Thermotolerance and Hsp70 profiles in adult and embryonic California native oysters, Ostreola conchaphila (Carpenter, 1857).ABSTRACT The oyster Ostreola conchaphila is endemic to shallow subtidal and low intertidal in·ter·tid·al adj. Of or being the region between the high tide mark and the low tide mark. in marine waters of the West coast of North America. The heat shock response of the species was characterized for both adult and early life stages. O. conchaphila expressed proteins that were the same molecular weights as and were immunologically related to the heat shock protein heat shock protein n. Any of a group of cellular proteins that are produced under conditions of heat stress and help to stabilize other cellular proteins exposed to high temperatures. (Hsp)70 family of proteins that have been described for the oyster, Crassostrea gigas. Hsp77 and Hsp72 were present in adult and larval larval 1. pertaining to larvae. 2. larvate. larval migrans see cutaneous and visceral larva migrans. tissue. Expression of Hsp69 occurred in adults that were heat shocked at 33-38[degrees]C, regardless of season or previous environmental temperature regimen. A heat shock at 34[degrees]C induced thermotolerance to otherwise lethal temperatures of 38.5[degrees] or 39[degrees]C. Early lifestage embryos (early cleavage. late cleavage, and ciliated cil·i·at·ed adj. Having cilia. Ciliated Covered with short, hair-like protrusions, like B. coli and certain other protozoa. The cilia or hairs help the organism to move. blastula blastula /blas·tu·la/ (blas´tu-lah) pl. blas´tulae [L.] the usually spherical structure produced by cleavage of a zygote, consisting of a single layer of cells (blastoderm) surrounding a fluid-filled cavity (blastocoele). stages) were unable to express Hsp69 or mount a heat shock response. Veliger ve·li·ger n. A larval stage of a mollusk characterized by the presence of a velum. [New Latin v larvae Larvae, in Roman religion Larvae: see lemures. expressed Hsp69 after a 34[degrees]C heat shock. The increased Hsp69 persisted for at least 12 h after 1 h of heat shock as opposed to the acquisition of thermotolerance, which had declined by 12 h after heat shock. KEY WORDS: Hsp70, thermotolerance, larvae, Olympia oyster. Native oyster. Ostrea conchaphila INTRODUCTION An important component of tolerance to stress at the cellular level involves the "heat shock response." This response involves changes in the levels of several families of heal shock proteins (Hsps) or molecular chaperones that stabilize protein conformation con·for·ma·tion n. One of the spatial arrangements of atoms in a molecule that can come about through free rotation of the atoms about a single chemical bond. and function in the face of denaturing stress, including thermal stress, thus providing protection (Lindquist 1986, Gething & Sambrook 1992, Morimoto el al. 1994). Typically part of the response involves selective expression of certain inducible Hsps, which in turn can provide an enhanced tolerance beyond lethal conditions (Parsell & Lindquist 1994, Mailhos et al. 1993, 1994). Three Hsp isoforms exist: two are constitutive, Hsp77 and Hsp72, and are routinely present in cells, whereas the expression of a third, Hsp69, is induced after thermal shock (Clegg et al. 1998, Feder & Hoff man 1999). The Hsp70 family is directly implicated im·pli·cate tr.v. im·pli·cat·ed, im·pli·cat·ing, im·pli·cates 1. To involve or connect intimately or incriminatingly: evidence that implicates others in the plot. 2. in thermal tolerance of several marine invertebrates, however, the temperature at which the heat shock response is activated not only differs between species, but can also vary within species as a result of environmental or physiologic history. For example, the temperature for induction (set point) of the heat shock response in the intertidal mollusks within the genera Tegula Teg´u`la n. 1. (Zool.) A small appendage situated above the base of the wings of Hymenoptera and attached to the mesonotum. , Mytilus, and Crassostrea is a function of acclimatization acclimatization Any of numerous gradual, long-term responses of an individual organism to changes in its environment. The responses are more or less habitual and reversible should conditions revert to an earlier state. to past thermal conditions (Tomanek & Somero, 1999, Buckley et al. 2001, Hamdoun et al. 2003). And, in at least one genus, Crassostrea, there is a cost to this acclimation acclimation /ac·cli·ma·tion/ (ak?li-ma´shun) the process of becoming accustomed to a new environment. ac·cli·ma·tion n. 1. : this increase of the Hsp induction set point is not balanced by an equivalent increase in the thermal tolerance limit (Hamdoun et al. 2003). Thus, thermally acclimatized Crassostrea may be less fit to tolerate stress. The North American North American named after North America. North American blastomycosis see North American blastomycosis. North American cattle tick see boophilusannulatus. Olympia oyster, Ostreola conchaphila, has been characterized as intolerant to both tidal exposure and temperature stress. A native to the coast of western North America from Sitka, Alaska to Panama (Harbo 1997), this comparatively small (<60 mm) and slow growing species is found attached to rock or other hardened substrates in bays and estuaries extending from low intertidal to shallow subtidal depths (Baker 1995). Like other oysters of the genus Ostrea and Ostreola, fertilization and embryonic development in O. conchuphila take place in the mantle cavity of females, thus early life stages arc captive to the same environmental conditions as brooding adults. Hopkins (1937) reported that Ostrea lurida (O. conchaphila) could not withstand temperatures outside the range of 0-30[degrees]C and this limited thermal tolerance range was described as the primary constraint to geographical distribution of the species. This upper thermal limit is considerably lower than that reported for its European congener congener /con·ge·ner/ (kon´je-ner) something closely related to another thing, as a member of the same genus, a muscle having the same function as another, or a chemical compound closely related to another in composition and exerting , Ostrea edulis (40[degrees]C) or the Japanese oyster, Crassostrea gigas (43-44[degrees]C), both of which inhabit intertidal to shallow subtidal environments, but do so over a broader geographical range (Clegg et al. 1998, Piano et al. 2002). In the present report, we characterize the heat shock response for O. conchaphila adults and veliger larvae from Tomales Bay, California. We present evidence for the induced expression of Hsp 69 after thermal stress, demonstrate that the response is short-lived, but does convey thermal tolerance, and discuss the ecological significance of this response. We present evidence that the response is not present in pre-gastrula embryos and appears to be more truncated in larvae than in adults. Lastly, we addruss the question: Is the Hsp70 family expression pattern in O. conchaphila genetically fixed and how does this response compare with the phenotypic plasticity observed in C. gigas and other marine invertebrates? MATERIALS AND METHODS Animal Collection and Maintenance Adult oysters were collected from Tomales Bay, California, at a tidal height of 0-1.0 ft and transported to the Bodega bo·de·ga n. 1. A small grocery store, sometimes combined with a wineshop, in certain Hispanic communities. 2. A warehouse for the storage of wine. Marine Laboratory within 1-2 h of collection. Animals were maintained in flow-through seawater tanks with aeration aeration /aer·a·tion/ (ar-a´shun) 1. the exchange of carbon dioxide for oxygen by the blood in the lungs. 2. the charging of a liquid with air or gas. aer·a·tion n. at 12-15[degrees]C and were led an algal algal pertaining to or caused by algae. algal infection is very rare but systemic and udder infections are recorded. See protothecosis. algal mastitis the algae Prototheca trispora and P. paste daily consisting of Chaetoceros, Tetraselinis, and Isochrysis (Reed Mariculture mariculture marine aquaculture. , Inc). Collections were conducted during June through July over successive years (2000 and 2001), with one collection during December 2000 for seasonal comparison. Adults were used in heat shock experiments within 3-5 days of collection. During recovery periods after heat shock, adults were maintained in the flow-through seawater tanks and fed as described above. Embryos and larvae were obtained from brooding females collected during the summer; the reproductive season in northern California extends from May to September, during which time females brood embryos for the first 10-12 days of embryonic development (Bonnot 1935, Baker 1995). To remove the Larvae from females, the right valve of the shell was removed and the mantle cavity was rinsed with 0.45-[micro]m filtered seawater (FSW FSW Friction Stir Welding FSW Flight Software FSW Full Spectrum Warrior (video game) FSW Family Support Worker FSW Female Sex Worker FSW Fox Sports World (cable TV channel) ) over an 85-[micro]m mesh screen to filter out large debris. The filtrate filtrate /fil·trate/ (fil´trat) a liquid or gas that has passed through a filter. fil·trate v. To put or go through a filter. n. , containing embryos or larvae was then passed through a 20-[micro]m filter to collect and concentrate the larvae. Collected embryos or larvae were cultured in aerated aer·ate tr.v. aer·at·ed, aer·at·ing, aer·ates 1. To supply with air or expose to the circulation of air: aerate soil. 2. FSW, 15[degrees]C, in 20 x 11 x 6-cm glass dishes at a concentration of approximately 200 embryos per milliliter milliliter /mil·li·li·ter/ (mL) (-le?ter) one thousandth (10-3) of a liter. mil·li·li·ter n. Abbr. . All experiments with embryos and larvae were conducted within 1 day of collection during which time the larvae were cocultured with Isochrysis as a larval food source. Temperature Data Logging Tomales Bay temperature data from the oyster collection site was obtained using a Hobo[c] Tid-Bit temperature sampler that was placed at a tidal height of 0 ft. Temperature readings were recorded at 2-min intervals between May and September 2001. Heat Shock and Lethal Temperature Determination For all adult heat shock experiments, animals (n = 10) were removed from ambient seawater and placed for one hour into pre-heated seawater in a 2L beaker beaker /beak·er/ (bek´er) a glass cup, usually with a lip for pouring, used by chemists and pharmacists. beaker a round laboratory vessel of various materials, usually with parallel sides and often with a pouring spout. that was, in turn, placed in a Neslab RTE (1) See runtime engine. (2) (Real-Time Executive) The operating system used in the HP 1000 series. See HP 1000. 211 waterbath that set to within 0.1[degrees]C of the experimental temperature. Adult oysters were heat shocked at temperatures ranging in one-degree increments from 32 to 39[degrees]C. After heat shock, adults were transferred back to ambient seawater, with daily feeding, to recover. Mortality was monitored daily for two weeks Embryo and larval heat shock experiments were conducted at a concentration of 200 embryos/ml in 20-mL scintillation scintillation /scin·til·la·tion/ (sin?ti-la´shun) 1. an emission of sparks. 2. a subjective visual sensation, as of seeing sparks. 3. vials by suspending the vials in the Neslab RTE211 water bath for one hour at the appropriate temperature. Four stages of development, early cleavage, late cleavage, ciliated blastula, and veliger larvae were used in the embryo/larval heal shock experiments. Embryos and larvae were provided a 24 h post-heat shock recovery period (monitored at 4 h, 12 h. and 24 h) at 15[degrees]C, then percent mortality was scored using a dissecting microscope. The criteria for viability vs mortality was stage dependent; early cleavage embryos were considered alive if cleavage had continued post-heat shock, while motility motility /mo·til·i·ty/ (mo-til´ite) the ability to move spontaneously.mo´tile Motility Motility is spontaneous movement. was used as the criteria for viability in embryos/larvae in the other three stages. Hsp70 Determinations The presence and quantities of Hsp70 proteins were determined by using oyster proteins separated by sodium dodecyl sulfate Sodium dodecyl sulfate (or sulphate) (SDS or NaDS) (C12H25NaO4S),is an anionic surfactant that is used in household products such as toothpastes, shampoos, shaving foams and bubble baths for its thickening effect and its ability to polyaco, lamide gel electrophoresis (SDS-PAGE SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ) with an Hsp70 monoclonal antibody monoclonal antibody, an antibody that is mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies are typically made by fusing a normally short-lived, antibody-producing B cell (see immunity) to a fast-growing . All procedures for sampling tissue and preparation for Hsp analyses were conducted according to Clegg et al. (1998) with modifications after Hamdoun et al. (2003). Briefly, gill samples were excised 48 b postheat shock, weighed, frozen in liquid nitrogen, and stored al -80[degrees]C. For electrophoresis, the pre-weighed gill tissue was placed in potassium gluconate potassium gluconate Potassium-Rougier (CA) Pharmacologic class: Mineral, electrolyte Therapeutic class: Electrolyte replacement, nutritional supplement Pregnancy risk category C buffer (KGB KGB: see secret police.KGB Russian Komitet Gosudarstvennoy Bezopasnosti (“Committee for State Security”) Soviet agency responsible for intelligence, counterintelligence, and internal security. : 5 mM MgS[O.sub.4], 5 mM Na[H.sub.2]P[O.sub.4], 40 mM Hepes, 70 mM gluconic acid gluconic acid /glu·con·ic ac·id/ (gloo-kon´ik) the hexonic acid derived from glucose by oxidation of the C-1 aldehyde to a carboxyl group. glu·con·ic acid n. , 150 mM sorbitol sorbitol /sor·bi·tol/ (sor´bi-tol) a six-carbon sugar alcohol from a variety of fruits, found in lens deposits in diabetes mellitus. , pH 7.5) at a concentration of 100 mg wet tissue per 1 ml of KGB. Samples were then homogenized ho·mog·e·nize v. ho·mog·e·nized, ho·mog·e·niz·ing, ho·mog·e·niz·es v.tr. 1. To make homogeneous. 2. a. To reduce to particles and disperse throughout a fluid. b. and diluted 1:1 with 2X SDS-PAGE sample buffer and heated to 100[degrees]C for 5 min. Volumes of solubilized gill were adjusted so that equivalent gill weights for each sample were loaded onto a 10% polyacrylamide gel pol·y·a·cryl·a·mide gel n. A hydrated polymer consisting of a long chain of amide groups, used as a medium for substances that undergo gel electrophoresis. , the proteins separated by SDS-PAGE, and protein bands subsequently transferred to nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp. . Note: gels were electrophoresed for 30 min after the tracking dye had migrated to the bottom of the gel; this provided separation of the Hsp70 isomers isomers (ī´sōmurz), n.pl 1. organic compounds having the same empirical formula–i.e. . Western blots were probed with a rat anti-HSP70 monoclonal antibody (MA3-001, Affinity Bioreagents) followed by a goal anti-rat horseradish peroxidase horse·rad·ish peroxidase n. An enzyme used in immunohistochemistry to label the antigen-antibody complex. conjugated conjugated adj. Conjugate. estrogens, conjugated Warning - Hazardous drug! C.E.S. secondary antibody (Sigma). Labeled proteins were detected using chemiluminescence chemiluminescence /chemi·lu·mi·nes·cence/ (kem?i-loo?mi-nes´ens) luminescence produced by direct transformation of chemical energy into light energy. (ECL (Emitter-Coupled Logic) A digital circuit composed of bipolar transistors in which the emitter ends are wired together. ECL gates switch faster than TTL gates, but consume more power. See TTL, I2L and bipolar. 1. , Pierce) in a UVP UVP Under Voltage Protection UVP Unique Value Proposition UVP Ultrasonic Vibration Potential UVP Ultraviolet Peroxide Epi Chemi II darkroom darkroom, n a completely lightproof room or cubicle that is used in the processing of photographic, medical, and dental films. See also safe light. . Densitometry densitometry /den·si·tom·e·try/ (den?si-tom´i-tre) determination of variations in density by comparison with that of another material or with a certain standard. of the bands on western blots was conducted using Laboratory Works image acquisition and analysis software (UVP Bio-imaging Systems). Larvae were sampled by removing 15-100 mg wet weight per time point from the culture vial and were concentrated on a 50-[micro]m nitex mesh screens. The larvae were washed with FSW, pelleted, frozen in liquid nitrogen, and stored at -80[degrees]C until all samples were collected. Prior to SDS-PAGE analysis, the larval pellets were resuspended in hypotonic hypotonic /hy·po·ton·ic/ (-ton´ik) 1. denoting decreased tone or tension. 2. denoting a solution having less osmotic pressure than one with which it is compared. lysis buffer (HLB HLB Hong Leong Bank HLB Hydrophilic-Lipophilic Balance HLB Horton Lees Brogden Lighting Design (company with studios in New York, San Francisco, Los Angeles, and Boston) HLB Hotels Licensing Board (Singapore) : 10 mM KCl, 1.5 mM Mg[Cl.sub.2], 10 mM Tris-HCl, pH 7.4) with protease inhibitors Protease Inhibitors Definition A protease inhibitor is a type of drug that cripples the enzyme protease. An enzyme is a substance that triggers chemical reactions in the body. to 200 mg/mL, sonicated and then diluted 1:1 with 2X SDS-PAGE sample buffer and analyzed as stated above for the adults. INDUCED THERMOTOLERANCE Induced thermotolerance experiments followed procedures outlined in Clegg et al. (1998) with adjustments for species temperature tolerance differences. Adults received a sublethal sublethal /sub·le·thal/ (-le´thal) insufficient to cause death. sub·le·thal adj. Not sufficient to cause death. beat shock (34[degrees]C, 1 h) and were allowed to recover at ambient for 24 h prior m a 1-h incubation at the preheat shock lethal temperature (38.5 or 39[degrees]C). Veliger larvae were subjected to a sublethal heat shock (34[degrees]C, 1hr) and subsequently incubated at the pro-heat shock lethal temperature (39[degrees]C) at 6, 8, or 12 h postsublethal heat shock. Controls for both adults and larvae were exposed to 38.5 or 39[degrees]C without a prior sublethal heat shock treatment. Survival of adults was assessed over a 3 wk period and larvae survival was monitored over a 24 h period. Data Analysis Protein levels were analyzed for statistical significance using one-way analysis of variance (Sigmastat software v2.03). RESULTS Collection-Site Temperature Profiles The temperature profiles at oyster collection sites in Tomales Bay for the 2 weeks leading up to each sampling are summarized in Figure 1. The profiles differed not only between winter and summer but also slightly between the two summer sampling periods. During the winter sampling, temperatures ranged from 0.7 to 16.5[degrees]C; however, greater than 90% of the degree-hours (the number of hours at a particular temperature) for the 2-week period fell within a 3-degree range, 8-10[degrees]C (Fig. 1). During the summer there was more variation in water temperature: the ranges were 12.629.6[degrees]C and 6.6-26.0[degrees]C for 2000 and 2001, respectively. The temperature ranges that encompassed 90% of the degree hours for the 2 week summer periods were also broader, 16-20[degrees]C and 14-20[degrees]C, respectively. Lethal Temperature Determination The lethal temperature fur O. conchaphila adult and larval life stages is between 38[degrees]C and 39[degrees]C. No adult oysters survived heat shock for 1 h at 39[degrees]C, and the range of survival subsequent to a 1 h exposure to 38[degrees]C was 50-87%, with no statistically significant seasonal difference (P > 0.1; Figure 2A). Brooded veliger larvae (from summer collections) mimicked the adult pattern; 70.7 [+ or -] 12.01% SD (range of 59-83%) of larvae survived a heat shock of 38[degrees]C while only 21.3 [+ or -] 5.7% SD (range of 15-26%) survived 39[degrees]C (Fig. 2B). [FIGURE 2 OMITTED] Adult Hsp Protein Profiles Ostrea conchaphila expresses the Hsp70 family of heat shock proteins: constitutive (Hsp77 and Hsp72) and inducible (Hsp69) forms co migrated with and were immunologically similar to the Hsp isomers described for C. gigas and O. edulis (Fig. 3; Clegg et al. 1998, Piano et al. 2002). Oysters maintained in ambient seawater (15[degrees]C) and individuals that were assayed 60 min after heat shock (60 min at 34[degrees]C) were indistinguishable in Hsp70 profiles. Both possessed Hsp72 and Hsp77 (Fig. 3). By 24 h post heat shock, however, the inducible isoform, Hsp69, as well as the two constitutive Hsp70s, were present (Fig. 3). The lowest heat-shock temperature at which Hsp69 was consistently induced ([T.sub.on]) was 34[degrees]C regardless of season, whereas the highest heat-shock temperature ([T.sub.off]) at which Hsp69 was consistently seen was 38[degrees]C (39[degrees]C was the lethal temperature), also regardless of season (Fig. 4). Hsp69 was maximal (assayed at 48 h post heat shock) when the heat shock temperature was between 35 and 38[degrees]C during the summer and 32-38[degrees]C during the winter, but there was no significant difference in the level of HspP69 expression for specific heat shock temperatures between seasons (Fig. 4). Likewise, the overall levels of the constitutive forms, Hsp72 and Hsp77, did not follow seasonal patterns, nor did they change significantly as a result of heat shock. [FIGURES 3-4 OMITTED] Once induced, Hsp69 remained present in adults for at least 3 days, but was maximal at the 24 and 48 h post-heal shock sampling times (Fig. 5). Quantities of Hsp69 were statistically indistinguishable at 24 and 48 h within all seasonal samples, and were significantly reduced by day 3 in two of the collections, summer, 2000 and winter, 2001. In all collections there was a significant decline in Hsp69 from 24-48 h post-heat shock levels to the 3-7 day levels (P < 0.05; Fig. 5). And, although not statistically significant, this declining trend was evident between 24 and 48 h and between 3 and 7 days in all collections (Fig. 5). [FIGURE 5 OMITTED] Long-Term Survival of Adults After Induced Thermotolerance Heat shock did induce thermotolerance in O. conchaphila but only over a narrow time span. Initial experiments showed that tolerance to the naive lethal temperature of 39[degrees]C was present at 24 h post-sublethal heat shock but not at 48 h None of the oysters that were heat shocked at 34[degrees]C and then exposed to 39[degrees]C at 48 h post-heat shock survived a week, whereas 100% that were exposed to 39[degrees]C at 24 h post heat shock, survived. Hsp69 levels did not reflect the decline in acquired thermal tolerance; at 24 and 48 h Hsp69 levels were not significantly different (P > 0.05; see Fig. 5). Only in one collection (summer 2000) was there a significant difference between Hsp69 levels at 3 days post-heat shock (Fig. 5). Because the duration of induced thermal tolerance was much shorter than that reported for other oysters (Clegg et al. 1998), we investigated whether long-term survival was compromised by post heat-shock exposure to lethal temperatures. Animals that were exposed to 38.5 or 39[degrees]C at 24 h after a 34[degrees]C sublethal heat-shock exhibited temperature dependent survival. Pre-heat shock animals that were placed directly from 12[degrees]C into lethal temperature seawater, 38.5 or 39[degrees]C. died also in a temperature dependent manner over the course of the experiment. All naive adults that were exposed to 39[degrees]C died within 6 days while those exposed to 38.5[degrees]C perished over tire course of 20 days, 36.7 [+ or -] 19.6% (SE) survived 10 days and 20.7 [+ or -] 10.4% survived to 20 days (Fig. 6A). In contrast, all of the adults that were heat shocked at 34[degrees]C prior to 38.5 or 39[degrees]C exposure at 24 h post-heat shock, survived 6 days (Fig. 6B). After 20 days 88.7 [+ or -] 5.7% of adults subjected to 38.5[degrees]C and 66.7 [+ or -] 9.5% of those exposed to 39[degrees]C survived, significantly higher survival than in the nonheat shock groups (P < 0.05. Fig. 6). Survival of the oysters exposed to the 38.5[degrees]C lethal temperature post-heat shock was significantly higher than that of the 39[degrees]C group after 20 days (P < 0.05). [FIGURE 6 OMITTED] Larval Heat Shock Response All four developmental stages, early cleavage, late cleavage, blastula, and prerelease pre·re·lease n. Something released before an official or scheduled date. adj. Of or relating to an interval preceding an official or scheduled release: veliger larva larva, in zoology larva, independent, immature animal that undergoes a profound change, or metamorphosis, to assume the typical adult form. Larvae occur in almost all of the animal phyla; because most are tiny or microscopic, they are rarely seen. , contained constitutive Hsp77 and Hsp72; however, only the veliger larval stage possessed Hsp69 after heat shock (Fig. 7). Hsp69 was present in veliger larvae by 6 h post-heat shock and had increased by a factor of four at 12 h post-heat shock (Fig. 8). In contrast to the temporal increase in Hsp69 over the first 12 h post-heat shock, thermaltolerance decreased (Fig. 9). Heat shocked larvae were more tolerant to a 39[degrees]C post-heat shock treatment at 6 h after heat shock than at 12 h post-heat shock. [FIGURES 7-9 OMITTED] DISSCUSSION The preheat shock and post-heat shock thermal tolerance profiles of Hsp70 isoforms in O. conchaphila adults are similar to that reported for a closely related oyster, Ostrea edulis. (see Pirano et al. 2002). Adults of O. conchaphila exhibited a pre-heat shock thermal limit between 38 and 39[degrees]C, whereas the thermal ceiling in O. edulis, was between 39 and 40[degrees]C (Pirano el al. 2002). Both species initiate expression of inducible Hsp69 at 32-33[degrees]C. and express Hsp69 maximally at 2448 h post beat shock (Figs. 2 and 5: Pirano et al. 2002). In the present study we have shown that exposure of O. conchaphila, to a sublethal temperature high enough to induce Hsp69 expression also induces tolerance to the pre-heat shock lethal temperature of 39[degrees]C that lasts for 24h. This induced thermal tolerance is not as dramatic as that seen in either Crassostrea or Mytilus species and, in part, explain the comparatively restricted geographical and bathymetric ba·thym·e·try n. The measurement of the depth of bodies of water. bath  y·met distribution of O. conchaphila Hopkins
1937).
Research into the heat shock response of intertidal molluscs supports the concept of thermal plasticity. Both Crassostrea and Tegula modulate temperature tolerance ranges anti thresholds of Hsp production based on acclimation to changes in environmental conditions such as seasonal temperature shifts or tidal height exposure (Roberts el al. 1997, Tomanek & Somero 1999, Buckley et al. 2001, Hamdoun et al. 2002). Hamdoun et al. (2003) demonstrated that seasonal increases in ambient temperature led to elevated levels of constitutive Hsps (Hsp77 and Hsp72), and to seasonal increases (2-3 degrees) in pre-heat shock thermal tolerance. Thus, C. gigas is capable of adjusting its thermal response to accommodate baseline environmental conditions. Ostreola conchaphila did not exhibit a significant seasonal change in constitutive Hsp levels, nor was there a shift in Hsp69 [T.sub.on] or change in the upper thermal tolerance limit although the mean summer temperature was twice that of the mean winter temperature (18.3 and 17.3[degrees]C vs 9.4[degrees]C). It appears that O. conchaphila, does not possess the capability of adapting to seasonal temperature shifts that is exhibited by other intertidal bivalves examined to date (Tomanek & Somero, 1999, Buckley et al. 2001, Hamdoun el al. 2003), reinforcing the idea that the distribution of Ostreola is primarily temperature determined. Ostrela conchaphila are protandic hermaphrodites Hermaphrodites half-man, half-woman; offspring of Hermes and Aphrodite. [Gk. Myth.: Hall, 153] See : Androgyny that exhibit a larviparous form of reproduction. Embryonic and early larval stages are brooded within the mantle cavity of female stage adults during the late spring and early summer, after which larvae are released as planktotrophic veligers (Santos 1992). Developmental stages arc thus captive to temperature conditions experienced by brooding adults, temperatures that can approach adult lethal limits (Korringa 1976b). The fact that the lethal temperature for early life stages of O. conchaphila is the same as that for adults (38.5-39[degrees]C) may be an adaptation to survive captive (brooded) developmental periods. Typically early life stages are less tolerant of environmental stresses than is the adult life stage. Our results show that embryonic stages prior to gastrulation Gastrulation The formation of the primordial gut, the archenteron, or digestive cavity of an early animal embryo. More generally, and originally, the term gastrulation referred to the process by which the gastrula stage of the embryo is formed. (early cleavage, late cleavage, ciliated blastula) were not able to mount a heat shock response, but that the veliger larvae were able to do so. This developmental stage discrepancy to mount a heat shock response indicates that the embryonic genome is not transcriptionally active during early developmental stages and that maternal message for Hsp69 is not present as has been described for other species (e. g sea urchin; for a review see Giudice et al. 1999). Interesting differences were observed between induced thermal tolerance and the presence of Hsp69 in veliger larvae that did not parallel tolerance and Hsp69 patterns seen in adults. While the quantity of Hsp69 in veliger larvae increased for the first 12 h post-heat shock, the ability to survive the naive lethal temperature (39[degrees]C) after heat shock declined between 6 and 12 h This could be explained by either a disconnect between the presence of Hsp69 and thermal tolerance in veliger larvae or by a general decline in condition, not necessarily heat shock associated, that lessens a larvae's ability to withstand stress. Larvae that are pre-maturely removed from brooding females, as was the case in the present study, typically do not survive to settlement (unpublished observations). Thus, young veliger larvae appear to require the environment of the mantle cavity; seawater is not sufficient for development until larvae have matured sufficient for natural release. Whether differences in the relationship between Hsp69 levels and thermaltolerance observed between adults and veliger larvae represent inherent life stage-specific physiologic states or arc related to early removal of larvae from the mantle cavity is not known. To delineate which is operative, the heat shock response of brooded larvae for which the parent has been heat shocked prior to larval release needs to be investigated. Regardless of the duration of the heat shock response and relationship to Hsp69 levels, larvae that possess Hsp69 do produce a heat shock response that is within the same tolerance range as adults.
Figure 1. Temperature profile for Tomales Bay. Hobo samplers were
used to collect temperatures at the tidal height and location of O.
conchapila for a 2-week period prior to each collection. A, temperature
range (range), mean, minimum (min), and maximum (max) temperatures
were calculated for each site. B, In addition, the total number number
of hours at each temperature (rounded to the nearest [degrees]C) over
the 2-week period was calculated.
Summer Winter Summer
2000 2001 2001
Mean ([degrees]C) 18.3 9.4 17.2
Range ([degrees]C) 17.0 15.7 19.4
Min ([degrees]C) 12.6 0.7 6.6
Max ([degrees]C) 29.6 16.5 26.0
ACKNOWLEDGMENTS The authors thank Karl Menard (Bodega Marine Lab) and Eric Larson (California Department of Fish and Game) for aid in obtaining adult O. conchaphila. The research was supported by the City of San Francisco
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Evolutionary and acclimation induced variation in the heat shock responses of congeneric marine snails (Genus Tegula) from different thermal habitats: implications for the limits of thermotolerance and biogeography Biogeography A synthetic discipline that describes the distributions of living and fossil species of plants and animals across the Earth's surface as consequences of ecological and evolutionary processes. . J. Exp. Biol 202:2925-2936. HEATHER M. BROWN, (1,2) ALISON BRIDEN, (1) TARYN STOKELL, (1) FRED J. GRIFFIN (1) * AND GARY N. CHERR (1,3) (1) Bodega Marine Laboratory, PO Box 247, Bodega Bay CA, 94923 (2) Current address: Department of Molecular, Cellular and Developmental Biology Developmental biology A large field of investigation that includes the study of all changes associated with an organism as it progresses through the life cycle. The life cycles of all multicellular organisms exhibit many similarities. , University of California, Santa Cruz The University of California, Santa Cruz, also known as UC Santa Cruz or UCSC, is a public, collegiate university, one of the ten campuses of the University of California. , CA, 95064 (3) Departments of Environmental Toxicology and Nutrition, University of California The University of California has a combined student body of more than 191,000 students, over 1,340,000 living alumni, and a combined systemwide and campus endowment of just over $7.3 billion (8th largest in the United States). Davis, Davis CA, 95616 * Correspondence: fgriffin@ucdavis.edu |
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