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INTRODUCTION
The rocky intertidal communities, modeled by the interaction between biotic and abiotic pressures (Silliman et al., 2011; Menge & Branch, 2001; Bertness et al., 2006; Silliman et al., 2011; Rechimont et al., 2013), undergo a gradient of physical factors between marine to terrestrial conditions (Palomo et al., 2019) determined by the height of the tides (Denny & Wethey, 2001).
Northern Patagonia rocky intertidal habitats are considered within the world’s most stressful, particularly because of the high desiccation rates (Bertness et al., 2006). These shores, subjected to the unobstructed and persistent dry winds of “The Roaring 40 s” (Crespi et al., 2018), present a low annual rainfall (180 mm/yr) and low humid ity (40%) (Bertness et al., 2006). In Patagonia, tides are semi-diurnal, with amplitudes of more than 8 m, reaching 12.5 m in some sites, exposing the intertidal for hours (Boraso de Zaixso & Zaixso, 2015). Hence as a result of the individual adaptations to these abiotic forces, the individuals present a specific position along the intertidal (biotic zonation) that determines the abundance, diversity, biomass, and species richness (Menge & Branch, 2001; Raffaelli & Hawkins, 2012).
Thermal tolerance is decisive in intertidal habitat selection (Somero, 2002). Ectotherms can be particularly susceptible to temperature varia tions (Porter & Gates, 1969), highlighting the importance of strategies to avoid desiccation. In this sense, sun-exposed and shaded microhabitat selection is extremely important for some species in terms of their distributional limits (Tomanek & Sanford, 2003; Pöhlmann et al., 2011). One important mechanism against a variety of stress conditions during tidal emersions, such as tem perature and oxygen deficiency, is the heat-shock response. It describes the activation of heat-shock proteins (Hsp), which act as chaperones stabiliz ing and preventing cytotoxic protein aggregates (Lindquist, 1986; Tomanek & Sanford, 2003; Jeno & Brokordt, 2014). Also, Hsp expression is a physiological defense indicator against endog enous proteins denaturation (Feder & Hofmann, 1999). Mollusks, being ectotherms, are excellent models for studying thermal tolerance and moni toring changes in environmental temperature. The expression of Hsp, under the action of different stressors, has been studied in several mol lusk species such as Ostrea edulis (Piano et al., 2005), Placopecten magellanicus, Argopecten ir radians (Brun et al., 2008), Mytilus galloprovin cialis (Franzellitti & Fabbri, 2005), Concholepas sp. (Roco, 2010), Haliotis tuberculata (Farcy et al., 2007) and Tegula sp. (Tomanek & Somero, 1999), observing an increase in its synthesis un der heat stress conditions.
The mollusks Tegula patagonica (d’Orbigny, 1835) and Trophon geversianus (Pallas, 1776) are abundant organisms that inhabit rocky ar eas along the Argentine coasts. Tegula patagonica is distributed from southern Brazil (23°03’ S; 43°21’ W) to Tierra del Fuego (54°48’30” S; 68°18’30” W) province in Argentina (Pastorino, 1994), mainly inhabiting the shallow subtidal and the low intertidal. On the other hand, T. gever sianus is recorded from the province of Buenos Aires (38°00’ S; 57°32’ W) to the Burdwood bank (54°30’ S; 60°30’ W) in the Namuncurá Reserve in the southern Atlantic Ocean (Griffin & Pastorino, 2005; Pastorino, 2005) and up to 42° latitude south in the Pacific Ocean, from mid in tertidal to shallow subtidal. Over the lower tides in the rocky shores, T. patagonica is a dominant herbivorous, essential in the ecosystem dynam ics (Miloslavich et al., 2016; Avaca et al., 2021; Nieto-Vilela et al., 2021), who scraps the biofoul ing adhered to laminar algae (Teso et al., 2009). In the mid intertidal areas, T. geversianus is one of the key predators in the community (Andrade & Ríos, 2007), being mussels their principal re source (Rechimont et al., 2013). Both gastropods are distributed on different intertidal levels (mi crohabitats) of the same rocky shore, while T. patagonica inhabits the low zone (Rechimont et al., 2013) and remains motionless during exposure to air, T. geversianus inhabits the mid intertidal level, seeking shelter in crevices during air ex posure.
There is extensive evidence about stressors in Patagonian intertidal habitats (Bazterrica et al., 2007; Bertness et al., 2006; Silliman et al., 2011). However, we have poor information about the strategies that organisms have to deal with them. The present study is intended to understand the responses of T. patagonica and T. geversianus to heat stress, as an input to field monitoring in a climate change scenario. Understanding the dynamic among heat stress and the organism responses can provide baseline information to monitor the impact of climatic change on South Atlantic rocky shores. We measured the heat-shock response, by assessing Hsp70 expression in the foot and gonad tissues of both rocky inter tidal species.
MATERIAL AND METHODS
Sampling site
Punta Loma, a Protected Area (42°48’ S; 64°53’ W) near Puerto Madryn, Patagonia Argentina (Fig. 1), presents semidiurnal tides. The intertidal zone is exposed to terrestrial con ditions for around 6 hours each tide cycle. The low, middle, and high intertidal levels are ex posed up to 2, 4 and 6 hours, respectively (Boraso de Zaixso & Zaixso, 2015). Air temperature presents an annual mean of 13.4 °C with an annual amplitude reaching 40 °C and daily variations of up to 30 °C (Rechimont et al., 2013). The year ly mean wind speed registered is 15±2.1 km/h, with extreme historical records higher than 90 km/h (EMA, Last visit 2019). The sea surface temperature presents an annual mean of around 13.5 °C, with maximum values of 20 °C at the end of the summer, and a minimum of 8 °C during the spring (Dellatorre et al., 2012).
To characterize the variation of air tempera ture in the intertidal, a digital temperature re corder was located in the middle zone, collecting data continuously between the summer seasons of 2018 and 2019 with a 60 minute interval and a 0.01 °C resolution. To verify if the air tem perature recorded influences the shell surface temperature of T. patagonica and T. geversianus gastropods, thermal images (n=18, nine pictures of a single individual of each species) were taken using a FLIR C2 thermal camera, in one extreme event with temperatures higher than 30 °C (Supplementary Figure 1). The images were taken over randomly visualized adults, follow ing the published mature sizes (Cumplido et al., 2010; Nieto-Vilela 2014). The images were then analyzed with the FLIR Tools software register ing, in each image, the mean temperature in the same defined body area.
Thermal stress experiments
Hsp70 expression was assessed in adults of T. patagonica (n=10) and T. geversianus (n=10), collected during summer 2018 from the low and mid intertidal levels. Snails were acclimated for 10 days at 12 °C in aquaria within a tempera ture-controlled room under a 12:12 h photope riod. Biofilm was used for feeding T. patagonica, and scorched mussels (Perumytilus purpuratus and Brachidontes rodriguezii) for T. geversianus. Aquaria were controlled daily, regarding animal conditions, feeding rate, water pH, temperature, salinity and oxygen levels. After the acclimatiz ing period, animals were randomly exposed to two experimental temperatures in seawater 200 ml jars (Tomanek & Somero, 2000): the control treatment T1=14 °C (similar to the annual aver age water temperature in the site 13.5 °C), and T2=20 °C, (resembling the maximum tempera ture recorded in the water of the study site, http://www.hidro.gov.ar/; Acker & Leptoukh, 2007). Experimental temperatures were increased by 1 °C every 1 h up to 14 °C or 20 °C respectively, adapting previous methodologies (Tomanek & Somero, 1999; Tomanek & Zuzow, 2010). After 48 h of treatment, each individual was immedi ately frozen at -80 °C until further processing.
Cell line
Human T47D cells were obtained from ATCC and maintained in DMEM/F12 without phenol red (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin with 10% fetal bovine serum (Gibco).
Hsp70 determination
Total cell extracts from T47D cells were pre pared by using RIPA lysis buffer including pro tease inhibitors (Giulianelli et al., 2019). Two tissues with different exposure to temperature and clear heat-shock response were selected: foot, the more exposed tissue and gonad, the less exposed tissue in the same stage of maturation (Lima et al., 2016; Smolina et al., 2016; Nieto-Vilela 2020; Sukhan et al., 2021). Around 20 mg of tissue were dissected from each animal and homogenized in ice-cold TEDGS 10% lysis buf fer (50mM Tris pH=7.4, 7.5mM EDTA, 0.5mM dithiothreitol, 10% glycerol, 0.25M sucrose), in cluding protease inhibitors. Total protein con centration was determined by Lowry method (Lowry et al., 1951). Equivalent amounts of pro tein (100 μg) from tissue lysates were separated on discontinuous polyacrylamide gels and de tected by Western Blot (Giulianelli et al., 2020). Briefly, protein samples were boiled for 5 min in 4X Laemmli buffer (250mM Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 8% beta-mercaptoethanol, 0.02% bromophenol blue) and protein separa tion was performed by electrophoresis on SDS-polyacrylamide gels (5% for stacking and 10% for the resolving gels, respectively, using the Mini-PROTEAN systems, BioRad). To allow quantita tive comparison among samples from different gels, we prepared a ‘standard sample’ by mix ing aliquots from all tissue lysates (Lima et al., 2016). The human T47D cell line was used as a positive control. Proteins were transferred to 0.45 μm nitrocellulose membranes (Santa Cruz Biotechnology). After transfer, membranes were blocked by 0.5% nonfat dried milk in TBS-T so lution and then incubated with Hsp70 (sc-33575, 1:600) or β-Actin (sc-47778, 1:600) antibodies (Santa Cruz Biotechnology) overnight at 4 ºC. The primary Hsp70 antibody recognizes both the cognate (Hsc70) and inducible (Hsp70) forms of mammalian Hsp70 with demonstrated cross-reactivity with Hsp70 present in other mollusk species (Long et al., 2015; Arribas et al., 2021). Membranes were then washed with TBS-T so lution, and incubated with peroxidase-conjugat ed secondary antibodies (Vector Laboratories, 1:4000). The luminescent signal was generated by a luminol-based method (Sigma), and the blots were exposed to a medical X-ray film (AGFA). Finally, the band intensities were determined by densitometry using ImageJ software (Schneider et al., 2012). Hsp70 levels in each sample were normalized related to β-Actin (loading control) expression (Nakano & Iwama, 2002; Evans & Somero, 2010; Choi et al., 2018; Giulianelli et al., 2020). Then, normalized values were quantified relative to the optical density of the standard sample band previously assessed for each gel.
RESULTS
The annual air mean temperature in the in tertidal for 2018 and 2019 was 13.99±4.79 °C with oscillations of 41.5 °C (Fig. 2A). The highest value registered was 36.3 °C in January, and the minimum was -4.7 °C in August. The highest air thermal amplitude recorded during the same day was more than 20 °C in February. Moreover, dur ing the summer (January-March) there was 32 °C of thermal variation. In addition, the monthly and seasonal mean temperatures recorded for each year were included as Supplementary Tables 1 and 2.
Fig. 2 Annual air temperature in the rocky intertidal of Punta Loma recorded during 2018 and 2019 and body temperatures of T. patagonica and T. geversianus after thermal images analysis. (A) Hourly temperature registered by thermal recorder placed in the middle intertidal level. The oscillation curve formed by the high density of points represents the time when the tide was high. (B) and (C) Thermal images (top) and associated digital pictures (bottom) of T. patagonica (scale bar: 1cm) and T. geversianus (scale bar: 2.5 cm), respectively, obtained at the middle intertidal level of Punta Loma area. (D) Average body temperature recorded for each species. ***, p<0.001.
The shell surface temperature that each gas tropod species undergoes was always lower than the air temperature. In the field T. patagonica were found exposed to the sun, whereas T. ge versianus were always found in shaded crevices (Fig. 2 B and C). A higher body temperature was observed in T. patagonica in comparison with T. geversianus (Fig. 2D).
Thermal induction of heat-shock protein ex pression
Hsp70 was detected in gonads and the foot of T. patagonica and T. geversianus. Immunoblots showed a single protein band at approximately 70 kDa, with a migration pattern similar to that of the human Hsp70 isoform (Fig. 3). Hsp70 was upregulated, approximately 3.8-fold in gonads and 2.2-fold in foot in T. geversianus snails exposed to 20 °C for 48 h, relative to the control expression (14 °C; Fig. 3A). No differences in Hsp70 expression were found in T. patagonica gonads or foot after water temperature increase (Fig. 3B). These results indicate a different dy namic in the Hsp70 expression in response to the water temperature increase in our experimental setting between both studied species.
Fig. 3 Effect of temperature on Hsp70 expression of T. geversianus and T. patagonica. T. geversianus (A) and T. patagonica (B) western blot of Hsp70 expression from total extracts of gonads (left) and foot (right) after 48 h of thermal stress. The band intensity ratios of Hsp70 expression relative to β-Actin were plotted. Human T47D cell extracts were used as positive control. A representative western blot using two individual samples for each temperature treatment is shown. **, p<0.01.
DISCUSSION
Our work is a first approach to study the physiological response to temperature in two widely distributed intertidal gastropods from Atlantic Patagonian rocky shores, where a dif ferent sensitivity is shown in species inhabiting distances less than a meter.
Temperature fluctuation registered through out the year and even during the same day reveals the importance of thermal regulation investment in intertidal inhabitants. Air temperature, which is a crucial factor for ectotherms physiology (Hofmann, 1999), would seem to affect the shell surface temperature of both species in different ways. Trophon geversianus and T. patagonica could be exposed to thermal variations of more than 20 °C during the same day. Thermal imag ing taken during an extreme heat event showed a difference in body temperature between both gastropod species, suggesting that T. patagonica might be more affected by high temperatures than T. geversianus. Future contributions will corroborate whether it is possible to use thermal imaging as a rapid indicator for tissue damage or heat stress before physiological analysis of bio chemical indicators or even lethal response.
A common strategy in sessile invertebrates to stay wet during low tides is to tightly close their shells (Pöhlmann et al., 2011). While for mobile invertebrates, achieving protected microenvi ronments is a well-documented strategy to avoid desiccation (Bertness et al., 2001). Nevertheless, in the present study, we found that T. geversian us remains hidden in crevices during the hottest periods, whereas T. patagonica remains exposed. In this case, the use of microhabitats for sun pro tection maintained lower body temperatures in T. geversianus, and could be described as an ef fective strategy to avoid desiccation.
In terms of physiological response to water temperature increase, our experimental results showed basal levels of Hsp70 expression in T. ge versianus, which were upregulated when expo sure temperature reached 20 °C, independently of the examined tissue. Conversely, T. patagonica was unable to increase Hsp70 synthesis after 48 h at 20 °C. Studies made with congeners of Tegula on different intertidal levels showed that T. funebralis was able to activate and complete the heat-shock response faster than its conge ner from the low-intertidal to subtidal species, T. brunnea (Tomanek & Somero, 2000). This fast reaction was described as a response for miti gating the unpredictable physical conditions in the intertidal against periods of heat stress. In the theory of “preparatory defense”, described for congeners of Lottia by Dong et al. (2008), the fully sun-exposed species (L. scabra and L. austrodigitalis) in the high intertidal zone, ex hibited high constitutive levels of Hsp70, while only L. austrodigitalis exhibited a high inducible synthesis of Hsp70 at extreme temperature. The species that inhabit the lowest intertidal zone (L. scutum and L. pelta) showed low levels of Hsp70, being still able to respond to heat stress by in creasing the synthesis of Hsp70. Furthermore, these results are in line with those found in the dogwhelk Nucella canaliculata, where the sub populations exposed to more severe environ mental conditions showed a pattern of inducible Hsp70 (Sorte & Hofmann, 2004). Our results suggest that the muricid T. geversianus, inhabit ing the middle intertidal and exposed longer to aerial conditions at low tides (Boraso de Zaixso & Zaixso, 2015), might have a successful heat stress defense strategy, maintaining a constitutive level of Hsp70 and responding to water temperature increase by Hsp70 upregulation. Contrarywise, given that T. patagonica is very common in shal low waters, up to 30 m depth, and it is scarce in the low intertidal (Rechimont et al., 2013), it would indicate that the species is more adapted to subtidal stability than intertidal habits. The T. patagonica population exposed to the current ex perimental conditions did not show an effective thermal defense by Hsp70 upregulation, suggesting a greater influence by the environmental temperature.
The heat-stress response evoked by T. gever sianus was evident at behavioral level, remaining hidden in crevices during high air temperature events, and also at physiological level increasing Hsp70 expression. Even though T. patagonica re mains exposed during low tides with high body temperatures, we could not find a physiological heat-stress response.
Our results are reference information that could be useful for future monitoring of global warming effects on marine biota, highlighting the importance of analyzing behavioral and physiological sentinels. Future experiments would help us to thoroughly explore the strategies of this keystone species. Keeping in mind the eco logical importance of both species studied in this work, T. patagonica as a leading herbivorous and T. geversianus as a key predator in the commu nity, inhabiting the subtidal and the low inter tidal level, we proposed both species as sentinels of climate changes monitoring in Southwestern Atlantic coasts.
Supplementary Information. Supplemental material for this article can be accessed here: http://revista.macn.gob.ar/ojs/index.php/RevMus/rt/suppFiles/778/0
