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

Mussels in the intertidal zone experience extremes in temperature, from baking in the sun in summer to freezing in winter.  They are subject to freshwater exposure during rainstorms, to risk of being dislodged by waves or battered by logs during storms, and to periodic interruption of feeding, gas exchange, and excretion through tidal cycling.

This section begins with several papers of a more general nature that use transcriptome-analysis techniques to give information on physiological responses

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Research study 1

Although much as been written on the physiology of mussels, most articles deal with  specific responses measured at specific times, and/or in laboratory conditions.  A more comprehensive approach is taken by researchers at Hopkins Marine Station, California who measure physiological rhythms in sea mussels Mytilus californianus through complete tidal cycles and at different intertidal levels, and the study is worth considering graph showing tidal levels and corresponding transcriptase activity in mussels Mytilus californianusas an introduction to the general topic of life in the intertidal zone. The researchers' approach is to analyse changing levels of gene expression through identification of transcriptome products.  Results show that mussels exist in 4 distinct physiological phases or states corresponding to 1) a respiration/metabolism phase, 2) a cell-division/growth phase, and 3) & 4) two stress-response phases linked to moderate and severe heat-stress events, respectively.  The first physiological state is characterised by genes involved in the tricarboxylic acid cycle (TCA), electron-transport chain, ATP synthesis, and so on.  The second state, that of cell division and growth, is characterised by genes involved in mitosis, cytokinesis, transcription, RNA processing, and the like.  Expression of these respective clusters is temporally separated. The magnitude and timing of the phases vary with intertidal position and thus with microhabitat changes correlated with state of tide and climatological conditions.  A sample of data to demonstrate this shows expression of the respiration/metabolism Eigengene cluster for high-level mussels through 3 tidal cycles (see graph).  Note the similarity of gene expression in the 3 tissue-types and also the poor correlation of Eigengene levels with either tidal cycles or air-emersion temperature cycles.  The authors suggest that this may owe to the restricted opportunities for these high-level individuals to feed, a situation that would tend to dampen metabolic activities.  Other data, not considered here, reveal patterns of expression of Heat-shock protein 70 and several protein-folding genes, as expected, correlated with body temperatures associated with tidal height.  This unique study is rich with data and provides the research community a standarised methodology with which to assess physiological changes in intertidal organisms, especially important in this time of global climate change.  Gracey et al. 2008 Current Biol 18: 1501.

NOTE  the 2 mussel beds studies are located within 1m of one another, one at 1.6m above MLLW (mean lower low water); the other, at 1.5m.  Individuals are removed at 20 time points over 3d and gill, digestive gland, and adductor-muscle tissues sampled for analysis   dots on graph indicate sampling times

NOTE a cDNA microarray is employed to monitor transcriptional changes.  A pattern analysis is used to identify Eigengenes, or key gene-expresssion patterns in the data.  The first of 4 of these gene-clusters (involved in respiration and metabolism) includes 76 genes and the second (cell-division and growth), 272 genes 

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Research study 2

A similar approach is used in a later study by a group of west-coast scientists to compare physiological variation in mussels Mytilus californianus at 2 sites in Oregon.  The researchers sample tissues from individuals periodically over a 6mo study period and analyse for transcription profiles using cDNA microarray methodology as in the foregoing Research Study 1 above.  Temperature records are made at each site using thermisters embedded in silicon-filled mussel shells (real-animal proxies) and chlorophyll-a measurements (proxies for phytoplankton).  The authors are particularly interested in small-scale gene-expression variability (amoung individuals and within tide-levels) compared with large-scale expression (as between sites).  For example, is intra-site variability greater or lesser than inter-site variability?  Tissues  are sampled for analyses of gene-expression profiles from different sites and tidal heights and, in a separate study, from individuals just prior to and following inundation by the afternoon high tide.  Body temperatures during the study period are about 2OC higher at Boiler Bay than at Strawberry Hill, while equivalent phytoplankton levels are 2-3 times lower at Boiler Bay than at Strawberry Hill.  Results of gene expression, as expected, show strong variation in levels in individuals inhabiting different tidal levels, most notably in genes relating to metabolic processes, but little variation is evidenced among individuals inhabiting the same level on the shore in populations even as far distant as 15m.  Overall, the authors identify 648 features displaying significant change in gene expression to at least one of collection-site and tide-height parameters.  Genes showing largest differences in response to emersion stress are, as expected, heat-shock proteins.  The data show interesting interactions between temperature and food availability, suggesting that mussels living in areas of low phytoplankton productivity, such as at Boiler Bay, may be living closer to their thermal physiological limits than previously thought.  The authors relate their study in this regard to predictions of impact of future climate change.  Place et al. 2012 Functional Ecology 26: 144.

NOTE  the sites are at Strawberry Hill at Cape Perpetua and Boiler Bay at Cape Foulweather, Oregon, separated by about 65km.  Mussel populations at Strawberry Hill are known from previous studies to differ from those at Boiler Bay in several aspects of physiology – greater reproduction, greater cellular response to stress, and higher growth potential – so the expectation is that gene-expression levels and patterns will reflect these differences 

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Temperature effects

  Topics dealing with life in the intertidal zone include temperature effects considered here, and HEAT-SHOCK PROTEINS, 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 body temperatures of a mussel Mytilus californianus during air exposure over a 10-h periodphotograph of a sea mussel Mytilus californianus with thermistor probe insertedSeasonal temperature changes for mussels may be extreme, but they generally come on slowly.  What about stress-effects from more abrupt changes occurring daily owing to tidal emersion and immersion?  This is shown for a single mussel Mytilus californianus on a summer’s day in San Juan Island, Washington during 10h air exposure.   Note the abrupt temperature drop of almost 15OC when the incoming tide covers the mussel.  The dark shell of the mussel acts like a “black body”.  Internal tissue temperatures are close to rock-surface temperatures and are consistently higher than air temperatures. Carefoot 1977 Pacific Seashores Univ Wash Press, Seattle 208 pp.



Mytilus californianus with
thermistor probe inserted about
3cm into the mantle cavity 0.8X

Research study 2

graph showing tissue temperatures of sea mussels over several seasons measured in animals on the Oregon coastA detailed thermal history is presented by researchers at the Hatfield Marine Science Center, Oregon for a population of Mytilus californianus located 1m above MLLW level.  Results show that average tissue temperatures range from 8oC in winter to 13oC in spring/summer. Several years of field observations, however, show individual temperatures reaching as high as 34oC in individuals at the +2m level in summer, or as low as 0oC when the bed is covered in snow.  Experiments with radiolabelled Isochrysis galbana as food show that periods of air exposure actually lead to increased assimilation efficiencies, likely because of the longer residence time and better processing of food in the gut.  The authors measure filtration and absorption rates, and present estimates for energy budgets for mussels at different levels on the shore.  These estimates suggest that mussels living higher on the shore have less scope for growth than ones lower on the shore.  Elvin & Gonor 1979 J Exp Mar Biol Ecol 39: 265.

NOTE  a hypodermic thermoprobe is used to record tissue temperatures of individual mussels

Research study 3

graph showing body temperatures of high- and low-level mussels Mytilus californianus over a tidal cycle in OregonA unique approach to assessing temperature adaptations in mussels Mytilus californianus is taken by researchers in Oregon.  The state of phospholipid vesicles in a membrane enables the health of the membrane, or the degree of its “order”, to be estimated in an organism.  Lower temperatures are known to cause the packing order of membrane phospholipids to increase, thus lowering membrane “fluidity”, while higher temperatures cause the packing order to decrease.  If these changes are too great the membrane may not function properly.  Under prolonged exposure to potentially membrane-disrupting temperatures, some organisms can adaptively modify the packing order  of their phospholipids, either by changing the phospholipid composition or by modifying membrane structure, processes collectively known as homeoviscous adaptation (HVA).  An important question relating to intertidal organisms such as mussels is whether HVA occurs in response to temperature cycling of the tides, or whether temporary membrane disorder, with associated physiological consequences, is simply tolerated.  This is investigated in the ctenidia of M. californianus both seasonally, involving mean temperature differences of about 10-32OC between winter and summer, and during summer when tidally induced body temperatures of high- and low-intertidal individuals in the same population differ markedly (see graphs on Right).

Results show that the packing order of gill phospholipids differs significantly between seasons in accordance with predictions, indicating both summer and winter acclimatisation.  Moreover, in 6-wk laboratory experiments to test the effects of simulated daily temperature cycling, as would occur from the tides, the authors are able to document compensatory HVA responses in high-level mussels, but not in low-level ones.  Overall, the authors conclude that sea mussels photograph of a sea mussel Mytilus californianus dissected open to show ctenidia and other body partsare able to adjust membrane order both seasonally as well as hourly, with the latter being done in relation to intertidal height occupied.  An interesting question posed by the researchers is whether membrane-structuring is done in advance of exposure to high temperature, or whether it is done while the mussel is experiencing the temperature increase.  Williams & Somero 1996 J Exp Biol 199: 1587.

NOTE  in a later paper, George Somero writes a thoughtful account of the value of physiological studies on thermal tolerances and types of genetic modifications that would lead to adaptations to acute and long-term increases in temperature, in increasing our understanding of how to predict the effects of climate change in marine organisms.  The paper is well worth reading. Somero 2010 J Exp Biol 213: 912.

Research study 4

graph showing tissue temperatures during a tidal cycle in high- and low-level mussels Mytilus californianus in OregonAnother example of how thermal landscapes can differ for sea mussels is shown in this study on Mytilus californianus living at low and high levels on an Oregon shore.  Thermistors embedded within the tissues of test individuals record similar temperature increases for both populations during the morning, with the low-level population being covered by the incoming tide at about 1020h.  Immersion of the high-level population does not occur until about 1220h.  Two features are of interest.  First, note the marked and precipitous temperature drops for both populations on contact with the incoming tide.  Adverse physiological effects of this kind of hot-to-cold temperature shock have only rarely been investigated for west-coast marine invertebrates.  Second, while rates of temperature rise are similar for the 2 populations, heat stress is cumulative, so the high-level population is being subjected to much greater potential temperature stress than the low-level population.  Note that tissue temperatures are above 18oC for 2.5h during emersion at the high site, but only for about 0.5h at the low site.  On another day at this site, or at other sites, thermal histories will be quite different.  Roberts et al. 1997 Biol Bull 192: 309.

NOTE  the two sites are vertically separated by 1m.  Temperature records are for July

Research study 5

schematic showing heat budget for a musselFor comparative studies of temperature effects on the physiology and behaviour of intertidal invertebrates it would be handy to have a deterministic model that employs all requisite environmental inputs to predict body temperatures.  This is done for intertidal mussels Mytilus spp. using inputs of solar radiation, air/ground temperature, wind speed, and so on to determine heat flux in a model mussel.  The balance of heat loss and gain determines the total heat stored and, therefore, its temperature.  In all, 6 kinds of heat transfer are required in the model, as shown in the accompanying illustration.

Tests of the model’s predictive ability are done in the field at locations on the Olympic Peninsula and San Juan Island, Washington with Mytilus californianus and M. trossulus. The mean deviation of the predicted temperatures from actual body temperatures is within about 1oC.  The model allows ready comparison of heat budgets of solitary mussels versus ones aggregated in beds, comparison of evaporative heat loss in mussels of different sizes, and so on.  Data for the first comparison show, as expected, that body temperatures of mussels in beds may be several degrees Celcius cooler than those of individuals living in open spaces. With respect to the latter, larger mussels are confimed to have a greater “thermal inertia”, which buffers them against rapid change in environmental conditions.  The author makes the point that because body temperature is controlled by several factors, then no one measurement of a single factor, such is air temperature, is likely to provide a good estimate of actual body temperature.  Helmuth 1998 Ecol Monogr 68: 51.

NOTE  for comparable studies on limpets see LIFE IN THE INTERTIDAL ZONE: TEMPERATURE STRESS

Research study 6

photograph of a mussel bed showing the considerable vertical extent of the shore occupiedAnyone who has spent time on the shore knows that the thermal landscape of, say, mussel beds, varies greatly depending upon intertidal height, aspect to the sun, slope of the shore, and many other factors.  Add to these the changing conditions over tidal cycles and season, and the thermal history of a mussel in one patch could differ greatly from another individual in a not so distant patch.  Such information may be important to researchers interested in the thermal biology of mussels and other shore organisms.  The author of the previous Research Study 5 has tested the efficacy of using climatological data to recreate the thermal history of mussels Mytilus californianus at 2 tidal heights under conditions of aerial exposure over a typical “climatological year”.  The author uses hourly weather data from federal meteorological databases for the area in question, the Olympic Peninsula, Washington, to develop heat budgets as described in the earlier study.  Results show: 1) that body temperatures estimated in this way may fluctuate by more than 20OC over a 12-h period, and 2) that mussels could be hotter in spring (Apr-Jun) than in summer (Jul-Sep), and colder in autumn (Oct-Dec) than in winter (Jan-Mar).  Both sources of variability owe primarily to the effect of tidal cycles in determining exposure times.  Thus, tidal cycles may be more important than seasonal differences in determining body temperatures of the mussels.  Helmuth 1999 Ecology 80: 15.

View of a mussel bed in Barkley Sound, British Columbia
showing the considerable vertical extent occupied

Research study 7

Is it possible that harmful effects of summer-time heat are ameliorated in mussels by gaping, allowing evaporative water loss to cool the tissues?  Studies on internal temperatures in sea mussels Mytilus californianus using “data-logger” thermistors “thermally matched” to the mussels’ own tissues photograph showing mussels Mytilus californianus in the sunshow that gaping does not significantly influence body temperature, at least not under simulated conditions in the laboratory. Fitzhenry et al. 2001 Am Zool. 41: 1444; Fitzhenry et al. 2004 Mar Biol 145: 339.

NOTE  for a complementary study on sea stars Pisaster ochraceus employing biomimetic data-loggers see Broitman et al. 2009 Oikos 118: 219

Mussels Mytilus californianus in the sun with
one individual, centre of photo, gaping 0.33X

Research study 8

data on species richness in mussel communities in California present day vs. historicalCan the effects of climate change be monitored by changes in diversity and abundance of  species in mussel-bed communities?  This important question is addressed by researchers at the University of California, Los Angeles by comparing diversity data collected in 2002 at 22 sites in California with historical data collected during the 1960-70s at the same sites.  Results indeed show significant declines in species numbers ranging from 24-80%, the last value equivalent numerically to about 140 species (see accompanying illustration).  Note that the authors have arbitrarily divided the array in 3 major geographic divisions for convenience, and mean values for species richness are presented for each. The data are, of course, correlative, but are strongly suggestive, with changes associated with the Pacific Decadal Oscillation being implicated as likely agents.  The work is part of a growing and important literature on possible climate-induced change in west-coast intertidal community structure.  Smith et al. 2006 Ecology 87 (5): 1153.

NOTE  the sites span about 1300km along the California coast

NOTE  major shifts in surface seawater temperature in both the north and south Pacific regions occurring on a 20-30yr cycle



Research study 9

map of west coast of North America showing sites for temperature monitoringtemperature mosaic for 10 west-coast intertidal sites for a 5-yr periodA species with large latitudinal distribution such as Mytilus californianus is subject to correspondingly wide geographical range in temperature.  Yet, to what extent can a temperature regime for a population of Mytilus be predicted from latitudinal position, or even, for that matter, from a few temperature measurements taken at the site in air or water over a relatively short period of time?  To investigate this, researchers install termperature-data loggers at 10 sites along the west coast from northern Washington to southern California, and record continuously over a 5yr period (see map).

Some of the results of this unique experiment are shown in the display on the Right and indicate the large-scale mosaic of body temperatures that exists in these populations. Temperature variability for a given location reflects a myriad of physical conditions that exist intertidally, including aerial exposure during low tide, degree of solar radiation, day length, upwelling occurrences, timing of low tide, and so on.  Results show that simulated mussel-body temperatures may vary in physiologically meaningful and often counter-intuitive ways over large sections of the species geographic range, and show the existence of “hot” and “cold” spots, where temperatures are hotter or colder than expected based on latitude. Overall, southern mussels exhibit more predictable body temperatures than northern mussels.  Body temperatures of these ectotherms are often quite different from temperature of surrounding air or substratum. Localised patterns of wave splash can overwhelm predicted trends in latitudinal temperature. Temperatures in excess of 30oC, when formation of heat-shock proteins is likely, also show a complex pattern, with sites having a relatively high probability of occurrence being interspersed with sites having a lower probability. None of the findings is unexpected, but to have the data recorded in such a long-term, comprehensive way, and presented so strikingly, is quite original, indeed.  In summary, rather than simple latitudinal gradients, these intertidal mussels experience a complex thermal mosaic.  With so many potential variables affecting the thermal state of an intertidal organism, the authors remind us of the importance when doing field research of quantitatively assessing biogeographic patterns in temperatures and other environmental variables at scales relevant to the organisms themselves.  Helmuth et al. 2006 Ecol Monogr 76: 461; see also Zippay & Helmuth 2012 Integr Zool 7: 312 for a general review of effects of temperature on mussels Mytilus.

NOTE  the thermocouple ends of these data loggers are not inserted into live mussels; rather, they are either encased in black-tinted expoxy plastic or inserted into real mussel shells filled with silicone.  In each case, the temperatures recorded mimic real temperatures with about 2oC accuracy

NOTE  more on heat-shock proteins (HSPs) can be found in another section of "life in the intertidal zone": LEARN ABOUT MUSSELS: HEAT-SHOCK PROTEINS

Research study 10

map of collecting sites for Mytilus study in San Francisco BayA few decades ago it seemed enough to take a few measurements of seawater temperatures then relate these to latitudinal distributions of an intertidal species without attending to the myriad conditions that exist at the microhabitat level.  A more detailed approach is taken by researchers from the University of South Carolina who investigate distributions of native mussels M. trossulus and their invasive counterparts M. galloprovincialis, and hybrids, in relation to spatial variation in microhabitat temperatures at several sites in San Francisco Bay (see map; this is the "hybrid zone" of overlapping distributions of the 2 species).  The researchers focus specifically on species inhabitance of shaded vs. sun-exposed regions and intertidal vs. subtidal areas at sites in San Francisco Bay over a 2yr graph comparing temperatures of "biomimetic" mussels under conditions of sun- and shade-exposed, and submergedperiod.  Results show, as one would expect, that M. trossulus is more abundant than M. galloprovincialis in shaded intertidal and in subtidal habitats.  Temperatures in these habitats are found to differ commonly by 5-10oC, with peak differences of up to 16.5oC (see graph). Translocation of individuals of both species and their hybrids from a single “donor” site (orange dot on map) to cages located in sun-exposed and sun-shaded intertidal, and subtidal locations at several other sites (yellow dots on map) leads to greater summer mortality after 100d in all habitats for all mussels combined.  Overall and as expected, survivorship is lowest for both species and their hybrids in the sun-exposed treatments. The researchers also find that the native species M. trossulus has lower survivorship that the invasive species in all habitats studied, a result that is both unexpected and unexplained.  The authors conclude that processes operating over broader geographic scales may be experimentally detectable over much narrower scales.  Such detailed analyses may likely reveal a mosaic of distribution of the 2 species where localised “hot spots” in otherwise cool M. trossulus-inhabited areas may be occupied by M. galloprovincialis, and vice versa for localized “cool spots” in otherwise warm M. galloprovincialis-inhabited areas.  Schneider & Helmuth 2007 Mar Ecol Progr Ser 339: 157.

NOTE  after collection of all mussels, a small portion of mantle tissue is snipped off for genetic identification of species and hybrid status. Species are identified through assay of the diagnostic genetic marker Glu-5’ after amplification by polymerase chain reaction

NOTE  temperature sensors placed in silicone-filled mussel shells create “biomimetic loggers” that provide temperature data for shaded and sun-exposed intertidal microhabitats

Research study 10.1

graph comparing ubiquitin-conjugated protein levels in 2 species of mussels Mytilusmap showing distributions of 2 west-coast mussel species Mytilus trossulus and M. galloprovincialisIn a later paper on a related topic researchers at University of California, Santa Barbara use levels of ubiquitin-conjugated proteins in ctenidial tissues to assess seasonal physiological stress in 2 mussel species Mytilus galloprovincialis and M. trossulus, and their hybrids in a zone of overlap at Bodega Bay, California (see map).  These and shell lengths are measured in the 3 groups 8 times over a 2yr study period to assess stress levels.  Temperature-loggers provide 1 hourly data continuously throughout this period.  The species are spatially separated in the Bay, with M. galloprovincialis dominating (91%) subtidally in one location and M. trossulus dominating (71%) in a nearby intertidal mudflat area.  Hybrids are only rarely present (18 and 9%, respectively, in the 2 habitats).  Results show significantly higher growth rates for M. galloprovincialis and hybrids than for M. trossulus.  As expected in view of the mid-distributional location of the study, just at the northern edge of range of the invasive M. galloprovincialis and at the southern edge of that of the native M. trossulus, seasonal highs in levels of ubiquitin-conjugated proteins are seen in winter for the former species, and in summer for the latter (see graphs).  Note the correspondence of highest levels of ubiquitin conjugates with highest and lowest annual temperatures for the 2 species in their respective habitats.  The authors discuss the relative scarcity of M. trossulus in the subtidal area, a region expected to provide equally favourable conditions for growth of this species as for M. galloprovincialis, and suggest that the competitive dominance of the latter invasive species may exclude the native species in the generally more crowded conditions of the subtital habitat.  In contrast, in the intertidal area, suitable substrata for byssus attachment is at a premium and predation levels are greater, leading to more sparse distributions and, as a possible result, to less intense interspecific competition for space.  Dutton & Hofmann 2008 Mar Biol 154: 1067.

NOTE  ubiquitin is a small protein (only 76 amino acids in length) present in the tissues of all organisms whoses function is to attach to other proteins, labeling them for movement to other locations in the cell and identifying damaged proteins for repair or for destruction and recycling.  During times of stress, such as from temperature as in the present study, more proteins are damaged and attached to, or conjugated, with ubiquitin, and turnover is generally greater.  The levels of these ubiquitin-conjugated proteins can thus act as proxies for levels of stress in an organism

NOTE  identified through genotyping of DNA material extracted from adductor muscles

Research study 11

graph showing gonad indices of mussels Mytilus californianus at 2 sites in Oregon at different intertidal heightsgraph showing gonadal indices in mussels Mytilus californianus at 2 locations in Oregon at different tidal heights over a 2-year periodThermal and other stresses related to longer exposure to air, such as drying and reduced feeding time, increase with increasing height in the intertidal zone.  The physiological costs of stress1 and their effects on growth and reproduction will be greater, then, in high-level mussels than in low-level ones.  This is investigated from the standpoint of gonadosomatic indices2 in two3 populations of mussels Mytilus californianus in central Oregon.  Results show, as predicted, that higher-dwelling individuals invest proportionally less energy in reproduction than lower-dwelling ones.  Spawning times and frequencies are affected by intertidal height at both sites, but perhaps less so at the Strawberry Hill site (see graph on Left). Here, both sexes spawn in late winter/early spring. In comparison, at Fogerty Creek (see graph on Right), high-level mussels spawn in early summer, while low-level ones spawn small batches of gametes continuously throughout the year. The authors conclude that mussels exhibit physiological trade-offs to stress and suggest that under the increased thermal stress predicted from climate change, may increasingly allocate energy from reproduction to physiological defenses.  Petes et al. 2008 Ecol Monogr 78: 387.

NOTE1 the researchers also investigate possible defense against stress, specifically, harmful oxygen radicals, of carotenoid pigments in the gonad, thought from earlier observations to be more prevalent in high-level mussels based on their more orange-coloured gonads.  However, the results are inconclusive and not considered here

NOTE2  gonadosomatic indices are calculated as gonad mass divided by gonad mass plus somatic-tissue mass, and are equivalent to the more commonly used “gonad indices” of other publications

NOTE3  the 2 sites are at Strawberry Hill and Fogerty Creek.  The former has somewhat higher mean daily temperatures than the latter.  Field temperatures are measured within “robomussels”, that is, dead shells containing thermal-loggers embedded in epoxy/resin

Research study 12

photograph of gaping mussels Mytilus californianus at Strawberry Hill, Oregon courtesy Gretchen Hofmannmap showing collecting sites for study on mussels genetics/physiology in OregonScientists in Oregon uniquely apply cDNA-microarray techniques to investigate genetic variability in physiological responses to emersion in sea mussels Mytilus californianus.  Specifically, the authors compare gene-expression profiles1 in gill tissues sampled at low-tide periods from mussels at 4 west-coast sites extending from Bamfield, British Columbia to Punta San Isidro, Baja California (see map on Right) with respect to emersion times2.  That these estimates provide a reliable index of temperature stress at each site is shown by the strong correlation of the emersion times with level of expression of heat-shock protein3 hsc71 (see graph lower Left). The findings, which are understandingly complex given the broad scope of the study, reveal that expression patterns for the majority of genes assessed vary significantly as a function of collection site.  Thus, mussels at each site respond differently to emersion time in the different areas sampled, with the biggest difference being manifested in the Strawberry Hill, Oregon site (a suspected "ecological hotspot"). Interestingly, thehistogram depiction of the effect of emersion times of mussels Mytilus californianus on levels of heat-shock proteins 2 sites exhibiting the most similar expression patterns are Bamfield, British Columbia and Jalama Beach, California.  While acknowledging that other factors, such as nutrient availability, predation effects, and so on, may be contributing to variation in gene expression, the authors conclude from their findings that physiological responses of M. californianus to temperature effects associated with emersion cannot be predicted simply from latitudinal location. The study is a wonderful integration of genomics, environmental physiology, and biogeography. It provides a glimpse of possible future directions of research in marine physiological ecology. Place et al. 2008 Mar Ecol Progr Ser 356: 1.

NOTE1  the profiles refer to gene clusters (cDNA microarrays) relating to the following 12 general areas of physiology: cell-cycle regulation/cell proliferation, protein rescue/protein folding, cell signaling, protein degradation, carbohydrate metabolism, lipid metabolism, apoptosis, cell adhesion, transcriptional regulation, protein biosynthesis, cellular trafficking, and cytoskeletal reorganisation

NOTE2  these are not measured directly; rather, are estimated from the cumulative amount of time the different mussel beds are emersed during daylight hours of the month preceding collections

NOTE2  more on heat-shock proteins (HSPs) in mussesl can be found in another section: LEARN ABOUT MUSSEL: HEAT-SHOCK PROTEINS

Research study 13

graph relating past chemical attributes of a mussel shell Mytilus californianus with seawater temperature experiencedWith all the attention being paid to microenvironmental temperature variability within mussel habitats, all of them employing large numbers of temperature-loggers, it is interesting that a group of researchers from several west-coast universities have undertaken, in a way, to ask the mussels directly.  They do this by outplanting specimens of Mytilus californianus into high and low intertidal positions in San Diego Harbor, leaving them for over a year with temperature-loggers, then serially microsampling and chemically analyzing their shells for temperature-related chemical parameters, such as delta18Ocalcite and Mg/Ca.  Seawater bathing the mussels is periodically sampled and analysed for concentrations of delta18Oseawater and Mg/Ca.  Results show a significant correlation of delta18Ocalcite values with equilibrium values predicted from the environmental data (see graph).  In contrast, Mg/Ca values are significantly correlated with growth rate and intertidal position, as more-or-less expected, but not with temperature.  The delta18O data, then, seem to provide a reliable proxy for past temperature events and, as summarised by the authors, make “M. californianus a valuable source of information for paleoceanographic reconstructions”.  Ford et al. 2010 Paleoceanography 25: PA1203.

NOTE  measured isotopic delta18Ocalcite values in the shell are compared with predicted values obtained from the seawater-sampling data. The method has been used previously with success with carbonate-secreting organisms such as molluscs and corals

NOTE  in a related study, also on the mussel Mytilus californianus, scientists are unable to correlate 9yr of directly measured environmental data (including pH) with B/Ca ratios measured in the shell to test the feasibility of B/C being used as a proxy for pH.  A single individual mussel shell is used in the study.  McCoy et al. 2011 Biogeosciences 8: 2567.

Research study 14

Previous studies have shown that differences in thermal tolerances are likely the chief explanation for the success of the native Mediterranean mussel Mytilus galloprovincialis in invading and displacing the native congenor M. trossulus in southern and central California.  The extent to which species-specific heat-stress genes are involved in this displacemnt is investigated by a research group at Stanford University, California.  The researchers develop an oligonucleotide microarray with probes that are able to recognise 4488 different genes in both species.  In acute heat-stress experiments, 1531 of these genes are expressed similarly in the 2 species, representing a conservation of the heat-stress-induced transcriptome within them, while 96 genes exhibit species-specific differences in expression.  The genes that differ are ones involved in oxidative stress, energy metabolism, proteolysis, ion transport, cell signaling, and others. Most telling is the difference in expression of the gene for heat-shock protein24, an important molecular chaperone that is highly induced in M. galloprovincialis (64-fold) but only slightly induced in M. trossulus.  Interestingly, and somewhat unexpected based on results of previous studies, another set of heat-shock proteins, Hsp70, is expressed similarly in the 2 species, with induction values ranging from 31-fold in M. galloprovincialis to 34-fold in M. trossulus.  The authors conclude that the different responses to acute heat-stress (especially noted in 3 oxidative-stress genes and one molecular-chaperone gene, Hsp24) may help explain, and predict, the invasive success of M. galloprovincialis during future climate change.  The study provides a unique and valuable insight into the molecular basis of invasion biology, and should be of great interest to other researchers. Lockwood et al. 2010 J Exp Biol 213: 3648.

NOTE  all mussels are acclimated to 13oC, a thermally realistic temperature for both species, then heat-ramped in 6oC per-hour increments from 13-32oC to simulate thermal heating on a hot day during low tide.  At intervals, gill tissues are removed from experimental and control animals of both species for transcriptional analysis

NOTE  heat-shock proteins are dealt with in their own section LEARN ABOUT MUSSELS: HEAT-SHOCK PROTEINS

Research study 15

A somewhat different approach is used by researchers at the California Polytechnic State University to investigate essentially the same subject as in the foregoing Research Study, that is, a comparison of heat-stress responses in mussels Mytilus galloprovincialis and M. trossulus but, in this case, not by measuring transcriptional mRNA gene-expression products, but by measuring proteomic responses.  As in the foregoing study, mussels are acclimated at 13oC for 4wk, then acutely exposed to temperatures between 24-32oC for 1h, then gill tissues sampled for analysis.  Results show 47 proteins that significantly change in abundance in M. galloprovincialis and 61 that change in M. trossulus.  As expected, the onset temperatures of greater abundance of several heat-shock proteins are higher in M. galloprovincialis than in the native species, reflective of a higher tolerance to thermal stress.  Levels of certain oxidative-stress proteins decrease at higher temperatures, but only in M. trossulus, indicating that its ability to combat heat-induced oxidative stress is less well-developed than in the warmth-tolerant M. galloprovincialis.  Thus, in these and several other related cellular processes, the warm-adapted invasive species exhibits a lower sensitivity to thermal stress, consistent with its expanding range in the warmer waters of California.  The study is the first detailed comparison of proteomic response to acute thermal stress in any pair of Mytilus congenors, and the results will be especially useful for researchers interested in other species undergoing similar biogeographic flux or in ones facing future trends in global warming.  Tomanek & Zuzow 2010 J Exp Biol 213: 3559.

NOTE  analysis of actual proteins produced, identified in gill tissue by 2-dimensional gel electrophoresis and tandem mass spectrophotometry

NOTE  the similarity of treatment protocols between this study and the foregoing Research Study above owes to a sharing of facilities between the 2 research groups, with one group doing transcriptomic analysis and the other, proteomic analysis

Research study 15.1

map showing distributional changes of mussels Mytilus galloprovincialis in California from 1997-2007Partly because of the time-scale involved it is not easy to demonstrate direct effects of global warming on distributions of marine-invertebrate species.  However, based on baseline data collected in the mid-90s, researchers at University of South Carolina and Irmo High School, South Carolina are in a position to test whether a hybrid zone formed between the invasive Mediterranean mussel Mytilus galloprovincialis and the native M. trossulus has shifted northwards as a result of global warming over this time.  The authors collect mussels from 15 locations (see map) spanning the distribution of the 2 species and their hybrids, and determine genetic makeups using the same methods as in the earlier study.  Results show, contrary to expectation that the warm-water preferring M. galloprovincialis and their hybrids with M. trossulus would have shifted northwards in response to global warming, they actually shifted southwards by about 200km (see map).   The distribution of the native cold-water species M. trossulus is little changed and the species is still rare or absent south of Monterey Bay.  The authors credit the reverse trend with a decadal event known as the Pacific Decadal Oscillation in which a previous 15yr warm phase shifted at the turn of the Century to a cold phase with minimal El Nino warming events.  The authors note that this distributional shift of M. galloprovincialis represents one of the largest and most rapid ever recorded for any species and greatly surpasses that expected through global warming.  Hilbish et al. 2010 J Biogeogr 37: 423.


Research study 15.2

diagram showing experimental design for study of heat and food effects in mussels Mytilus galloprovincialis and M. trossulusResearchers at the University of North Carolina investigate the effects of food and temperature on survival of mussels Mytilus galloprovincialis and M. trossulus obtained from a commercial supplier in Coupeville, Washington. The researchers’ expectation is that high food availability will ameliorate thermal stresses during air exposure. Experiments are conducted in 2 laboratory tidal tanks using heat lamps to yield 3 body temperatures (20, 25, and 30oC; 30 mussels of each species per treatment), with an additional treatment batch remaining continuously submerged at 12oC (see diagram). Once daily the water is pumped out of the tanks for 6h to simulate low-tide conditions. Food consists of a commercial algal paste reconstituted in seawater to form a 10% solution, and is provided in low amount to one set of mussels and in 10-fold greater amount to the other set. Biomimetic loggers placed within each mussel graph showing survival of mussels Mytilus galloprovincialis and M. trossulus at 30oC under conditions of high- and low-food availabilityaggregation provide continuous thermal records. Survival, measured after 9wk, is >85% in all treatments save for 30oC, so only the 30oC data are shown here (see graph). Note that survival in the hot (30oC) treatments is significantly better in high-food conditions than in low-food conditions for both species (57% vs. 33%, respectively, for M. galloprovincialis, and 38 vs. 16 for M. trossulus. Overall, thermal tolerance is greater for M. galloprovincialis (an invasive species) than for M. trossulus, a finding corroborated in several other studies. The authors note that theirs is the first study to examine interaction of emersed body temperature and food supply on survival of these 2 mussel species. Schneider et al. 2010 J Thermal Biol 35: 161.

NOTE species comprising the paste are Chaetoceros-B, Phaeodactylum tricornutum, and Nannochloropsis oculata. High-food water contains approx. 550µg l-1 chlorophyll, while low-food water contains about 58µg l-1

NOTE these are mussel shells each packed with silicon and an iButton data logger, expected to record body temperatures within 1.5oC of living mussels in the field

Research study 16

photograph showing open-coast rocky shorelineIn a time of growing concern about climate change, west-coast scientists are becoming increasingly attentive to the potential impact of ocean warming on the health and vitality of intertidal invertebrate communities. But what kinds of effects are anticipated? With all the microenvironmental variability associated with rocky intertidal shorelines, can effects of temperature increments associated with global warmed be even measured?   A heat-budget modeling approach is employed by a group of South Carolina scientists who use biomimetic mussels with implanted temperature sensors, positioned at 3 intertidal elevations on a rocky shore at Bodega Bay, California to assess potential effects of changes in air temperature, solar irradiation, wind speed, wave height, and time of low tides on “body” temperature of the mussel mimics.  The results show that there is little consistency among the different sites.  Even substantial increases in air temperature of 2.5oC may be mitigated by moderate wind velocities, the timing and heights of low tides, and other factors.  At some sites increases in air temperature primarily affect organisms at high intertidal levels, while at other sites the effects are felt across all tidal heights.  At other times, “body” temperatures may be high even when air temperatures are not because of solar irradiation, or low even when air temperatures are high because of convective cooling and wave splash.  Local mists, pockets of high-humidity, and aspect and shading associated with local topography would all be expected to contribute to variability.  Based on the strong influence of, and interaction between, local oceanographic and weather processes, the authors suggest that the timing and magnitude of warming will be highly variable at different coastal sites.  Moreover, a single environmental parameter such as air temperature or water temperature cannot be relied upon to predict reliably future physiological stresses associated with climate change.  Helmuth et al. 2011 J Exp Mar Biol Ecol 400: 191.

NOTE  more information on environmental data logging can be found in Research Study 9 above.  The present study is really a collaboration of ideas and techniques initiated in several earlier studies by the senior author

Research study 17

simulated mussel bed with mussel-positions notedhistogram showing daily maximum temperatures of simulated mussels in bedWhat is the degree of variation in individual body temperature of mussels in a typical west-coast mussel bed, and to what extent does within-site variability in temperature and tolerance to thermal stresses influence the persistence of an intertidal mussel population?  These important questions relating to the ecological effects of climate change are addressed in a study of sea mussels Mytilus californanus along a 336m length of rocky shoreline adjacent to Hopkins Marine Station, Pacific Grove,  California, an area characterised as being moderately wave-exposed.  The researchers use several approaches in the study, the main 2 of which are noted here.  The first is to construct a simulated mussel bed of 23cm diameter using 114 mussel shells, each emptied of its occupant and replaced with a silicone sealing compound and, in 38 of these, an embedded temperature logger.  Temperatures are recorded every 20min over a 29d period.  The second approach involves selection of 2 sites for close examination of temperature variation in individual mussels, one at a cool, wave-exposed part of the shore (the “exposed” bed); the other, at a relatively warm, protected part of the shore (the “protected” bed).  The 2 beds are separated by 24m. In each site about 20 mussels are removed and replaced with shells containing silicone and implanted temperature logger.  Temperatures are recorded every 20min over  2 replicate periods of 24 and 28d.  A number of other experiments are done on thermal tolerance of individual mussels.  Results for the simulated 23cm-diameter bed show lower maximum temperatures on the edge than in the middle (see graph upper Right), unexpected in view of previous predictions, and likely owing to more convective cooling by wind at the edges than in the more thermally insulated centre of graph showing mussel survival after aerial thermal stressthe bed.  Overall site-results show that within-site variation in temperature over the 4wk study period is similar and oftentimes greater than that seasonally recorded over a 1600km length of Pacific shoreline (11-40oC).  Moreover, the authors discover a much larger inter-individual variation in thermal tolerance than expected, something not apparently noted previously by physiologists.  The graph on lower Right shows mortality of mussels in relation to aerial thermal stress.   In the authors’ view, such within-site variability may actually buffer the effects of long-term temperature trends or short-term extreme-temperature events on the proportion of population killed by heat stress.  The researchers discuss ways that future intertidal ecologists can best incorporate localised temperature variability into their predictions of effects of climate change.  Denny et al. 2011 J Exp Mar Biol Ecol 400: 175.

NOTE  known from other studies to approximate closely the thermal properties of of live mussel flesh (see Helmuth & Hofmann 2001 heat-shock proteins)

NOTE  the athours remark that their highly variable temperature readings are obtained within what ecologists would normally consider a “single site”

Research study 18

graph showing circadian rhythm of gene expression in mussels Mytilus californianusgraph showing circadian fluctuation of RORB (a "clock" gene) in musselsGiven that temperature is likely to be the single most important influence on biological rhythms in a mussel, the question arises as to which daily event will have the most influence on behaviour and physiology:  the 24h diel cycle of light and dark, or the superimposed 12.4h semidiurnal cycle of the tides?  This interesting question is investigated by researchers at the University of Southern California in a most unique way.  They expose mussels Mytilus californianus in the laboratory to a simulated intertidal environment of 12h cycle of light and dark, and 6h cycle of alternating high and low tides at constant temperature of 17oC.  On Day 4 (80h time point) the mussels are warmed during low tide by 7oC.  Samples of ctenidia are taken from 4 individuals every 2h for 96h, including 8 high tides and 8 low tides, then analysed for relative transcript abundance.  Results show that >40% of the transcriptome (representing 2,756 transcripts) exhibits rhymicity of gene expression and, of this amount, over 80% of the transcripts (about 2, 365) follow a circadian cycle with a 22-28h period (see graph on upper Left), while only 20% or 236 transcipts oscillate with a tidal rhythm of 10-14h.  Note in the graph that highest gene expression occurs just following the start of dark phase of the 24h cycle.  Note also in the sample data for RORB the relatively small effect on the circadian rhythm of a 6h exposure to 7oC+ warm air (graph upper Right).  Thus, contrary to expectation, the tidal cycle of air exposure and water emersion is not the dominant driver of rhythmic gene expression in M. californianus. A small field experiment provides supportive evidence for this.  The authors identify several transcripts, including 2 isoforms of carbonic anhydrase, whose expression can be linked to hypoxia associated with the onset of low tide, so the methodology holds considerable promise for future research. Connor & Gracey 2011 Proc Nat Acad Sci 108 (38): 16110. 

NOTE  represented by the sum total of RNA molecules present (= transcriptome), and providing a measure of gene activity (expression).  At this juncture, the reader is invited to guess which of circadian light-dark or tidal cycle will have the greatest effect.  Hint: follow the advice of your old physics prof…incorporate all parameters, reason it out carefully, then choose the opposite answer!

NOTE  considered by the authors as an ortholog of the mammalian “clock” gene RORB, one of several circadian oscillators that activate the transcription of a range of target genes in mammals, plants, and certain invertebrates

Research study 19

graph showing relationship of levels of carnitine-conjugated fatty acids to tidal cycle in mussels Mytilus californianusAs if our understanding of tidal effects on metabolism in mussels were not already challenged enough by such things as heat-shock proteins, isotopic carbon/oxygen, membrane phospholipids, and other biochemical entities, researchers at the University of Southern California, Los Angeles have raised the “bar” to an even higher level.  They have done this by creating a simulated tidal environment in the laboratory with alternating high and low tides of 6h duration, and populating it with mussels Mytilus californianus freshly collected from the field.  They then sample gill tissue from the mussels at 2h intervals over a 72h period and quantify levels of 169 metabolites. 

graph showing relationship of glucose levels in mussels Mytilus californianus with tidal cycleOf these, 24 are found to cycle significantly with a 12h period linked to the tidal cycle.  Seven of the 24 peak during high tide, while 17 peak during low tide.  Eight of the low tide-peaking ones are carnitine-conjugated metabolites that appear to be involved in anaerobic energy metabolism.  The tight fit to the tidal cycle of 2 of these, propionycarnitine and acetylcarnitine, is shown in the graph at upper Left.  Glucose, the primary substrate for anaerobiosis, also declines significantly during each low-tide period (see graph on Right). During high-tide periods the lipid sphingosine appears in the gills and is thought by the authors to be acting possibly as a signaling molecule. The study is novel and should be of great interest.  Much of what has been discovered is new not just to west-coast “mussel science”, but also globally (e.g., M. edulis, and further investigation will be required to determine how this new information adds to our present understanding.  Connor & Gracey 2012 Amer J Physiol Regul Integr Comp Physiol 302: R103.

NOTE  using techniques of liquid chromatography and gas chromatography-mass spectrometry

NOTE  carnitine is an ammonium compound synthesized from amino acids lysine and methionine, and is involved in the transport of long-chain fatty acids to the mitochondria during their oxidation to generate  metabolic energy

Research study 20

In an attempt to explain the invasive superiority of introduced Mediterranean mussels Mytilus galloprovincialis over the indigenous M. trossulus in California, a group of scientists compare proteomic responses of each species to long-term temperature stress.  After 4wk acclimation to 7, 13, and 19oC, cytoskeleton composition and expression of certain energy-metabolism proteins in the ctenidia changes in both species in response to the higher temperatures, but moreso in the native species than in the heat-tolerant invasive species.  The colder-water-inhabiting Mytilus trossulus also produces abundant protein chaperones at the 19oC acclimation temperature, while M. galloprovincialis does not, indicating different upper temperature tolerances.  Conversely, at the cooler temperatures both species exhibit changes in cytoskeletal proteins accompanied by increased amounts of oxidative stress-proteins and molecular chaperones, but with M. galloprovincialis exhibiting significantly more change than M. trossulus.  The authors note that these metabolic adaptations all cost energy, and this may help explain the competitive advantage shown by the invasive M. galloprovincialis in southern California since its introduction there in the early 1900s.  They also predict further expansion of its northern range, with concomitant pull-back of the southern-most limits of M. trossulus, during future anthropogenic global warming.   Fields et al. 2012 J Exp Biol 215: 1106.

NOTE  these changes reflect increased rates of filtration and respiration owing to increased ciliary activity

NOTE  these are special “repair”-type proteins that function to reconfigure the amino-acid structure in damaged proteins

Research study 21

graph showing time spent with valves closed in mussels Mytilus californianus under laboratory conditions of immersed/emersed and at different temperaturesIs the behavioral repertoire of mussels Mytilus spp. of opening and closing of the shell valves sensitive enough to use as an indicator of thermal stress?  This is tested by researchers at Hopkins Marine Station, California for Mytilus californiansus, M. trossulus, and M. galloprovincialis, by exposing individuals of each species to gradually increasing seawater temperatures (13, 25, and 33oC) and air temperatures (13 and 33oC) over 1 or 3d, and monitoring the shell opening/closing behaviour along with complementary analyses of stores of glycogen in the shell-adductor muscles.  Results show a threshold effect at 33oC in both media, but especially in air, where mussels mostly gape (see sample data for M. californianus in graph on Left).  Valve closures under control conditions (13oC) in M. trossulus are 2.5 times longer on average than in M. californianus and 3.3 times longer than in M. galloprovincialis. A lack of correlation of closure times with decreased levels of adductor-muscle glycogen or other contractile functions in the muscle suggests that valve gaping is primarily in response to the need to maintain oxygen flow to the ctenidia.  Post-treatment effects of elevated temperatures on survival reveal significant differences among the species, with M. trossulus experiencing lowest survival through the 90d recovery period following exposure to elevated body temperature in seawater.  Artificially sealing the valves closed for 1 or 5h to simulate an anti-predator response during the 90d recovery period, leads to no significant effect on survival in any of the species.  The authors discuss their data at length.  Dowd & Somero 2013 J Exp Biol 216: 502.

NOTE  the propensity of mussels to gape in warm weather has long been noted in the literature, but has never been tested in such a rigorous and well-documented way  

Research study 22

graphs showing mortality in juvenile mussels Mytilus trossulus at different temperaturesAn interesting contribution to the subject of stress tolerance in mussels is made by researchers at the Bamfield Marine Sciences Centre, British Columbia who investigate temperature/desiccation effects on 1-2mm juveniles of Mytilus trossulus.  The juveniles are collected from the field shortly after settlement in Barkley Sound, British Columbia during spring/summer.  Larvae typically settle preferentially onto filamentous algae or byssus threads of adults in the mid-intertidal zone where they will be air-exposed for 5-8h per tide cycle.  Results from a series of 6h-duration laboratory exposures to temperature range 10-45oC (in 5oC increments) reveal a critical dose between 30-35oC (see upper graphs on Left).  A second set of finer-scale experiments reveal that the critical thermal-stress level is between 33-35oC, with the LT50 = 34oC (see upper graphs on Left).  Temperature recordings from the field indicate, however, that these lethal limits are almost never encountered by these new recruits.  Results from a similar series of 6h desiccation experiments show that half the juveniles die at a Vapour Pressure Deficit of about 1 (see lower graph on Left). Measurements of VPD in the field during the season of larval settlement histograms showing ontogenetic shift in juvenile mussels Mytilus trossulus exposed to different levels of desiccation(May-Aug 2011) reveal many days where a VPd of 1 is exceeded, suggesting that desiccation stress for these 1-2mm juveniles may be a significantly greater source of mortality than temperature.  Later in life, at a size range of 3-8mm, risk of death from desiccation is much reduced, even at relatively high levels of VPD (see histograms on Right).  This ontogenetic shift in tolerance to drying has interesting behavioral implications, because it is at the approximate size of 3mm that the youngsters abandon their preferred settlement habitat of moist filamentous algae and crawl to the much drier conditions of the adult rock habitat.   Jenewein & Gosselin 2013  Mar Ecol Progr Ser 481: 147.graph showing mortality of juvenile mussels Mytilus trossulus under different conditions of desiccation

NOTE  Lethal Temperature50 is the temperature at which 50% of the test individuals are dead.  For their desiccation results, the authors use the term LD50 , or Lethal Death50

NOTE  rather than the usual RH% units seen in such studies, the authors use Vapour Pressure Deficit (measured in kPa). VPD is the difference between saturated and actual vapour pressure and, in effect, indicates the evaporative power of the air.  It is more informative for the purposes of this study than Relative Humidity, or how much moisture is in the air, that alone gives no indication of the rate of evaporation.  VPD is also more sensitive to temperature change than is RH.  Note that a low VPD value is least desiccatory, while a high value is most 

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