the centres of their ranges and declining densities towards their edges.  The edges are assumed to be more stressful to the organisms, especially the more southernmost one and, should therefore be sensitive indicators to a variety of stressors including, for example, those associated with global warming.  This is examined in a latitudinal survey of M. californianus, where map showing collecting sites for mussels Mytilus californianus used in study of heat-shock proteins relative to latitudinal location on the west coast of North Americapopulation densities and abundances of heat-shock proteins are co-related across the southernmost half of the species’ range.  The map shows collection sites for M. graph showing abundance of mussels Mytilus californianus and relative heat-shock protein levels in their tissues in relation to latitudinal distributioncalifornianus from southern British Columbia to the species’ southernmost limit of distribution in mid-Baja California. The species' northernmost distribution extends into the Aleutian Islands, Alaska. 

The accompanying plot shows average densities of M. californianus and relative Hsp70 concentrations for 17 of the 19 sites.  Note that were the authors’ original hypotheses to be upheld, we would expect to find lowest levels of Hsp70 at the centre of distribution and highest levels at the most stressful southernmost limit of distribution. While the first part is true in that Hsp70 levels are low at 39-42oN latitude at the centre of the distribution, there is another set of low levels around 49-52oN latitude, and highest levels are found in mussels at about 45oN latitude. Overall, the data are highly variable. In view of the lack of significant relationship between levels of Hsp70 and latitudinal position or population density, the authors caution against generalisations about the relationship between environmental stress and range limits of a species without prior knowledge of species-specific differences in both locality and latitudinal effects.  Sagarin & Somero 2006 J Biogeogr 33: 622.

NOTE  the authors include whelks Nucella ostrina in the study but, as results for both species are similar and there may have been some N. emarginata mistakenly included in the collections, only data for mussels are included here.  Hsp70 analyses are done on gills of summer-collected mussels taken from the lower parts of their intertidal range.  Hsp70 is expressed in units relative to standards run at the same time as the unknowns

NOTE  density data are taken from another publication: Sagarin & Gaines 2002 J Biogeog 29: 985.

Research study 7

What costs are imposed by heat stress in a mussel?  For example, what specific metabolic costs are imposed by production of heat-shock proteins (HSPs) and repair of thermally stressed proteins?  These considerations are addressed by researchers at California State University, Long Beach by subjecting mussels Mytilus californianus to chronic heat stress over periods of up to 8wk in both field and laboratory, then assessing effects on growth and survival.  In other experiments some individuals are previously acclimated to high temperatures, while others are fed less.  The researchers’ specific interest is whether chronically but sublethally heat-stressed individuals will exhibit a trade-off between increased thermal tolerance and growth or condition.  Results are as predictd, with chronically heat-stressed individuals growing less in tissue mass than unstressed ones, and individuals provided with less food exhibiting poorer condition indices and survival than ones provided normal rations.  Prior thermal conditioning results in greater survival following acute exposure to more extreme temperatures, indicating the expression of HSPs.  Finally, individuals receiving less food have poorer condition and survival than ones fed more, suggesting that they are less able to mitigate costs of thermal stress through physiological defenses.  The authors discuss their results from the perspective of climate-change effects.  Fitzgerald-Dehoog et al. 2012 Biol Bull 223: 205.

NOTE  no measurable shell growth occurs in any treatment, so soft-tissue growth is used instead

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