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 (e.g., Fabry et al.,
2008; Sokolov et al., 2009), specific information obtained from field
investigations has been limited, particularly with regard to gastropod snails
(Gazeau et al., 2013). Thus, the current study was performed to address this
issue within an extreme hydrothermal environment.
Map showing the sampling sites. *DX: Euplica sp. sampling
site at Da-xi (24.9413∘ N, 121.90390∘ E); *GF:
Euplica sp. sampling site at 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: northwest (Source: Google Maps).
The shallow hydrothermal vents of interest are located east of Kueishan (KS)
Islet, Taiwan, near the southern end of the Okinawa Trough (Fig. 1). The
vents emit yellow or white plumes, with temperature and pH varying in the
ranges of 78–116 ∘C and 1.52–6.32 and 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 < 50 ppm 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 diffuse mainly from north to south
due to ebb tide and move 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 diffusion is closely correlated with diurnal tides, benthic
organisms would face the lowest pH twice per day but for no more than 4 hours each time.
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 kinds of sea anemones, hexacoral Tubastraea aurea, serpulid polychaete, a
chiton, the snail Nassarius sp., and the dove snail Anachis misera. These vent organisms naturally
inhabit acidic and toxic environments. High concentrations of trace metals
in various tissues of the crab X. testudinatus are reported, and the levels are not
above those found in other crabs collected from different habitats (Peng et al., 2011).
We herein test the hypothesis that populations of A. misera distributed around vents
exposed to varying degrees of plumes would exhibit a different
ecophysiological performance compared to the non-vent dove snail, Euplica sp., a
common species in coastal waters off 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 the protein expression profile
of each snail by cluster analysis, similarities among samples can be
determined and classified. This method has been applied to laboratory and
field pollution studies, such as studies on blue mussels exposed to polyaromatic
hydrocarbons and heavy metals (Knigge et al., 2004) and on Sydney rock oysters
inhabiting an acid sulfate runoff estuary (Amaral et al.,
2012).
Materials and methods
Sampling sites and collection of snails
Anachis misera was collected around a shallow-water vent in Kueishan Islet, Taiwan (Fig. 1), including the north (N), east (E), south (S), southwest (SW), and
northwest (NW) sites during the period of 28 June to 1 July 2011. The
sampling vent emitted white plumes, and another vent with yellow plumes was
nearby to the northeast. The distance of the collection sites to the vent
center was 10–16 m, and the water depth was in the range of 14.5–17.5 m.
Snails of Euplica sp. were sampled from Da-xi (DX) and Geng-fang (GF), northeastern
Taiwan, between July and September 2012.
Sampling locations and environmental parameters of vent sites in the
Kueishan Islet and northeastern Taiwan. Data are shown as mean ± SD
(range of parameter).
Site
White vent
Yellow vent
Da-xi (DX)
Geng-fang (GF)
Latitude
24.8341∘ N
24.8355∘ N
24.9413∘ N
24.9046∘ N
Longitude
121.96196∘ E
121.96371∘ E
121.90390∘ E
121.87200∘ E
Depth ( m)
17.0
9.5
3.0
3.0
Fluid flux (m3 h-1)
18.5
21.0
-
-
Temperature (∘C)
55.0
115.0
27.2 ± 0.2 (27.0–27.4)
27.4 ± 0.5 (27.0–27.9)
pH
4.0
2.3
8.13 ± 0.06 (8.1–8.2)
8.13 ± 0.06 (8.1–8.2)
Sampling locations were identified by scuba divers equipped with GPS.
Temperature was determined by a thermometer inserted into the seawater
samples. The flow rate was measured by a Hydro-Bios digital flowmeter (Model
438 110). The pH was measured by a pH meter (Radiometer, Copenhagen,
Denmark). Each environmental parameter was determined with one or three
replicates, and the results are shown in Table 1. The collected snails were
preserved in dry ice in the field. Upon returning to the laboratory, they
were deep-frozen at -70 ∘C for later use.
Measurements of snail morphological traits
Shell traits, i.e., shell length and width, shell thickness of the body whorl
(T1) and the penultimate whorl (T2), as well as the total weight of the intact
individual, were measured (Fig. 2). Shell thickness was determined through
enlarged X-ray radiographs which were produced by exposing snail shells to
X-rays at 80 kVp and 1 mA for 116.7 ms. The distance between
the X-ray source and the objects was 50 cm. The shell images were further
drawn with 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
compare the least square means (LSM) for each variable (i.e., shell width,
total weight, shell thickness T1 and T2) among sites with shell length as the
covariate. The relationships of shell length to shell width and the shell
thickness of T1 and T2 were calculated using linear regression analysis. If
the relationship of total weight and shell length was curvilinear, linear
regression slopes were obtained and compared after data were logarithmically
transformed.
Proteomic study
The protein expression profiles of Anachis snails were determined by
one-dimensional sodium dodecyl sulfate–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 were centrifuged at 13 000 g for 10 min at 4 ∘C. The homogenous
supernatant was collected, and the protein concentration was determined by
Bradford assay, using bovine serum albumin as the standard.
Shell morphology and X-ray photos of Anachis misera around
the vent off Kueishan Islet and Euplica sp. from non-vent control
sites of Da-xi and Geng-fang. (a) A. misera from the east
site; (b) A. misera from the south site; (c)
A. misera from the southwest site; (d) A. misera
from the northwest site; (e) Euplica sp. from Da-xi;
(f) Euplica sp. from Geng-fang; (g) X-ray photos
of A. misera from the south site(left) and the northwest site
(right). Scale bar: 5 mm; SL: shell length; SW: shell width; T1: thickness
of body whorl; T2: thickness of penultimate whorl.
The stacking and resolving gels were prepared with percentages of 5
and 12 % (Hoefer SEM 260 system, Amersham Pharmacia). After loading 25 µg
protein in each sample lane, electrophoresis was run for 30 min at 120 V and
then for 4 h at 180 V. The gels were stained with Coomassie blue G-250 (Candiano
et al., 2004).
Stained gels were scanned and transformed into digitalized images using Image
Scanner (Amersham Pharmacia). The Multi Gauge software v2.2 (Fujifilm) was
utilized for protein quantification. The protein bands were assigned band
numbers, and their intensity levels were calculated as their relative area to
the total protein area on the gel. A cluster analysis of the Bray–Curtis
similarity (BCS) indices (Primer 6.0) was employed to compare the expression
of overall protein patterns among snail individuals (Clarke and Warwick,
2001). In addition, the contribution of each protein band was further
estimated by principal component analysis (PCA).
Environmental parameters and shell traits of Anachis misera
around the vent off Kueishan Islet. Data are shown as mean ± SD (range
of paramenter). SL: 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 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 (∘C)
27
27
27
27
26
pH
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)
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)
T1 (µm)
-
199 ± 56 (136–285)
225 ± 69 (109–481)
200 ± 56 (118–290)
168 ± 49 (79–276)
T2 (µm)
-
188 ± 44 (109–248)
200 ± 51 (112–328)
205 ± 55 (117–354)
180 ± 55 (79–325)
Results
Morphological traits of Anachis snails from vent sites
The temperature ranges of the sampling sites were from 26 to 27 ∘C (Table 2). Spatial variability in pH among sites was clearly observed, with the
lowest pH being 7.22 ± 0.03 at the north site (p< 0.01).
Anachis snails were found around the vent, except at the most acidic north site.
Shell lengths of the snails ranged from 6.88 to 11.01 mm. Several snails
with an eroded apex were observed at the east and northwest sites (Fig. 2).
Protein expression profiles of Anachis snails from vent sites
Based on the protein expression results, 16 protein bands were selected for
further Bray–Curtis similarity (BCS) analysis (Fig. 3). The classification
of snails fell into three clusters (Fig. 4). Snails from the high-pH south
site were all within one cluster. In contrast, snails from the remaining
sites were indistinguishable from other clusters. In the process of the
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., bands 8, 1, 15, and 12) and second (i.e., bands 15, 13, 12,
1, and 11) principal components.
Based on the cluster results, the Anachis snails were classified into groups of
V-South (pH 7.78–7.82) and V-Rest (pH 7.31–7.83). Their shell traits were subsequently compared to non-vent Euplica snails (pH 8.10–8.20).
Comparison of the shell traits of dove snails among vent and
non-vent sites
Shell traits of the Anachis and Euplica snails were listed in Table 3. A positive
correlation between shell length and shell width was observed in all
populations (Fig. 5). A difference in shell shape (shell width : shell length), with vent
populations having more globular shells, was also found, as shown by the significant
difference in regression slopes. ANCOVA with shell length as the
covariate showed the mean values of shell width to be significantly different among
sites (p< 0.01), with a descending order of GF > DX
> V-South and V-Rest (Table 3).
Gel electropherogram with molecular markers of Anachis misera. Number: protein
band serial number.
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 (range of parameter): shell
length; SW: shell width; TW: total weight; T1: thickness of body whorl; T2:
thickness of penultimate whorl. Least square (LS) means that differ
significantly from each other are indicated by different letters.
Site
V-South
V-Rest
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)
LS mean 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)
LS mean of TW (mg)
118.07 ± 8.30 b
117.59 ± 8.98 b
105.94 ± 4.28 b
149.66 ± 5.70 a
T1 (µm)
225 ± 69 (109–481)
232 ± 31 (141–299)
385 ± 113 (243–653)
536 ± 171 (201–852)
LS mean of T1 (µm)
255 ± 73 c
228 ± 70 d
446 ± 76 b
514 ± 71 a
T2 (µm)
200 ± 51 (112–328)
234 ± 36 (148–304)
241 ± 104 (147–588)
343 ± 124 (157–702)
LS mean of T2 (µm)
256 ± 56 b
230 ± 52 c
295 ± 60 a
324 ± 55 a
The relationships of shell length to total weight were curvilinear for both
Anachis and Euplica snails (Fig. 6). The slopes among sites were significantly different,
and the mean body weight of the GF population was significantly greater than
that of the others (Table 3 and Fig. 6).
Results from the combined principal component analysis (PCA) and
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:
variable of protein bands.
Relationship between shell length and shell width of
Anachis and Euplica snails from different sites. Different
letters following the values in the legend indicate
that the regression lines differ significantly (p< 0.05).
Relationship between shell length and total weight of
Anachis and Euplica snails from different sites. Different
letters following the values in the legend indicate that the logarithmic
transformed regression lines differ significantly (p< 0.01).
Positive correlations between the shell length and shell thickness of body
whorl (T1) and penultimate whorl (T2) were only observed in non-vent GF and
DX populations. Their slopes were significantly different for T1 only
(Fig. 7). The mean shell thickness of T1 and T2 varied among sites (Table 3).
Anachis snails from vent sites were thinner in T1 compared to the
non-vent Euplica snails (p< 0.001), with a descending
order of GF > DX > V-South > V-Rest. A
similar trend was also found in T2. Within each vent site, shell thickness
between T1 and T2 was insignificantly different (paired t test,
p> 0.05). By comparison, T1 and T2 of the snails from V-Rest
were 89.4 and 89.8 %, respectively, of those of the V-South snails. In
the comparison of vent and non-vent sites, T1 and T2 of the Anachis
snails from V-Rest were 44.4 and 71.0 %, respectively, of those of the
Euplica snails from GF. Clearly, both measurements of shell
thickness decreased under acidic environments.
Discussion
This was the first study to compare morphological traits of snails under
varying shallow-vent stresses with populations previously classified by
biochemical responses. A difference in shell shape (shell width : shell length),
with vent populations having more globular shells, was found. Snails from V-Rest (pH
7.31–7.83) exhibited a 10.6 and 10.2 % decrease in shell thickness of
the body whorl (T1) and penultimate whorl (T2), respectively, compared to snails
from V-South (pH 7.78–7.82). Compared to non-vent sites (pH 8.10–8.20),
T1 and T2 of the Anachis snails from V-Rest showed a 55.6 and 29.0 % decrease
in T1 and T2, respectively, relative to Euplica snails from GF. Our shallow-vent-based results were, in general, consistent with laboratory, controlled,
and deep-sea vent studies, i.e., shell organisms are susceptible to acidic
environments.
Application of proteomic-based approach to shallow-vent snails
The proteomic-based method has been used in environmental toxicology to
characterize an 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 studies in which the blue mussel
Mytilus edulis was exposed to polyaromatic hydrocarbons and heavy
metals (Knigge et al., 2004), to crude oil (Mi et al., 2006), to
polychlorinated biphenyls, and polycyclic aromatic hydrocarbons extracted from Baltic Sea sediments (Olsson et al., 2004) and
studies in which the mussel Mytilus galloprovincialis was exposed to
a tributyltin-polluted area (Magi et al., 2008).
The application of the proteomic approach to the 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
proteins from the gill revealed a clear difference among sites, which
indicates that specific adaptations of B. azoricus depend on local conditions.
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
following the values in the legends indicate that the regression lines differ
significantly (p < 0.05).
It is known that large spatial and temporal variations in environmental
parameters, such as temperature, pH, and hydrothermal fluid composition are
detected around vent environments with regard to dissolved oxygen, methane,
and sulfide concentrations, etc. The pH of the hydrothermal fluids within our
sampling vent and the surrounding seawater was determined on 31 May 2011,
with pH ranges from 2.29 to 5.11 and 5.51 to 6.15, respectively (Zeng et al.,
2013). The diffusion activities of vent plumes were also evaluated through
the environmental factors of temperature, pH, and Eh (Han et al., 2014). The
diffusive plume is mainly affected by the wind, sea waves, and tides. If
ocean currents in the east–west direction are not considered, sea currents
around the vents are from north to south during ebb tide, whereas, in flood
tide, the opposite direction dominates. Our proteomic results indicated that
snails from the south site were distinguished from those at the rest of the
sites; this is consistent with the diffusion of local vent fluids.
Comparison with other dove snail studies
Anachis avara is a common dove snail (Family: Columbellidae) living
off 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 6–7 months. The
estimated life span was less than 2 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 (SL)
(mm) and dry tissue weight or shell weight (g) has been reported as Y=-0.025+0.003 SL (R2=0.88; N=26) and Y=-2.39+1.04 lnSL
(R2=0.92), respectively (Bertness and Cunningham, 1981). By comparison,
in this study, shell lengths of A. misera and Euplica sp.
ranged from 6.88 to 11.01 and 3.40 to 7.56 mm, respectively (Tables 2 and
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 values 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 the body whorl (T1) and penultimate whorl (T2) were only found
in non-vent populations with an 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.
Comparison with other ocean acidification studies
To date, ocean acidification studies have been conducted mostly in the
laboratory or controlled environments for a short period of time. The
results indicate that exposure to future global change scenarios (Caldeira
and Wickett, 2003; Sokolov et al., 2009) may alter the tolerance of
calcifying species and, ultimately, their fitness and survival through
complex physiological and ecological pathways. Based on data from the
literature, it is concluded that the effects of acidified seawater on species
growth occurred at higher pH than those on species reproduction (mean pH10
was 7.73 vs. 7.63 and mean pH50 was 7.28 vs. 7.11) (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 high
reductions in recruitment in the acidified stations (Cigliano et al., 2010;
Ricevuto et al., 2012; Milazzo et al., 2014). In experiments in which juvenile
pen shells Pinna nobilis were 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 compared to those at pH 8.1 was
reported (Basso et al., 2015). In studies on the limpet Patella caerulea within and outside
the Ischia vent, shell formation and dissolution are both observed at a 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 absent at normal-pH sites. The
nassariid gastropods Nassarius corniculus and Cyclope neritea adapted to the Ischia vent were smaller than those
found in normal-pH conditions and had a higher mass-specific energy
consumption but a significantly lower whole-animal metabolic energy demand
(Garilli et al., 2015). Compared with deep-sea vent studies, on the
northwest of Eifuku Volcano, Mariana Arc, the vent mussel Bathymodiolus brevior, inhabiting low-pH
environments (pH 5.36–7.29), exhibited a shell thickness and daily growth
increments in shells of only about half of that of mussels living in environments with pH > 7.8
(Tunnicliffe et al., 2009).
Under low pH (7.7 vs. 8.0), the periwinkle Littorina littorea
increased less in weight and were shorter than snails grown in
normal conditions
(Melatunan et al., 2013). 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), of the larvae of the Mediterranean
pteropods Cavolinia inflexa (Comeau et al., 2010), and of the
mussels Mytilus edulis (Gazeau et al., 2010) and Mytilus californianus (Gaylord et al., 2011). Along a gradient of pH (5.78–8.30)
and salinity (3.58–31.2 p.s.u) in the Sungai Brunei Estuary, Malaysia,
whelk Thais gradata exposed to acidified sites possessed heavier
shells, and the degrees of erosion were negatively related to water pH and
calcium concentration (Marshall et al., 2008). At low pH (7.7), a 2.45 %
change in shell shape (shell width : shell length) towards a more globular
shell and a decrease in the outer lip shell thickness of up to 27 % in
Littorina littorea were observed (Melatunan et al., 2013).
In this study, the comparison of A. misera from V-South (pH
7.78–7.82) and V-Rest (pH 7.31–7.83) revealed that the change in shell
ratio was 3.4 % and shells were more rounded in the V-Rest group. In
addition, snails of V-Rest exhibited a 10.6 and 10.2 % decrease in shell
thickness of the body whorl (T1) and penultimate whorl (T2), respectively,
compared to the V-South snails. In the comparison of vent and non-vent sites,
T1 and T2 of the Anachis snails from V-Rest were 44.4 and
71.0 %, respectively, of those of the Euplica snails from GF (pH
8.1–8.2). Our shallow-vent-based results were, in general, consistent with
other laboratory, controlled, and field studies, i.e., shell organisms are
susceptible to acidic environments.
It is known that vent systems are not entirely representative of future ocean
changes, not only because of the temporal variability in pH but also because
of the existence of other toxic elements. However, vents' acidifying
environments are sufficiently large on spatial and temporal scales
for a valid comparison. It is a naturally occurring system to assess the effects of ocean acidification on the whole
life cycle and across multiple generations of target organisms.