Current Zoology, 2017, 63(5), 487–493 doi: 10.1093/cz/zow110 Advance Access Publication Date: 7 January 2017 Article Article Limited proteomic response in the marine snail Melarhaphe neritoides after long-term emersion a,b a,c a,b Angel P. DIZ , Margarita A LVAREZ-RODRI´GUEZ ,Monica R. ROMERO , a,b a,b, Emilio ROLA´ N-ALVAREZ and Juan GALINDO * aDepartment of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, Vigo, 36310, Spain, bToralla Marine Science Station (ECIMAT), Universidad de Vigo, Vigo, 36331, Spain, and cInstitute of Marine Research (IIM), CSIC, Vigo, 36208, Spain *Address correspondence to Juan Galindo. E-mail: [email protected]. Received on 6 July 2016; accepted on 29 October 2016 Abstract Rocky intertidal organisms are commonly exposed to environmental gradients, promoting adapta- tions to these conditions. Emersion time varies along the intertidal range and in the supralittoral zone is frequently larger than a single tidal cycle, even lasting for weeks. The planktonic-dispersing gastropod Melarhaphe neritoides is a common species of the high shore, adapted to reduce water loss in order to survive during long-term emersion. In this study, we investigated the molecular re- sponse, at the proteome level, of M. neritoides collected in high-shore tide pools to a series of emersion periods, from 8 to 24 days, in laboratory conditions. We compared this response to indi- viduals maintained submerged during this period, because this was their original habitat. We also included a reversion treatment in the study, in which emersed individuals were returned to the sub- merged conditions. Although we detected an increase in overall protein concentration with longer emersion periods, contrary to general expectation, the two dimensional electrophoresis (2DE)- based proteomic analysis did not show significant differences between the treatments at the level of individual protein spots, even after an emersion period of 24 days. Our results suggest that the metabolism remains unaltered independent of the treatment carried out or the changes are very subtle and therefore difficult to detect with our experimental design. We conclude that M. neri- toides could be equally adapted to emersion and submersion without drastic physiological changes. Key words: adaptation, 2DE proteomics, environmental gradient, intertidal zone, metabolic rate depression. Intertidal species from rocky shores are exposed to striking environmen- Adaptation to emersion is an important feature of many inter- tal gradients, some of these changes are predictable because of the peri- tidal species, including littorinids, as they are exposed to this condi- odicity associated with the tides while others are more variable tion twice a day during low tide, and sometimes for days or weeks if (Raffaelli and Hawkins 1996; Hochachka and Lutz 2001; Somero they inhabit the supralittoral zone (McMahon 1988). At the molecu- 2004; Alpert 2005). These organisms often develop different adapta- lar level an organism needs to balance its ATP consumption and tions to those conditions that represent an important metabolic chal- production rates during emersion (Storey and Storey 2004). A mech- lenge for them (Sokolova and Po¨ rtner 2001; Somero 2002, 2004). anism involved in this response in a wide range of organisms, includ- Littorinid snails are a particularly interesting group because they include ing marine invertebrates, is a strong metabolic rate depression taxa adapted to several environmental conditions and the physiological (MRD), controlled by reversible protein phosphorylation and basis of some of these adaptations has been studied from different changes in ribosomal aggregation state (Larade and Storey 2002; angles (Adey and Hayek 2005,reviewedinRolan-Alvarez et al. 2015). Storey and Storey 2004). Therefore, regulation through protein VC The Author (2017). Published by Oxford University Press. 487 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Downloaded [email protected] https://academic.oup.com/cz/article-abstract/63/5/487/2870066 by CSIC user on 18 June 2018 488 Current Zoology, 2017, Vol. 63, No. 5 phosphorylation can affect the activity of a wide range of metabolic observed at the proteome level and detected by 2DE-based prote- enzymes involved in ATP production (Larade and Storey 2002; omic analysis. Here we evaluate this response at the proteome level Storey and Storey 2004). Despite this, there have not been many at- and discuss its ecological and evolutionary implications. tempts to characterize the molecular response to emersion by two di- mensional electrophoresis (2DE)-based proteome analysis (e.g., Fields et al. 2014), a methodology that allows resolving proteins with differences in post-translational modifications (Rabilloud and Material and Methods Lelong 2011). For example, differences in protein phosphorylation Emersion treatments can be detected by this technique because it causes a change in the Samples of Melarhaphe neritoides were collected in April 2013 at isoelectric point, shifting the relative position of the protein spot in the exposed rocky shore of Silleiro in Galicia (426’45"N, the 2DE gels. On the other hand, it has been generally recognized 853’58"W; NW Spain). Approximately 500 adult individuals of that intertidal organisms, especially those that inhabit the suppralit- the high-shore morph (3–5 mm shell height) were randomly col- toral zone, are able to survive long periods of emersion by activating lected within tide pools from a 3-m2 area at the limit of the barnacle different anaerobic pathways (Sokolova and Po¨ rtner 2001; Larade Chthamalus stellatus fringe (upper intertidal zone). The snails were and Storey 2009; Storey et al. 2013), while an aerobic metabolism kept in seawater and transported to the marine laboratory facilities is prevalent in the lower shore. Those changes should also be re- at ECIMAT (Estacion de Ciencias Marinas~ de Toralla, Vigo, Spain). flected at the proteome level (Diz et al. 2012), there are many 2DE- In the laboratory, groups of 30 random similarly sized individuals based proteomic studies that have recurrently reported the identifi- were placed in bags created with 12 Â 12 cm squares of 1-mm nylon cation of a wide range of enzymes involved in different metabolic mesh. Then, each bag was introduced in a glass aquarium pathways (e.g., Wulff et al. 2008; Diz et al. 2012a, 2012b; Rocher (18 Â 10 Â 6 cm) with an open sea water system (14.2 6 0.8 C) and et al. 2015). kept submerged continuously for 30 days of acclimatization to la- Melarhaphe neritoides (L.) is a small littorinid gastropod (3– boratory conditions prior to the experimental treatments. The 5 mm), highly abundant and widely distributed, from the NE Atlantic shores to the Mediterranean (Fretter and Graham 1980). It photoperiod was set to a 14/10-h light/dark cycle and the snails is a dioecious species and has a planktonic larval phase of 4–8 weeks were able to graze on the algae growing on the nylon mesh during (Johannesson 1992; Cronin et al. 2000). The long distance dispersal this time. Note that specimens of M. neritoides have been main- by the larvae has been associated with the absence of genetic struc- tained alive (and showing shell growth) in similar conditions for 9 ture at a geographical scale using allozymes (Johannesson 1992), al- (Cuna~ et al. 2011) and 15 months (Garcıa et al. 2013), respectively. though this has not been properly evaluated with other molecular In order to study the response at the proteome level induced by markers. Melarhaphe neritoides is considered a high-shore species emersion we exposed the individuals to this environment for differ- although it is found across the entire rocky intertidal range ent periods (8, 16, 24 days), all after the 30-day acclimatization (McGrath 1997). The settlement of the larvae occurs in the lower period in seawater. We chose these lengths in days for the treatments shore, then the juveniles graze on the microalgae growing on the because we know that this species had a quick recovery, actively rock and when they reach a certain size the snails start migrating crawling, after 11 days of exposure to emersion (Britton 1995) and upshore (Fretter and Manley 1977). It also has been reported that also could survive for months in these conditions (Lysaght 1941). In the settlement can occur, at lower frequency, in tide pools from the this experiment, we performed 5 different treatments and 3 biolo- upper shore and these recruits will be part of the high-shore subpo- gical replicates per treatment (Figure 1) as follows: 1) "Submersion" pulations (Fretter and Graham 1980; McGrath 1997). At this inter- (Sub) treatment, the snails were kept submerged in seawater for 24 tidal level, M. neritoides is commonly sheltered in crevices to avoid days, 2) "Emersion 8 days" (Em8) treatment, the snails were kept desiccation (Fretter and Manley 1977; Raffaelli and Hughes 1978; submerged for 16 days and subjected to emersion for 8 days, 3) Britton 1995) and can be exposed to emersion for weeks (Britton "Emersion 16 days" (Em16) treatment, submerged for 8 days and 1992); it is proven to survive this stress for at least 5 months 16 days of emersion, 4) "Emersion 24 days" (Em24) treatment, sub- (Lysaght 1941). Lower shore and high-shore subpopulations from jected to emersion for 24 days, 5) "Reversion" (Rev) treatment, the the same locality have been found to be morphologically different remaining snails from treatment Em16 were submerged again for 16 (shell size and shape) although they lacked genetic differentiation days, this included only 2 biological replicates. After each treatment based on AFLPs (amplified fragment length polymorphisms) (Cuna~ all the snails were snap frozen in liquid nitrogen and kept at À80 C et al.