Reply: We Added on Paragraph to Explain the Proteomic-Based Method in “Introduction”

j. Hall-Spencer (Referee) [email protected] Received and published: 18 December 2014 This paper shows that Anachis misera shells get more globular and are more eroded at sulphurous volcanic vents off Taiwan where the pH can fall to 7.22, and that protein expression co-varies with pH. The first paragraph of the introduction has issues that are symptomatic of those that could be improved here and in the rest of the paper. The ocean acidification literature should be updated from Fabry et al. 2008 using more comprehensive meta-analyses such as that of Kroeker et al. 2013. The suggestion that field evidence on the effects of OA on gastropods is limited misses some key works e.g. Cigliano et al. 2010 who looked at juvenile gastropod settlement along CO2 gradients, Marshall et al. 2008 and others who have looked at gastropod shell parameters along coastal pH gradients an work on gastropods at Ischia and Vulcano using locations with elevated CO2 levels but without the confounding effects that are clearly present off Kueishan (Rodolfo-Metalpa et al. 2012; Milazzo et al. 2014; Langer et al. 2014). Reply: Thanks for the comments. We already updated the references and discussion in the revised version as suggested. The authors might be better playing down the link with ocean acidification but instead concentrating on effects of extreme hydrothermal environments on mollusc shell growth and relating this to how extreme events in the pst may have altered mollusc shell morphology or calcification mineralogy in general. Reply: We added one different dove snail, Euplica sp. for comparison with the vent snails and focus our findings on the shallow vent environments. This paper lacks the level of carbonate chemistry detail required for a field-based examination of ocean acidification. The lack of geochemical data, lack of control or reference site data with normal pH values (ca 8.1) and some poor Figures (e.g. Fig 2) means that the paper has several points that could be improved. Reply: We could not find Anachis misera in any other coastal environments in Taiwan. In the revised version, for the comparative purpose, we analyzed two populations of Euplica sp. collected in northeastern Taiwan where is in the west of Kueishan Islet about 10km apart. Additional environmental information of the vents was listed in Table and the quality of Fig. 2 was improved too. I have not reviewed the proteomics part of the paper as this is outside my expertise, but I would like a new version of the manuscript to review why and how protein expression typically alters in marine molluscs under environmental stress. Reply: We added on paragraph to explain the proteomic-based method in “Introduction”. Our Anachis snails collected from 5 vent sites were classified to two groups, i.e. V-South (pH 7.78-7.82) and V-Rest (pH 7.31-7.83) based on the analyses of protein expression profiles. Then the effects of naturally acidified seawater on shell traits of A. misera were quantified following. Application of proteomic approach was also discussion in the section of “Discussion”. Interactive comment on Biogeosciences Discuss., 11, 17207, 2014.

K. S. Tan (Referee) [email protected] Received and published: 30 December 2014 This interesting paper examines shell characteristics and gross protein expression in populations of subtidal columbellid gastropods living in habitats influenced by shallow hydrothermal vent discharge. My main concern is that the authors have not identified a control site at the onset with a population that is not affected (at least relatively speaking) by the vent discharge. If we do not know how the population parameter varies at a control site, it would be difficult to determine if differences seen of the same parameter measured in another population at another site are caused by the conditions there. Based on pH data in Table 1, the two sites S and SW might be suitable control sites, and the two other affected sites should then be compared with the control sites. The authors in fact might want to consider pooling the data from S and SW. Reply: Thanks for the comments. We could not find Anachis misera in any other coastal environments in Taiwan. In the revised version, for the comparative purpose, we analyzed two populations of Euplica sp. collected in northeastern Taiwan where is in the west of Kueishan Islet about 10km apart. Our Anachis snails collected from 5 vent sites were classified to two groups, i.e. V-South (pH 7.78-7.82) and V-Rest (pH 7.31-7.83) based on the analyses of protein expression profiles. Then the effects of naturally acidified seawater on shell traits of A. misera were quantified following. Other issues: 1. Figure 1 is lacking in detail and would suggest the collecting localities (N, E, S, SW and NW as stated in the M & M section) and the locations of white and yellow vents be indicated clearly on a large-scale map of Kueishan Island. It would also help to indicate the prevalent current (Kuroshio) direction affecting the snail populations. What does the asterisk shown in the inset represent? Reply: Fig. 1 was revised as suggested. 2. It is not stated how the pH of the environment given in Table 1 was measured at the different localities. How many readings were taken for each location over how long a time? Provide the sample size and period of sampling. Reply: The description on variable measurements was added in “Materials and Methods”. 3. The shapes of the two shells shown in Figure 2 appear to be sufficiently different to represent different species. How did the authors confirm that they are indeed one species? Reply: Species identification on the dove snails (A. misera and Euplica sp.1) have be confirmed by COI and 16S rRNA genes as shown in the following figures. The snails collected in the Kueishan Islet belong to the same species.

GF 7 DX 56 84 GF C9 GF C4 85 GF C7 GF C11 Euplica sp.1 GF 22 98 DX 46 GF 27 99 96 GF 24 DX 67 94 DSH E4 Euplica sp.3 24 100 DSH E8 Euplica turturina 16 Mitrella ligula Mitrella cf. tuberosa Mitrella bicincta 86 Alia carinata 45 28 Amphissa versicolor 52 54 Amphissa columbiana 18 Amphissa reticulata DX 55 TI chu13 72 TI a1 TI a5 100 TI a2 Anachis misera TI a3 63 TI chu12 TI a4 TI a6 TI a7 DX C2 DX 57 100 DX 37 Euplica sp.2 95 GF 12 0.05 Maximum Likelihood tree of Columbellidae using GTR+G model based on the COI gene sequence.

GF C11 GF C9 GF 7 DX 46 43 GF 22 Euplica sp.1 DX 56 GF 27 42 95 78 GF 24 43 DX 67 98 GF C4 GF C7

49 DSH E8 DSH E4 98 Euplica sp.3 DSH E5 Euplica turturina TI a6 51 TI a7 TI a5 100 TI a4 TI a3 Anachis misera TI a1 52 TI a2 DX 55 99 Mitrella burchardi 12 Mitrella bicincta 20 Euplica scripta DX 57 98 98 DX 37 Euplica sp.2 78 GF 12 Pseudamycla formosa Mitrella ligula 100 DX C2

0.05 Maximum Likelihood tree of Columbellidae using GTR+G model based on the 16S gene sequence. 4. The authors seem to refer to the extent of erosion of the shell apex as a criterion (Results section, section 3.1 2nd paragraph) but it is not explicitly stated how this was ascertained. The number of individuals whose apices of the shells were eroded and the extent of erosion of the apex could be another parameter used to determine the effect of pH on the columbellid population. Reply: It is subjective to determine the eroded condition. So, we revised the MS only report some eroded snails from East and Northwest sites (shown in Fig. 2). 5. Figure 6: what does the letters A and B represent? I would suggest that the authors compare the thickness and total animal weight of the shells of each size class separately, instead of standardizing the data, which has the effect of compressing the differences. Given that the mean shell lengths are between 9.0 and 9.2, comparing all shells between 8 and 10 mm in length might provide a cleaner conclusion. Reply: The data were re-analyzed completely. So, the part was deleted already. 6. The Discussion section would benefit from re-organization of the paragraphs (which I find to be rather disconnected from each other), focusing on the implications of the results obtained, i.e., the change in shell morphometry caused by low pH, and the possible reasons behind the protein patterns obtained (this is presently not addressed in the Discussion), in relation to the known ecology of Kueishan Island. The discussion can then be extended to the greater realm of ocean acidification and how the results here contribute towards the existing understanding of this phenomenon. The paragraphs in the Discussion are rather unconnected in thought and does not read well. For instance, in section 4.1, the first paragraph reviews the evidence for reduction in shell growth in acidic conditions, but the results of the Kueishan Island study are not discussed. In the second paragraph, shell shape is suddenly brought into the picture, and the authors assert that a rounded shell shape is less vulnerable to crab predation, but it is unclear how this is related to the authors’ results, when there is no data presented on crab predation of Anachis at the study site. The next paragraph jumps to conditions in the deep sea, and the last paragraph finally refers to the results from this paper, which should really be moved to the first paragraph. Similarly, the second section (comparison with other Anachis studies) would be more appropriately positioned earlier in the Discussion section. Reply: The discussion was extensively revised as suggested. We separated it to 3 parts, including 1. Application of proteomic-based approach to shallow vent snails; 2. Comparison with other dove snail studies; 3. Comparison with other ocean acidification studies. 7. The authors should also address the question of whether columbellids are particularly adapted to acidic environments compared to other gastropods or mollusks in the Discussion. Reply: The discussion was added but adaptation mechanism is uncertain. Interactive comment on Biogeosciences Discuss., 11, 17207, 2014.

Effects of low pH stress on shell traits of the dove snail, Anachis 刪除: and proteomes misera inhabiting in shallow vent environments off Kueishan Islet, Taiwan

Y. J. Chen, J. Y. Wu, C. T. A. Chen, and L. L. Liu Department of Oceanography, National Sun Yat-sen University, Kaohsiung 804, Taiwan

Received: 12 October 2014 – Accepted: 24 November 2014 – Published: 29 November 2014

Correspondence to: L. L. Liu ([email protected])

Published by Copernicus Publications on behalf of the European Geosciences Union.

Abstract 刪除: a The effects of naturally acidified seawater on shell traits were quantified through the 刪除: species, comparison of dove snails (Family: Columbellidae) Anachis misera from vent environments and Euplica sp. from non-vent sites in northeastern Taiwan. Samples 刪除: (Family: Columbellidae)

5 of A. misera were collected around the shallow vent (24.8341°N, 121.96191°E), 刪除: were quantified i including the East, South, Southwest and Northwest sites. An absence of Anachis 刪除: n five snails was found in the most acidic North site (pH 7.19-7.25). Based on the similarities of protein expression profiles, the Anachis snails were classified to two 刪除: -based environments off groups, i.e. V-South (pH 7.78-7.82) and V-Rest (pH 7.31-7.83). Comparing their Kueishan Islet, Taiwan.

10 shell traits to the non-vent Euplica sp. from Da-xi (DX) and Geng-fang (GF) (pH 刪除: A 8.1-8.2), difference in shell shape (shell width:shell length) as vent populations 刪除: observed being more globular than that of non-vent ones was found. The means of shell width were significantly different among sites (p<0.01), with a descending order of 刪除: 22 GF > DX > V-South, V-Rest. The relationships of shell length to total weight were 刪除: , and the size structure 15 curvilinear for both Anachis and Euplica snails. The logarithmic transformed differed among the remaining slopes differed significantly among sites and the mean body weight of GF East, South, Southwest and population was greater than others (p<0.01). Positive correlations between shell Northwest sites

length and shell thickness of body whorl (T1) and penultimate whorl (T2) were only 刪除: If a positive correlation observed in non-vent GF and DX populations. Anachis snails from vent sites were between shell length and shell

20 thinner in T1 and T2 compared to the Euplica snails from non-vent sites (p<0.05). width or total weight existed, Within each vent group, shell thickness between T1 and T2 was insignificantly the coefficient of

different. Between vent groups, T1 and T2 from V-Rest showed a decrease of determination (R2) of the

10.6% and 10.2%, respectively, compared to V-South ones. The decrease of T1 equations was low, i.e.,

and T2 between vent Anachis snails and non-vent Euplica snails was as great as 0.207-0.444. Snails from the 25 55.6% and 29.0%, respectively. This was the first study to compare snail’s ... [1] 刪除: (thickness of body morphological traits under varying shallow vent stresses with populations prior ... [2] 刪除: (thickness of classified by biochemical responses. As a whole, the shallow vent-based findings ... [3] provide additional information from subtropics on the effects of acidified seawater 刪除: the Northwest site on gastropod snails in natural environments. 刪除: 6.3

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刪除: In a similar vein, based ... [4] 刪除: new

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1 Introduction

Although current evidence indicates that organisms with a CaCO3 skeleton, e.g., mollusks, echinoderms and corals, are likely to be among the most susceptible to ocean acidification (such as in Fabry et al., 2008; Sokolov et al., 2009), specific 5 information obtained from field investigations has been limited, particularly in gastropod snails (Gazeau et al., 2013). Thus, the current study was performed to address this issue within an extreme hydrothermal environment. The shallow hydrothermal vents locate east of Kueishan (KS) Islet, Taiwan, near the southern end of the Okinawa Trough (Fig. 1). The vents emit yellow or white 10 plumes with temperature and pH varying in the ranges of 78-116 °C and 1.52-6.32 vs. 30–65°C and 1.84–6.96, respectively. The gas bubbles are comprised of 90–99%

CO2, 0.8–8.4% H2S, <0.03% SO2, and <50ppm HCl (Chen et al., 2005). The diffusive plumes are affected by the wind, sea waves, and tides (Chen et al., 2005; Han et al., 2014). Based on the observed data, the emitted fluids diffused mainly 15 from north to south due to ebb tide and moved from southeast to northwest during the

spring tide. In addition, the fluids are also directed by the Kuroshio current flowing along the coast of Kueishan Islet to the north throughout the year. Because the 刪除: and diffusion is closely correlated with diurnal tides, benthic organisms would face the lowest pH twice per day but for no more than four hours each time. 20 Near the yellow vents, the crab Xenograpsus testudinatus is the only benthic macrofauna (Jeng et al., 2004). In contrast, around the white vents, benthic invertebrates include the crab X. testudinatus, two sea anemones, hexacoral Tubastraea aurea, serpulid polychaete, a chiton, snail Nassarius sp., and the dove snail Anachis misera. These vent organisms naturally inhabit acidic and toxic 刪除: shell 25 environments. High concentrations of trace metals in various tissues of the crab X. testudinatus are reported and the levels are not beyond other crabs collected from different habitats (Peng et al., 2011). We herein test the hypothesis that populations of A. misera distributed around 刪除: nachis

vents exposed to varying degrees of plumes would exhibit different ecophysiological 刪除: snails 30 performance compared to the non-vent dove snail, Euplica sp., a common species in coastal waters of northeastern Taiwan. A proteomic-based method was used to classify the samples of A. misera collected around the vent-based environments. This approach involves measuring changes in many proteins. Through the comparison of protein expression profile of each snail 35 by cluster analysis, similarities among samples can be determined and classified. This method has been applied to laboratory and field pollution studies, such as blue mussels exposed to polyaromatic hydrocarbons and heavy metals (Knigge et al., 2004), and Sydney rock oysters inhabiting in acid sulfate runoff estuary (Amaral et al.,

2012).

2 Materials and methods 刪除:

2.1 Sampling sites and collection of snails 刪除: Field study 5 Anachis misera was collected around a shallow-water vent in Kueishan Islet, Taiwan 刪除: The sampling area (Fig. 1), including the north (N), east (E), south (S), southwest (SW), and northwest (NW) sites during the period of June 28 to July 1, 2011. The sampling vent emitted 刪除: with white plumes

white plumes where another vent with yellow plumes was nearby northeasterly. The 刪除: distance of the collection sites to the vent center was 10-16m, and the water depth was 刪除: 10 in the range of 14.5-17.5m. Snails of Euplica sp. were sampled from Da-xi (DX) and Geng-fang (GF), northeastern Taiwan, between July and Sept. 2012. 刪除: Anachis snails were Sampling locations were identified by SCUBA divers equipped with GPS. collected around the vent (121º Temperature was determined by a thermometer inserted into the seawater samples. 96′19.1″E, 24º83′41.4″N), Flow rate was measured by a hydrobios digital flow meter (Model 438 110). The pH including the directions of 15 was measured by a radiometer PHM 85 system. Each environmental parameter was north (N), east (E), south (S), determined with one or three replicates and the results were shown in Table 1. The southwest (SW), and northwest collected snails were preserved in dry ice in the field. Upon returning to the (NW) during the period of laboratory, they were deep-frozen at -70oC for later use. June 28 to July 1, 2011. 2.2 Measurements on snail morphological traits 格式化: 縮排: 第一行: 1.5 字元 20 Shell traits, i.e., shell length and width, shell thickness of body whorl (T1) and penultimate whorl (T2), as well as total weight of the intact individual, were measured 刪除: the

(Fig. 2). Shell thickness was determined through enlarged X-ray radiographs which 刪除: determined were produced by exposing snail shells to X-ray with the settings of 80kVp and 1mA 刪除: measured for 116.7ms. The distance between the X-ray source and the objects was 50cm. 25 The shell images were further drawn outlines using GIMP version 2.8 which is an open source imaging system (http://www.gimp.org/). For statistical analysis, an ANCOVA (analysis of covariance) was used to 刪除: The relationship compare the least square means (LSM) for each variable (i.e., shell width, total weight, between shell length and shell shell thickness T1 and T2) among sites with shell length as the covariate. The width or total weight was 30 relationships of shell length to shell width, shell thickness of T1 and T2 were evaluated by the linear calculated using linear regression analysis. If the relationship of total weight and regression analysis. The data shell length was curvilinear, linear regression slopes were obtained and compared of pH and shell thickness were after data are logarithmic transformed. analyzed using a one-way 2.3 Proteomic study ANOVA and Tukey’s 35 The protein expression profiles of Anachis snails were determined by multiple-comparisons test. one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (1-D SDS-PAGE). The foot tissue was taken and homogenized with lysis buffer (0.5 M Tris-HCl, pH 7.4, 10% SDS, 0.5 M DTT) for proteomic analysis. Homogenates

o were centrifuged at 13000g for 10 min at 4 C. The homogenous supernatant was 刪除: spot collected, and the protein concentration was determined by Bradford assay, using 刪除: a bovine serum albumin as the standard. 刪除: spot The stacking and resolving gels were prepared in the percentages of 5% and 12% 5 (Hoefer SEM 260 system, Amersham Pharmacia). After loading 25ug protein in 刪除: spot

each sample lane, electrophoresis was run for 30min at 120V then 4h at 180V. The 刪除: different gels were stained with Coomassie blue G-250 (Candiano et al., 2004). 刪除: 1 Stained gels were scanned and transformed into digitalized images using Image Scanner (Amersham Pharmacia). The Multi Gauge software v2.2 (Fujifilm) was 刪除: 00

10 used for protein quantification. The protein bands were assigned band numbers, and 刪除: which is close to the their intensity levels were calculated as their relative area to the total protein area on very acidic yellow plumes the gel. A cluster analysis of the Bray-Curtis Similarity (BCs) Indices (Primer 6.0) north of the sampling white

was employed to compare the expression of overall protein patterns among snail vent individuals (Clarke and Warwick, 2001). In addition, the contribution of each 刪除: 15 protein band was further estimated by principal component analysis (PCA). 刪除: more commonly seen at

3 Results 刪除: (pH 7.33) than at the 3.1 Morphological traits of Anachis snails from vent sites South (pH 7.80) Temperature ranges of the sampling sites were from 26 to 27oC (Table 2). Spatial 刪除: 3 20 variability in pH among sites was clearly observed, and the lowest one was 7.22±0.03 at the North site (p<0.01). Anachis snails were found around the vent, except for the 刪除: Shell lengths of the most acidic North site. Shell lengths of the snails ranged from 6.88 to 11.01mm. snails were from 6.88 to Several snails with eroded apex were observed in the East and Northwest sites (Fig. 11.01mm (Table 1). Based 2). on the length-frequency 25 3.2 Protein expression profiles of Anachis snails from vent sites histograms, it is found that the Based on the protein expression results, 16 protein bands were selected for further relative proportions of size Bray-Curtis Similarity (BCs) analysis (Fig. 3). And the classification of snails fell classes differed among sites into three clusters (Fig. 4). Snails from the high pH South were all within one (Fig. 4). Positive correlation cluster. In contrast, snails from the remaining sites were indistinguishable in other between shell length and shell 30 clusters. With further determination on the contribution of each protein variable, the width was only observed in the data were characterized by principle component analysis (PCA). The first to the South and Northwest samples, fifth principal components accounted for 35.4, 28.5, 13.2, 8.8, and 4.2% of the total and their slopes were variance, respectively. The separation was mainly contributed by the first (i.e., significantly different (Fig. 5). bands 8, 1, 15, and 12) and second (i.e., bands 15, 13, 12, 1, and 11) The same pattern was also 35 principal-components. observed in the relationship Bsed on the cluster results, the Anachis snails were classified into groups of between shell length and total V-South (pH 7.78-7.82) and V-Rest (pH 7.31-7.83). Their shell traits were weight (Fig. 6). It is noticed compared to non-vent Euplica snails (pH 8.10-8.20) subsequently. that the coefficient of determination (R2) of the ... [5] 5

3.3 Comparison of shell traits of dove snails among vent and non-vent sites 刪除: thickness Shell traits of the Anachis and Euplica snails were listed in Table 3. A positive 刪除: Shell shape (shell correlation between shell length and shell width was observed in all populations. width:shell length), Difference in shell shape (shell width:shell length), with vent populations being more standardized 5 globular, was also found, as shown by the significant difference in regressions’ slopes. By ANCOVA with shell length as the covariate, the mean values of shell width were 刪除: s significantly different among sites (p<0.01), with a descending order of GF > DX > 刪除: (thickness of body V-South, V-Rest (Table 3). whorl:shell length),

The relationships of shell length to total weight were curvilinear for both Anachis 刪除: (thickness of 10 and Euplica snails (Fig. 6). The slopes among sites were significantly different and penultimate whorl:shell length) the mean body weight of GF population was significantly greater than others (Table 3 刪除: S & Fig. 5). Positive correlations between shell length and shell thickness of body whorl (T1) 刪除: at the Northwest site and penultimate whorl (T2) were only observed in non-vent GF and DX populations. (pH 7.33) exhibited 15 Their slopes were significantly different for T1 only (Fig. 7). The mean shell significantly greater shape thickness of T1 and T2 varied among sites (Table 3). Anachis snails from vent sites ratio (more globular) than were thinner in T1 compared to the non-vent Euplica snails (p<0.001), with a those of the South site (pH descending order of GF > DX > V-South > V-Rest. A similar trend was also found 7.80) (Fig. 7).

in T2. Within each vent site, shell thickness between T1 and T2 was insignificantly 刪除: each sampling 20 different (paired t-test, p>0.05). By comparison, T1 and T2 of the snails from 刪除: In contrast, among sites, V-Rest were 89.4% and 89.8%, respectively, of the V-South ones. With the both measures of standardized comparison of vent and non-vent sites, T1 and T2 of the Anachis snails from V-Rest shell thickness decreased under were 44.4% and 71.0%, respectively, of the Euplica snails from GF. Clearly, both acidic environments. measurements on shell thickness decreased under acidic environments. 25 刪除: Northwest

4 Discussion 刪除: 93.7 This was the first study to compare morphological traits of snails under varying 刪除: 90.6 shallow vent stresses with populations prior classified by biochemical responses. Difference in shell shape (shell width:shell length), with vent populations being more 刪除:

30 globular was found. Snails from V-Rest (pH 7.31-7.83) exhibited a 10.6% and 刪除: 3.3 Protein

10.2% decrease in shell thickness of body whorl (T1) and penultimate whorl (T2), expression profiles respectively, compared to snails from the V-South (pH 7.78-7.82). Comparing to In the protein expression non-vent sites (pH 8.10-8.20), T1 and T2 of the Anachis snails from V-Rest showed a profiles of Anachis snails, 16 55.6% and 29.0% decrease in T1 and T2, respectively, to Euplica snails from GF. protein spots were selected for ... [6] 35 Our shallow vent-based results were, in general, consistent with laboratory, controlled 刪除: Our results showed that and deep-sea vent studies, i.e., shell-organisms are susceptible to acidic environments. Anachis snails survived and 4.1 Application of proteomic-based approach to shallow vent snails ... [7] Proteomic-based method has been used in environmental toxicology to characterized 刪除: More specifically, snails in the acidic Northwest site ... [8] 6

organism’s responses to specific treatments with various gradients (Bradley et al. 2002; Jackson et al. 2002). It has been applied to laboratory and field studies, such as blue mussel Mytilus edulis exposed to polyaromatic hydrocarbons and heavy metals (Knigge et al., 2004), to crude oil (Mi et al., 2006), to PCBs and PAHs 5 extracted from Baltic Sea sediments (Olsson et al., 2004) and mussel Mytilus galloprovincialis exposed to a tributyltin-polluted area (Magi et al., 2008). Application of proteomic approach to vent mussel Bathymodiolus azoricus has been conducted with samples collected from three distinct hydrothermal vent fields in the Mid-Atlantic Ridge (Companya et al., 2011). The expression profiles of 35 10 proteins from the gill revealed clear separation among sites, which indicates that specific adaptations of B. azoricus depend on local conditions. It is known that large spatial and temporal variations in environmental parameters are detected around vent environments, such as temperature, pH and hydrothermal fluid composition in terms of dissolved oxygen, methane and sulphide concentrations 15 etc. The pH of the hydrothermal fluids within our sampling vent and surrounding seawater had been determined on 31 May, 2011, the pH ranges from2.29 to 5.11 and 5.51 to 6.15, respectively (Zeng et al., 2013). The diffusion activities of vent plumes were also evaluated through environmental factors of temperature, pH and Eh (Han et al., 2014). The diffusive plume is mainly affected by the wind, sea waves and tides. 20 If ocean currents in the east-west direction are not considered, sea currents around vents are from north to south during ebb tide, while in flood tide the opposite direction dominates. Our proteomic results indicated that snails from the South were distinguished from the rest sites which are consistent with the diffusion activities of local vent fluids. 25 4.2 Comparison with other dove snail studies Among the dove snails (Family: Columbellidae), Anachis avara is a common one living on the coast of the eastern United States (Scheltema, 1968; Hatfield, 1980). At Bear Cut, FL, the population of A. avara showed seasonal fluctuation in its size structure (Hatfield, 1980). It reached a mean terminal size of 10.50 mm (8.00-13.29 30 mm) and matured quickly at the age of six to seven months. The estimated life span was less than two years. It is suggested that the fluctuation in size structure was primarily the result of seasonal recruitment, and the abundance was probably determined by predation. In Anachis fluctuate, the regression equation of shell length (mm) and dry tissue 35 weight or shell weight (g) had been reported, i.e., Y = -0.025+0.003SL (R2=0.88; N=26) and Y=-2.39+1.04lnSL (R2=0.92), respectively (Bertness and Cunningham, 1981). By comparison, in this study, shell lengths of A. misera and Euplica sp. were from 6.88 to 11.01mm and 3.40 to 7.56mm, respectively (Tables 2 & 3). Variations

in size structure among sites were also obvious. Positive correlations between shell length and shell width or total weight in both dove snails was present, but the R2 of the equations were low in A. misera (0.07-0.28) compared to Euplica sp. (0.94-0.98) (Figs. 5 and 6). Positive correlations between shell length and shell thickness of 5 body whorl (T1) and penultimate whorl (T2) were only found in non-vent populations with the R2 of 0.43-0.64 (Fig. 7). Although differential recruitment and acidic stress are potential factors to account for low or even no correlation between the above shell traits in vent A. misera, further study is needed to address this question. 4.3 Comparison with other ocean acidification studies 刪除: 1

10 To date, ocean acidification studies have been conducted mostly in the laboratory 刪除: most or controlled environments for a short period of time. The results indicate exposures 刪除: As a result, it has been to future global change scenarios (Caldeira and Wickett, 2003; Sokolov et al., 2009) firmly may alter the tolerance of calcifying species and, ultimately, their fitness and survival through complex physiological and ecological pathways. Based on literatures’ data, 刪除: conclu

15 it is concluded that effects of acidified seawater on species growth were at higher pH 刪除: ded that

than those on species reproduction (mean pH10 was 7.73 vs 7.63 and mean pH50 was 7.28 vs 7.11, respectively) (Azevedo et al., 2015). Studies conducted in the natural vent system at Ischia, Italy indicated that the settlement and colonization of mollusks and microfauna showed highly reductions in 20 recruitment in the acidified stations (Cigliano et al. 2010; Ricevuto et al., 2012). In the experiments of juvenile pen shell Pinna nobilis transplanted to Ischia for 45 days, decreases in survival, growth, and oxygen consumption were found. A 22% decrease in survival rate for specimens transplanted at pH 7.7 comparing to those at pH 8.1 was reported (Basso et al., 2015). Studies on the limpet Patella caerulea 25 within and outside the Ischia vent, shell formation and dissolution are both observed in low pH site where enhanced shell production counteracts shell dissolution (Hall-Spencer et al., 2008; Rodolfo-Metalpa et al., 2011; Langer et al., 2014). In contrast, shell dissolution is absence in normal pH site. Compared with deep-sea vent studies, in the northwest Eifuku volcano, Mariana arc, the vent mussel, 30 Bathymodiolus brevior inhabiting low pH environments (pH 5.36-7.29), exhibited shell thickness and daily growth increments in shells of only about half of the ones with pH>7.8 (Tunnicliffe et al., 2009). Under low pH (7.7 vs. 8.0), periwinkle Littorina littorea increased less in weight and were shorter than snails grown in current conditions (Melatunan et al., 2013). 35 Similar results have been obtained for other calcifying organisms, e.g., the reduction in shell growth of the oysters Crassostrea gigas (Lannig et al., 2010) and Crassostrea virginica (Beniash et al., 2010), larvae of the Mediterranean pteropods Cavolinia inflexa (Comeau et al., 2010), and the mussels Mytilus edulis (Gazeau et al., 2010)

and Mytilus californianus (Gaylord et al., 2011). Along a gradient of pH (5.78-8.30) 刪除: Marine snails possessing and salinity (3.58-31.2psu) in the Sungai Brunei estuary, Malaysia, whelk Thais shells with a more elongated gradata exposed to acidified sites possessed heavier shells and the degrees of erosion shape are found to be more was negatively related to water pH and calcium concentration (Marshall et al., 2008). vulnerable to crab predation, 5 At low pH (7.7), a 2.45% change in shell shape (shell width:shell length) towards possibly due to higher more globular and a decrease in the outer lip shell thickness of up to 27% in Littorina handling efficiency compared littorea were observed (Melatunan et al., 2013). with a more globular shell (Cotton et al., 2004). In this study, comparison of A. misera between V-South (pH 7.78-7.82) and ... [9] V-Rest (pH 7.31-7.83), the change of shell ratio was 3.4%, to more rounded in the 刪除: L. 10 V-Rest group. In addition, snails of V-Rest exhibited a 10.6% and 10.2% decrease 刪除: This reduction in shell in shell thickness of body whorl (T1) and penultimate whorl (T2), respectively, thickness may increase the compared to the V-South snails. With the comparison of vent and non-vent sites, T1 organism’s susceptibility to and T2 of the Anachis snails from V-Rest were 44.4% and 71.0%, respectively, of the crushing predators (Boulding Euplica snails from GF (pH 8.1-8.2). Our shallow vent-based results were, in and Van Alstyne, 1993; 15 general, consistent with other laboratory, controlled and field studies, i.e., Trussell and Etter, 2001). As shell-organisms are susceptible to acidic environments. shell thickness is reduced It is known that vent systems are not entirely representative of future ocean under low pH and elevated changes because of not only the temporal variability in pH, but also the existence of ... [10] other toxic elements. However, vents’ acidifying environments are sufficiently large 刪除: compared to the South site (pH 7.80), snails from the 20 in spatial and temporal scales. Still, it is a naturally applicable system to assess the ... [11] effects of ocean acidification on the whole life cycle and across multiple generations 刪除: shape of target organisms. 刪除:

刪除: in the ranges of 0.5-4.3 Acknowledgments. We are grateful to the anonymous reviewers for their 25 constructive comments which have substantially improved the manuscript. We 刪除: to become

thank Mr. Siou-Yan Lin, Mr.. Chiu-Yeh Liao, and Ms. Yalan Chou, Dun-Ru Kang for 刪除: d conducting experiments. We also thank Dr. Kotaro Tsuchiya for species 刪除: (Fig. 6) identification on dove shells and Dr. Hui-Ling Lin for using the X-ray machine. This study was supported by the Ministry of Science and Technology, Republic of 刪除: in the acidic Northwest site 30 China (NSC99-2321-B-110-006; NSC 102-3113-P-005 -005 -002; MOST ... [12] 103-3113-M-005-001), the Asia-Pacific Ocean Research Center, and the Center for 刪除: 6.4 Emerging Contaminants Research, National Sun Yat-sen University, supported by the 刪除: 9.6 Ministry of Education, Taiwan. 刪除: snails from 35 References 刪除: the Amaral, V., Thompson, E.L., Bishop, M.J. and Raftos, D.A.: The proteomes of 刪除: deep-sea vent Sydney rock oysters vary spatially according to exposure to acid sulfate runoff. Marine and Freshwater Research, 63, 361–369, 2012. 刪除: 4.2 Comparison with other Anachis studies ... [13] 9

Azevedo, L.B., De Schryver, A.M., Hendriks, A.J., and Huijbregts, M.A.J.: Calcifying species sensitivity distributions for ocean acidification. Environmental Science & Technology, 49, 1495−1500, 2015. Basso, L., Hendriks, I.E., Rodríguez-Navarro, A.B., Gambi, M.C., and Duarte, C.M.: 5 Extreme pH conditions at a natural CO2 vent system (Italy) affect growth, and survival of juvenile pen shells (Pinna nobilis). Estuaries and Coasts, DOI 10.1007/s12237-014-9936-9, 2015. Beniash, E., Ivanina, A., Lieb, N. S., Kurochkin, I., and Sokolova, I. M.: Elevated 刪除: Boulding, E. G., and level of carbon dioxide affects metabolism and shell formation in oysters Van Alstyne, K. L.: 10 Crassostrea virginica. Marine Ecology Progress Series, 419, 95−108, 2010. Mechanisms of differential Bertness, M. D., Cunningham, C.: Crab shell-crushing predation and gastropod survival and growth of two architectural defense. Journal of Experimental Marine Biology and Ecology, 50, species of Littorina on 213-230, 1981. Brian P Bradley, , wave-exposed and on Bradley, B.P., Shrader, E.A., Kimmer, D.G. and Meiller, J.C.: Protein expression protected shores. Journal of 15 signatures: an application of proteomics. Marine Environmental Research, 54, Experimental Marine Biology and Ecology, 169, 139−166, 373-7, 2002. Caldeira, K., and Wickett, M.: Anthropogenic carbon and ocean pH. Nature, 425, 365, 1993. 2003. 刪除: Candiano, G., Bruschi, Candiano, G., Bruschi, M., Musante, L., Santucci, L., Ghiggeri, G. M., Carnemolla, M., Musante, L., Santucci, L., 20 B., Orecchia, P., Zardi, L., and Righetti, P. G.: A very sensitive colloidal Ghiggeri, G. M., Carnemolla, Coomassie G-250 staining for proteome analysis. Electrophoresis, 25, B., Orecchia, P., Zardi, L., and 1327–1333, 2004. Righetti, P. G.: A very Chen, C. T. A., Wang, B. J., Huang, J. F., Lou, J. Y., Kuo, F. W., Tu, Y. Y., and Tsai, sensitive colloidal Coomassie H.S.: Investigation into extremely acidic hydrothermal fluids off Kueishantao G-250 staining for proteome 25 Islet, Taiwan. Acta Oceanologica Sinica, 24, 125–133, 2005. analysis. Electrophoresis, 25, Cigliano, M., Gambi, M.C., Rodolfo-Metalpa, R., Patti, F.P. and Hall-Spencer, J.M.: 1327–1333, 2004. Effects of ocean acidification on invertebrate settlement at volcanic CO2 vents. 刪除: Caldeira, K., and Marine Biology, 157, 2489–2502, 2010. Wickett, M.: Anthropogenic Clarke, K.R., Warwick, R.M., 2001. Change in Marine Communities: An Approach to carbon and ocean pH. Nature, 30 Statistical Analysis and Interpretation, second ed. Primer-E, Plymouth. 425, 365, 2003. Comeau, S., Gorsky, G., Alliouane, S., and Gattuso, J. P.: Larvae of the pteropod Cavolinia inflexa exposed to aragonite undersaturation are viable but shell-less. 刪除: Cotton, P., Rundle, S., Marine Biology, 157, 2341−2345, 2010. and Smith, K. E.: Trait Companya, R., Antúnez, O., Bebiannoa, M. J., Cajaraville, M. P., and Torreblanca, A.: compensation in marine 35 2-D difference gel electrophoresis approach to assess protein expression profiles gastropods: shell shape, in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal vents. avoidance behavior, and Journal of Protemoics, 74, 2909-2919, 2011. susceptibility to predation. Fabry, V. J., Seibel, B. A., Feely, R. A., and Orr, J. C.: Impacts of ocean acidification Ecology, 85, 1581−1584, 2004. 10

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Renzone, G. and Cosulich, M.E.: Interdisciplinary study for the evaluation of biochemical alterations on mussels Mytilus galloprovincialis exposed to a tributyltin-polluted area. Analytical and Bioanalytical Chemistry, 391, 671–8, 刪除: Bioanal Chem 2008. Analytical and Bioanalytical Chemistry 5 Marshall, D.J., Santos, J.H., Leung, K.M.Y. and Chak, W.H.: Correlations between gastropod shell dissolution and water chemical properties in a tropical estuary. Marine Environmental Research, 66, 422–429, 2008. Melatunan, S., Calosi, P., Rundle, S. D., Widdicombe, S., and Moody, J.: Effects of ocean acidification and elevated temperature on shell plasticity and its energetic 10 basis in an intertidal gastropod. Marine Ecology Progress Series, 472, 155–168, 2013. Mi, J., Apraiz, I. and Cristobal, S.: Peroxisomal proteomic approach for protein profiling in blue mussel (Mytilus edulis) exposed to crude oil. Biomarkers, 12, 47–60,2007. 15 Olsson, O., Bradley, B.P., Gilek, M., Reimer, O., Shepart, J.L. and Tedengren, M.: Physiological and proteomic responses in Mytilus edulis exposed to PCBs and PAHs extracted from Baltic Sea sediments. Hydrobiologia, 514, 15–27, 2004. Peng, S. –H., Hung, J. –J., and Hwang, J. –S.: Bioaccumulation of trace metals in the submarine hydrothermal vent crab Xenograpsus testudinatus off Kueishan 20 Island, Taiwan. Marine Pollution Bulletin, 63, 396–401, 2011. Ricevuto, E., Lorenti,M., Patti, F.P., Scipione, M.B., and Gambi, M.C.: Temporal trends of benthic invertebrate settlement along a gradient of ocean acidification at natural CO vents (Tyrrhenian Sea). Biologia Marina Mediterranea, 19, 49-52, 2 刪除: Biol. Mar. Mediterr. 2012. 25 Rodolfo-Metalpa, R., Houlbreque, F., Tambutte, E., Boisson, F., Baggini, C., Patti, F. P., Jeffree, R., Fine, M., Foggo, A., Gattuso, J.-P., and Hall-Spencer, J. M.: Coral and mollusc resistance to ocean acidification adversely affected by warming. Nature Climate Change, 1, 308–312, 2011. Scheltema, A. H.: Redescriptions of Anachis avara (Say) and Anachis translirata 30 (Ravenel) with notes on some related species (Prosobranchia, Columbellidae). Breviora, 304, 1-19, 1968. Sokolova, I. M., and Portner, H. O.: Physiological adaptations to high intertidal life involve improved water conservation abilities and metabolic rate depression in 刪除: Trussell, G.C., and Etter, Littorina saxatilis. Marine Ecology Progress Series, 224, 171−186, 2001. R. J.: Integrating genetic and 35 Tunnicliffe, V., Davies, K. T. A., Butterfield, D. A., Embley, R. W., Rose, J. M., and environmental forces that Chadwick, Jr. W.: Survival of mussels in extremely acidic waters on a shape the evolution of submarine volcano. Nature Geoscience, 2, 344-348, 2009. geographic variation in a Zeng, Z., Wang, X., Chen, C.T.A., Yin, X., Chen, S., Ma, Y., and Xiao Y.: Boron marine snail. Genetica, 112−113, 321−337, 2001. 12 isotope compositions of fluids and plumes from the Kueishantao hydrothermal field off northeastern Taiwan: Implications for fluid origin and hydrothermal processes. Marine Chemistry, 157, 59–66, 2013. 刪除:

Table 1. Sampling locations and environmental parameters of vent sites in the Kueishan Islet and northeastern Taiwan. Data are shown as Mean±SD and ranges. 刪除: Table 1. Environmental parameters and shell traits of Site White vent Yellow vent Da-xi (DX) Geng-fang (GF) 5 Anachis misera around the Latitude 24.8341°N 24.8355°N 24.9413°N 24.9046°N vent off Kueishan Islet. Data

Longitude 121.96196°E 121.96371°E 121.90390°E 121.87200°E are shown as Mean±SD and ranges. T1: thickness of body Depth (m) 17.0 9.5 3.0 3.0 whorl; T2: thickness of

Fluid flux (m3/hr) 18.5 21.0 - - penultimate whorl; -: no data; Means that differ significantly Temperature (oC) 55.0 115.0 27.2 ± 0.2 (27.0 - 27.4) 27.4 ± 0.5 (27.0 - 27.9) from each other are indicated pH 4.0 2.3 8.13 ± 0.06 (8.10-8.20) 8.13 ± 0.06 (8.10-8.20) by different letters

刪除:

Table 2. Environmental parameters and shell traits of Anachis misera around the vent off Kueishan Islet. Data are shown as Mean±SD and ranges. SH: shell length; SW: shell width; TW: total weight; T1: thickness of body whorl; T2: thickness of penultimate whorl; Means that differ significantly from each other are indicated 5 by different letters.

Site North (N) East (E) South (S) Southwest (SW) Northwest (NW) Plume distance (m) 15.6 10.0 10.5 12.0 16.0 Depth (m) 15.0 14.5 14.2 15.7 17.4 Temperature (oC) 27 10 27 27 27 26 7.22 ± 0.03 (7.19 - 7.25) 7.66 ± 0.08 (7.59 - 7.75) 7.80 ± 0.02 (7.78 - 7.82) 7.80 ± 0.03 (7.78 - 7.83) 7.33 ± 0.02 (7.31 - 7.35) pH c b a a c No. snails (N) 0 7 65 33 36

SL (mm) - 9.23 ± 0.63 (8.23 - 9.97) 9.01 ± 0.89 (6.88 - 11.01) 9.14 ± 1.11 (6.93 - 10.84) 9.13 ± 0.56 (7.81 - 10.40)

SW (mm) - 4.54 ± 0.32 (4.16 - 5.05) 4.42 ± 0.29 (3.65 - 4.96) 4.41 ± 0.30 (3.71 - 5.16) 4.30 ± 0.72 (3.86 - 4.93)

TW (mg) - 125 ± 18 (104 - 152) 121 ± 22 (67 - 188) 137 ± 23 (91 - 213) 113 ± 20 (75 - 153)

15 T1 (um) - 199 ± 56 (136 - 285) 225 ± 69 (109 - 481) 200 ± 56 (118 - 290) 168 ± 49 (79 - 276)

T2 (um) - 188 ± 44 (109 - 248) 200 ± 51 (112 - 328) 205 ± 55 (117 - 354) 180 ± 55 (79 - 325)

Table 3. Shell traits of Anachis misera around the vent off Kueishan Islet and Euplica sp. from non-vent control sites of Da-xi and Geng-fang. Data are shown as Mean± SD and ranges. SH: shell length; SW: shell width; TW: total weight; T1: thickness of body whorl; T2: thickness of penultimate whorl; Least square (LS) means that 5 differ significantly from each other are indicated by different letters.

Site V-South (S) V-Rest (R) Da-xi (DX) Geng-fang (GF) Snail sp. Anachis misera Anachis misera Euplica sp. Euplica sp.

No. snails (N) 65 76 16 30

SL (mm) 9.01 ± 0.89 (6.88 - 11.01) 9.14 ± 0.84 (6.93 - 10.84) 7.33 ± 1.34 (5.92 - 10.58) 9.61 ± 1.75 (6.74 - 13.19)

SW (mm) 4.42 ± 0.29 (3.65 - 4.96) 4.37 ± 0.29 (3.71 - 5.16) 4.14 ± 0.87 (3.40 - 6.34) 5.50 ± 1.01 (3.62 - 7.56)

LSMean of SW (mm) 4.42 ± 0.32 c 4.33 ± 0.35 c 4.77 ± 0.40 b 5.27 ± 0.38 a

TW (mg) 121 ± 22 (67 - 188) 124 ± 24 (75 - 213) 84 ± 70 (39.3 - 294.3) 195 ± 105 (42 - 436)

LSMean of TW (mg) 118.07 ± 8.30 b 117.59 ± 8.98 b 105.94 ± 4.28 b 149.66 ± 5.70 a

T1 (um) 225 ± 69 (109 - 481) 232 ± 31 (141 - 299) 385 ± 113 (243 - 653) 536 ± 171 (201 - 852)

LSMean of T1 (um) 255 ± 73 c 228 ± 70 d 446 ± 76 b 514 ± 71 a

T2 (um) 200 ± 51 (112 - 328) 234 ± 36 (148 - 304) 241 ± 104 (147 - 588) 343 ± 124 (157 - 702)

LSMean of T2 (um) 256 ± 56 b 230 ± 52 c 295 ± 60 a 324 ± 55 a

120o 121o 122o 123o 26oN N

China East China Sea 5 ugh Tro *DX o awa 25 Okin *GF Kueishan Islet

5 Km 24o Taiwan 10

23o Kueishan Islet

N NW #Y 1Km E SW 15 S

Figure 1. Map showing the sampling sites. *DX: Euplica sp. from Da-xi (24.9413°N, 刪除: 121.90390°E); *GF: Euplica sp. from Geng-fang (24.9046°N, 121.87200°E); □: Anachis misera from the white vent (24.8341°N, 121.96196°E); #Y: yellow vent (24.8355°N, 121.96371°E); N: north; E: east; S: south; SW: southwest; NW: 20 northwest (Source: Google map). 刪除: Map showing the collection site of Anachis

misera (Source: Google map).

(B) (A) (C)

(D) (E) (F) 10

(G) 15

SL T2

20 SW

Figure 2. Shell morphology and x-ray photos of Anachis misera around the vent off 刪除: Kueishan Islet and Euplica sp. from non-vent control sites of Da-xi and Geng-fang. (A) A. misera from the East; (B) A. misera from the South; (C) A. misera from the 25 Southwest; (D) A. misera from the Northwest; (E) Euplica sp. from Da-xi; (F) Euplica sp. from Geng-fang; (G) x-ray photos of A. misera from the South (left) and the Northwest (right). Scale bar: 5mm; SL: shell length; SW: shell width; T1: thickness of body whorl; T2: thickness of penultimate whorl. 刪除: Biometric measurements of the shell of Anachis misera.

SL: shell length; SW: shell

width; T1: thickness of body

whorl; T2: thickness of penultimate whorl; Left: Snail

from the South; Right: Snail

from the Northwest.

Marker South Southwest (kD) Marker 133 134 135 25 26 27

170 5 130 95 72 1 55 2 3 4 10 43 5 34 6 7 8 26 9 10 11

17 12 15

20 14 15 16

10 25

Figure 3. Gel electropherogram with molecular markers of Anachis misera. Number: 刪除:

protein band serial number. 刪除: Length frequency distribution of Anachis misera

around the vent off Kueishan

Islet. N=sample size.

刪除:

20 Figure 4. Results from the combined principal component analysis (PCA) and cluster analysis. The cluster analysis of Bray–Curtis similarity (BCs) indices using standardized overall protein expressions of Anachis snails from different sampling sites. E: east, S: south; SW: southwest; NW: northwest; 1-16: protein spot variable. 25

刪除: Relationship between shell length and shell width of

Anachis misera from different

sites. S: standard error of the regression; Different letters

indicate that the regression

lines differ significantly

(p<0.05). 20

◆ DX y=0.646x-0.59, N=16; R2=0.982; p<0.0001 a △ GF y=0.058+0.566x, N=30; R2=0.960; p<0.0001 b ＊ V-South y=2.882+0.17x, N=65; R2=0.281; p<0.0001 c V-Rest y=3.463+0.10x, N=75; R2=0.068; p=0.01 c 8 5

10 5

Shell width (mm) 3 15 5 6 7 8 9 10 11 12 13 14

Shell length (mm)

Figure 5. Relationship between shell length and shell width of Anachis and Euplica snails from different sites. Different letters indicate that the regression lines differ 20 significantly (p<0.05). 刪除: Relationship between shell length and total weight of Anachis misera from different sites. S: standard error of the

regression; Different letters indicate that the regression

lines differ significantly

(p<0.01).

◆ DX logW = -0.986 + 3.298 logL; N=16; R2 = 0.946; p<0.0001 b △ GF logW = -0.822 + 3.121 logL; N=30; R2 = 0.941; p<0.0001 a ＊ V-South logW = 1.288 + 0.827 logL; N=65; R2 = 0.219; p<0.0001 b

V-Rest logW = 1.494 + 0.618 logL; N=75; R2 = 0.096; p<0.007 b 500 5 400

300

10 200 Total weight (mg) 100

15 0

567891011121314 Shell length (mm)

Figure 6. Relationship between shell length and total weight of Anachis and Euplica 刪除: 20 snails from different sites. Different letters indicate that the logarithmic transformed regression lines differ significantly (p<0.01).

刪除: Standardized shell traits of Anachis misera. (A) Shell

shape; (B) Thickness of body whorl (T1); (C) Thickness of

penultimate whorl (T2). Data are shown as Mean±SD and

ranges. Means that differ significantly from each other

are indicated by different

letters (p<0.05).

刪除:

900900 ◆ DX y=55.256x-20.281, N=16; R2=0.425; p=0.006 b △ GF y=69.132x-127.695, N=30; R2=0.501; p<0.0001 a

5 700700

500 500

刪除: Gel electropherogram 300300 with molecular markers of

10 Thickness of T1 (um) 100 Anachis misera. Number: 100 protein band serial number. 700700 ◆ DX y=60.618x-202.687, N=16; R2=0608; p=0.0004 a 567891011121314△ GF y=56.769x-202.105, N=30; R2=0641; p<0.0001 a 刪除: 500500 分分分分 15 300300

100ThicknessT2 (um) of 100 5 6 7 8910 11 12 13 14 5 6 7 8 9 10 11 12 13 14 Shell length (mm) 20 Figure 7. Shell thickness of Anachis and Euplica snails. (A) Thickness of body whorl (T1); (B) Thickness of penultimate whorl (T2). *: V-South; : V-Rest; Different letters indicate that the regression lines differ significantly (p<0.05).

Figure 8. Results from the combined principal component

analysis (PCA) and cluster

analysis. The cluster analysis

of Bray–Curtis similarity (BCs) ... [14] 23

第 2 分: [1] 刪除 user 2015/3/1 8:33:00 PM If a positive correlation between shell length and shell width or total weight existed, the coefficient of determination (R2) of the equations was low, i.e., 0.207-0.444. Snails from the Northwest site (pH 7.33) exhibited a more globular shape than those of the South ones (pH 7.80). Standardized shell thickness

第 2 分: [2] 刪除 user 2015/3/1 8:35:00 PM (thickness of body whorl:shell length)

第 2 分: [3] 刪除 user 2015/3/1 8:35:00 PM (thickness of penultimate whorl:shell length)

第 2 分: [4] 刪除 user 2015/2/28 2:23:00 PM In a similar vein, based on the 16 examined protein spots, protein expression profiles of snails in the South were distinct. With further characterization by principle component analysis, the separation was mainly contributed by the first (i.e., spots 8, 1, 15, and 12) and second (i.e., spots 15, 13, 12, 1, and 11) principal-components.

第 5 分: [5] 刪除 user 2015/3/1 10:27:00 AM Shell lengths of the snails were from 6.88 to 11.01mm (Table 1). Based on the length-frequency histograms, it is found that the relative proportions of size classes differed among sites (Fig. 4). Positive correlation between shell length and shell width was only observed in the South and Northwest samples, and their slopes were significantly different (Fig. 5). The same pattern was also observed in the relationship between shell length and total weight (Fig. 6). It is noticed that the coefficient of determination (R2) of the equations was low, i.e., 0.207-0.444 (Figs. 5 and 6).

第 6 分: [6] 刪除 user 2015/3/1 9:46:00 AM 3.3 Protein expression profiles In the protein expression profiles of Anachis snails, 16 protein spots were selected for cluster analysis (Fig. 8). Based on Bray-Curtis Similarity (BCs) Indices, classification of the snails falls into three clusters. Snails from the high pH South were all within one cluster. In contrast, snails from the remaining sites were indistinguishable in other clusters. With further determination of the contribution of each protein variable, the data were characterized by principle component analysis (PCA). The first to the fifth principal components accounted for 35.4, 28.5, 13.2, 8.8, and 4.2% of the total variance, respectively. The separation was mainly contributed by the first (i.e., spots 8, 1, 15, and 12) and second (i.e., spots 15, 13, 12, 1, and 11) principal-components.

第 6 分: [7] 刪除 user 2015/3/5 7:12:00 AM Our results showed that Anachis snails survived and differed in their ecophysiological performance under varying degrees of low pH stress.

第 6 分: [8] 刪除 user 2015/3/5 7:12:00 AM More specifically, snails in the acidic Northwest site (pH 7.33) possessed thinner shells and were more globular in shape than those of the South (pH 7.80) (Fig. 7). At the biochemical level, the protein expression profiles of snails from the South were distinguished from the others (Fig. 8). Overall, the effects of vent environments on snails at physiological and biochemical levels were comparable.

第 9 分: [9] 刪除 user 2015/3/5 1:18:00 PM Marine snails possessing shells with a more elongated shape are found to be more vulnerable to crab predation, possibly due to higher handling efficiency compared with a more globular shell (Cotton et al., 2004).

第 9 分: [10] 刪除 user 2015/3/5 1:39:00 PM This reduction in shell thickness may increase the organism’s susceptibility to crushing predators (Boulding and Van Alstyne, 1993; Trussell and Etter, 2001). As shell thickness is reduced under low pH and elevated temperature, acquiring a more globular shape could enable snails to compensate better (Melatunan et al., 2013). Compared with deep-sea vent studies, in the northwest Eifuku volcano, Mariana arc, the vent mussel, Bathymodiolus brevior inhabiting low pH environments (pH 5.36-7.29), exhibited shell thickness and daily growth increments in shells of only about half of the ones with pH>7.8 (Tunnicliffe et al., 2009). Along the Mid-Atlantic Ridge, the expression profiles of 35 proteins from the gill of Bathymodiolus azoricus revealed clear separation among sites, which indicates that specific adaptations of B. azoricus depend on local conditions (Companya et al., 2011). Moreover, it has been reported that snails of Melaraphe neritoides, Patella caerulea, and Patella rustica distribute in shallow vents off Ischia (pH 6.53) (Hall-Spencer et al., 2008). However, shell traits of these snails were not evaluated.

第 9 分: [11] 刪除 user 2015/3/5 1:37:00 PM compared to the South site (pH 7.80), snails from the East, Southwest, and Northwest (pH 7.66, 7.80, and 7.33, respectively) changed

第 9 分: [12] 刪除 user 2015/3/5 1:43:00 PM in the acidic Northwest site

第 9 分: [13] 刪除 user 2015/3/5 1:03:00 PM 4.2 Comparison with other Anachis studies Among the Anachis species (Family: Columbellidae), Anachis avara is a common one living on the coast of the eastern United States (Scheltema, 1968; Hatfield, 1980). At Bear Cut, FL, the population of A. avara showed seasonal fluctuation in its size structure (Hatfield, 1980). It reached a mean terminal size of 10.50 mm (8.00-13.29 mm) and matured quickly at the age of six to seven months. The estimated life span was less than two years. It is suggested that the fluctuation in size structure was primarily the result of seasonal recruitment, and the abundance was probably determined by predation. In Anachis fluctuate, the regression equation of shell length (mm) and dry tissue weight or shell weight (g) had been reported, i.e., Y = -0.025+0.003SL (R2=0.88; N=26) and Y=-2.39+1.04lnSL (R2=0.92), respectively (Bertness and Cunningham, 1981). By comparison, in this study, shell lengths of A. misera were from 6.88 to 11.01mm (Table 1). Variations in size structure among sites were also obvious. In addition, if a positive correlation between shell length and shell width or total weight in A. misera was present, the R2 of the equation was low, i.e., 0.207-0.444 (Figs. 5 and 6). The standard error of the regression varied from 0.04 to 4.50, which indicated that the precision of the regression models was low, particularly in the relationship of shell length and body weight. Although differential recruitment and acidic stress are potential factors to account for such discrepancy, further study is needed to address this question.

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Figure 8. Results from the combined principal component analysis (PCA) and cluster analysis. The cluster analysis of Bray–Curtis similarity (BCs) indices using standardized overall protein expressions of Anachis snails from different sampling sites. E: east, S: south; SW: southwest; NW: northwest; 1-16: protein spot variable.