ORIGINAL RESEARCH

published: 23 September 2021

doi: 10.3389/fmars.2021.688894

Why Are Barnacles Common on

Intertidal Rocks but Rare in Rock
Pools? Effect of Water Temperature,
Salinity, and Continuous
Submergence on Barnacle Survival in
Indian Ocean Rock Pools
Nutcha Buasakaew 1 , Benny K. K. Chan 2* and Kringpaka Wangkulangkul 1*
1
Division of Biological Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand, 2 Biodiversity Research
Center, Academia Sinica, Taipei, Taiwan
Edited by:
Mandar Nanajkar,
Council of Scientific and Industrial
Barnacles are less common in rock pools of the rocky intertidal zone than on open rock
Research (CSIR), India surfaces adjacent to those pools. Rock pools on the Andaman coastlines of Thailand
Reviewed by: showed diurnal variations in water temperature, salinity, pH, and dissolved oxygen level,
Guoyong Yan,
peaking in the afternoon. Multivariate analysis showed that water temperature and
Hong Kong University of Science and
Technology, Hong Kong, SAR China salinity (not pH and dissolved oxygen) can affect barnacle abundance in rock pools.
Sumit Mandal, The present study tests the hypothesis that a lack of recruitment or pool environmental
Presidency University, India
conditions (continuous submergence, water temperature, and salinity extremes) affect
*Correspondence:
Benny K. K. Chan
the abundance of barnacles (Chthamalus malayensis and Amphibalanus amphitrite) in
chankk@gate.sinica.edu.tw rock pools. During the recruitment season, recruits were found in a number of rock
Kringpaka Wangkulangkul
pools, but at significantly lower abundances than those in adjacent open rock surfaces.
kringpaka.w@psu.ac.th
In a laboratory experiment, C. malayensis and A. amphitrite that were continuously
Specialty section: submerged had a lower survival (80%) than the controls (simulated tide conditions; >90%
This article was submitted to survivals). Under different water temperature and salinities treatments, barnacles had low
Marine Evolutionary Biology,
Biogeography and Species Diversity, survival (<50%) in the low salinity (0◦ /oo ) and high temperature treatment (40◦ C). The
a section of the journal present study suggests that C. malayensis and A. amphitrite can recruit into rock pools,
Frontiers in Marine Science
and the interplay of continuous submergence, high water temperature and low salinity in
Received: 31 March 2021
rock pools can bring about mortality in barnacles.
Accepted: 24 August 2021
Published: 23 September 2021 Keywords: acorn barnacles, intertidals, rock pools, salinity, submergence, Thailand, Andaman Sea
Citation:
Buasakaew N, Chan BKK and
Wangkulangkul K (2021) Why Are INTRODUCTION
Barnacles Common on Intertidal
Rocks but Rare in Rock Pools? Effect The factors affecting species distribution constitute an interesting topic in ecological studies. The
of Water Temperature, Salinity, and
rocky intertidal zone can comprise a variety of microhabitats, including vertical rocks, horizontal
Continuous Submergence on
Barnacle Survival in Indian Ocean
rocks, shaded and exposed surfaces, rock pools, and crevices (Williams and Morritt, 1995; Seabra
Rock Pools. et al., 2011). These microhabitats differ mainly in their levels of heat stress, which affect species
Front. Mar. Sci. 8:688894. distribution (Williams and Morritt, 1995; Wang et al., 2020). Rock pools are depressions on
doi: 10.3389/fmars.2021.688894 rocky shores that retain water during low tide. Rock pools are foraging habitats and refugia from

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Buasakaew et al. Rarity of Barnacles in Rock Pools

predation and physical stresses for many intertidal species can tolerate a range of water temperatures (thermal windows)
(Moran, 1985; Delany et al., 1998; Laure et al., 2009). The and salinities (Sherwood et al., 2012; Diaz et al., 2021). When
physico-chemical environment of rock pools, comprising water barnacles experienced extremely high temperatures, heart beats
temperature, salinity, pH, and dissolved oxygen levels, is largely increase, and they subsequently enter a coma state (opercular
regulated by the tides (Metaxas and Scheibling, 1993; Chan, valves open without responses under stimulations). Barnacles
2000; Legrand et al., 2018). This environment also varies with will die if the coma condition persists for a long period (Chan
the volume, surface area, depth, and tidal position (intertidal vs. et al., 2006). Gonads will also be impacted by high temperatures
supratidal) of pools (Metaxas and Scheibling, 1993; Chan, 2000). and result in a lower number of larvae produced (Fraser and
When pools are disconnected from the sea, temperature, salinity, Chan, 2019). Moreover, intertidal barnacles that live on rock
pH, and dissolved oxygen concentration can be affected by solar surfaces experience tidal inundations, but those that live in
radiation from direct sunlight and bedrocks, water evaporation, pools experience continuous submergence. It is possible that
and the pool fauna and flora (Morris and Taylor, 1983). Salinity in continuous submergence is not the best living condition for
tropical rock pools can be as low as 0◦ /oo during heavy rain and as intertidal barnacles.
high as 40◦ /oo during hot days (Chan, 2000; Firth and Williams, The present study aims to investigate if and how
2009). Intertidal pools on the mid-low shores experience less recruitment and the pool physico-chemical environment
variation in physico-chemical parameters compared to supratidal determine barnacle distribution in rock pools. We tested the
pools. They often have higher species richness and biodiversity hypothesis that barnacles are not common in rock pools on
than their supratidal counterparts (Moschella et al., 2005; Firth the Andaman rocky shores due to (1) a lack of recruitment,
et al., 2013, 2014; Evans et al., 2016). Sponges, ascidians, hydroids, (2) continuous submergence, and (3) the effects of extreme
and gobies are often found in intertidal pools (Evans et al., 2016). salinity and temperature on barnacle survivals in pools
Barnacles (Suborder Balanomorpha) are common inhabitants was tested.
of intertidal rocky shores in Thailand (Pochai et al., 2017;
Sukparangsi et al., 2019; Chan et al., 2020). They are MATERIALS AND METHODS
habitat modifiers and can regulate the abundance and
distribution of other organisms (Branch, 1976; Amnuaypon Study Shores
and Wangkulangkul, 2018). In the temperate Atlantic, barnacles All rocky shores sampled were located in the Andaman Sea,
are rarer in rock pools than on open rock surfaces (Singletary Thailand (Figures 1D,E). The barnacle abundance and the
and Shadlou, 1983; Firth et al., 2014; Evans et al., 2016; Hall et al., physico-chemical environment of pools and the differences
2019). Two studies have been conducted in temperate regions of in barnacle abundance between pools and on rock, were
the Atlantic Ocean to examine factors affecting the distribution examined on shores with a number of rock pools in Laem
of barnacles in rock pools. Magre (1974) revealed that Balanus Panwa (7◦ 47′ 57.7"N 98◦ 24′ 30.1"E), Pleum Sook (7◦ 53′ 13.7"N
(Semibalanus) balanoides settle in rock pools, and its settlement 98◦ 26′ 31.7"E), Laem Sai (7◦ 37′ 45.7"N 99◦ 14′ 01.2"E), Whahin
density decreases with pool depth. Singletary and Shadlou (1983) Island (6◦ 47′ 14.0"N 99◦ 45′ 38.3"E), and Tanyong Po (6◦ 35′ 27.1"N
qualitatively monitored physical and biological parameters in 99◦ 56′ 53.9"E). Lanta Island (7◦ 35′ 23.2"N 99◦ 01′ 50.2"E) was
rock pools and found that B. balanoides can settle in pools at high visited only to take diurnal measurements of the physico-
volumes, but these settlers soon die from hypoxia. Chemicals chemical parameters of the rock pools. Lanta Island is suitable
released by pool algae can also affect the survival of barnacle for such diurnal measurement because the accommodation
spats (Singletary and Shadlou, 1983). Predation by snails, which site is very close to the field sites, which allows overnight
are common in pools, might also cause mortality (Singletary and monitoring possible. Recruitment surveys of barnacles on rocks
Shadlou, 1983). and in pools were conducted in Tanyong Po and Had Sai
Chthamalus malayensis Pilsbry, 1916 (Chthamalidae) Yao (6◦ 37′ 13.5"N 99◦ 57′ 10.2"E). All shores are wave-exposed
(Figure 1A) and Amphibalanus amphitrite (Darwin, 1854) or semi-exposed. Tide is semi-diurnal with a maximum tidal
(Balanidae) (Figure 1B) are the most common acorn barnacles range of ∼2.5 m. Shale was the predominant type of rock in the
on intertidal rocky shores off the tropical Andaman coast of study areas.
Thailand (latitude ∼6.4◦ -10◦ N) in the Indian Ocean (Tsang
et al., 2012). C. malayensis is common on mid-high shores, while Physico-Chemical Environment of Rock
A. amphitrite occurs on low shores (Chen et al., 2014; Pochai Pools and Barnacle Abundance
et al., 2017). Rock pools are common in the intertidal zone of the In September 2019, 20 pools were selected in each of the five
Andaman coast. Barnacles (C. malayensis and A. amphitrite) are sampled shores and their physico-chemical environments were
common on open intertidal rock surfaces, but rare inside rock surveyed. Pools were selected based on the following criteria: (1)
pools (Figure 1C). Factors affecting the distribution of barnacles no more than 12-cm deep (to reduce the effect of heterogeneity
in tropical rock pools can be different from the results previously within the habitat), (2) no cobbles (cobbles can be stirred by water
addressed in the temperate Atlantic Ocean. In the tropics, currents and create unstable habitat within the rock pool), and (3)
barnacles are rare in rock pools, which may be due to a lack in the intertidal zone (mid-shore level), at the same, tidal level as
of recruitment in pools. If barnacles can recruit in rock pools, the barnacle zone.
then extreme variations in salinity and temperature inside the The pool depths were measured using a ruler (±10 mm).
pool waters might affect barnacle survival. Marine invertebrates Each pool was photographed from the top of the rock

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Buasakaew et al. Rarity of Barnacles in Rock Pools

FIGURE 1 | (A) Chthamalus malayensis is a common barnacle on mid shores of the Andaman rocky shores. (B) Amphibalanus amphitrite are common on low shores
of the Andaman intertidal zone. (C) On the Andaman Coast of Thailand, barnacles are common on open rock surfaces adjacent to rock pools, but relatively rare inside
pools. (D) Study region on the Andaman coast of Thailand, magnified in (E). (E) Seven study shores spanning from the northern to southern coasts on the Andaman
coast of Thailand. (D,E) Courtesy of NOAA Bathymetric Data Viewer.

pools, then its surface area was calculated using the image Diurnal Variation in Physico-Chemical
analysis software ImageJ©. Temperature was measured using Parameters in Rock Pools
a glass thermometer (±0.1◦ C). Salinity was measured using a To examine diurnal variation in the water temperature, salinity,
refractometer. pH was measured by a Portable Digital pH Meter dissolved oxygen, and pH of rock pools (methods for measuring
(±0.1). Dissolved oxygen was measured by an OHAUS ST20 physico-chemical parameters as in section Physico-Chemical
portable DO meter (±0.1 mgl−1 ). A quadrat (10 × 10 cm) Environment of Rock Pools and Barnacle Abundance), six rock
was randomly placed inside each rock pool. The percentage pools (dimension criteria stated in section Physico-chemical
cover of C. malayensis and A. amphitrite was scored for Environment of Rock Pools and Barnacle Abundance) were
each quadrat. selected from a rocky shore on Lanta Island on September 16–
The effect of physico-chemical parameters of rock pools 18, 2020 (late monsoon season). It rained during some periods of
(water temperature, salinity, pH, and dissolved oxygen) on the measurement. The water temperature, salinity, dissolved oxygen,
abundance of barnacles (C. malayensis and A. amphitrite) was and pH of pools were measured hourly during low tide (when
analyzed using Multivariate Analysis (PRIMER 6.0 software). pools were exposed to the air) and every 3 h during high tide
The relationship between barnacle abundance and physico- (when pools were submerged).
chemical parameters was assessed using the BEST routine in
PRIMER (Clarke et al., 2008). The BIO-ENV function in the Comparison of Barnacle Abundance
BEST routine was used to calculate the smallest subset of Between Rock Pools and Adjacent
environmental variables that explained the greatest percentage Emergent Rocks
of variation in the ordination patterns using the Spearman Rank On each shore, whenever a rock pool within the selection
Correlation method. criteria in section Physico-Chemical Environment of Rock Pools

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Buasakaew et al. Rarity of Barnacles in Rock Pools

and Barnacle Abundance was found, a quadrat (10 × 10 cm) March to May 2020 because of logistical difficulties due to the
was randomly placed inside of that rock pools. A quadrat was COVID-19 lockdown.
established on the adjacent emergent rock surface (∼20 cm from Permutational Multivariate Analysis of Variance with 999
the rock pool). One quadrat was established for each pool due to permutations was performed to test the effect of Habitat (two
pool size limitations. Ten to 20 pools were sampled from each levels), Species (two levels), and Shore (five and three levels for
shore. In each quadrat, the percentage cover of C. malayensis and C. malayensis and A. amphitrite, respectively) on the percentage
A. amphitrite was scored. cover of barnacles. As suggested by Anderson et al. (2008),
Euclidean distance resemblance matrices (with a dummy variable
added) were constructed from untransformed data for all
Barnacle Recruitment in Rock Pools and univariate analyses. Data were pooled across the shores for the
on Adjacent Rock Surfaces first analysis, and Habitat and Species were treated as fixed factors
A quadrat (5 × 5 cm) was established to monitor recruitment and crossed. For the second analysis, data from each species
in rock pools and their adjacent rock surfaces (∼20 cm from were analyzed separately and Habitat was crossed with Shore
the pools). Barnacle recruits (defined as having a rostral-carinal (random factor). C. malayensis was found on all shores, whereas
diameter of 0.5–1 mm) in each quadrat were scored. It is A. amphitrite were not found in samples from Laem Panwa or
difficult to distinguish between recruits of C. malayensis and A. Whahin Island. Hence, there was a difference in the number
amphitrite, so recruits of both species were pooled. Recruitment of shores between species. PERMANOVA was also performed
monitoring was conducted monthly for 12 months, except from to evaluate the effects of Habitats (fixed, two levels), Months

FIGURE 2 | Variation in water temperature, salinity, pH, and dissolved oxygen with pool size dimensions (depth × surface area) from five study shores. Note that none
of the physico-chemical parameters were significantly correlated with pool size dimensions.

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Buasakaew et al. Rarity of Barnacles in Rock Pools

TABLE 1 | Range of variations in the physico-chemical environment of rock pools.

Shores Range of water parameters in pools

Temperature Salinity Dissolve oxygen pH Rock temperature

(◦ C) (◦ /oo ) (mg L-1 ) (◦ C)

Laem Panwa 27.5–28.4 10.0–34.5 1.0–3.9 7.4–7.8 27.7–28.4

Pleum Sook 27.3–28.3 32.0–35.0 0.9–2.4 7.3–7.6 26.8–28.3
Lanta Island 27.1–33.0 5.0–39.0 1.6–16.2 6.8–9.3 No data
Laem Sai 30.6–34.1 32.7–35.7 1.8–9.0 6.0–6.1 30.3–37.6
Whahin Island 31.2–37.2 33.0–35.0 4.5–11.0 8.6–9.7 32.1–41.4
Had Sai Yao 31.0–35.0 33.0–43.0 No data No data No data
Tanyong Po 28.9–37.5 30.6–46.0 3.6–8.6 6.0–6.1 27.2–30.9

Additional single measurements of salinity were conducted on 10 rock pools which has very high salinity in Tanyong pool, 6 rock pools in Had Sai Yao. Range recorded in these two
sites were greater than the paired dataset in Figure 2.

(fixed, nine levels), and Shore (random, two levels) on the

TABLE 2 | Results of Bio-Env analysis in the BEST routine of PRIMER 6, showing
density of recruits. Pairwise comparisons were conducted on the variation in the correlation coefficient with different subsets of
significant terms. physico-chemical variables.

Physico-chemical variables Spearman correlation

Effect of Continuous Submergence on coefficients
Barnacle Survival
Small pieces of rock with barnacles attached were collected by a Water temperature, salinity 0.30
chisel and hammer. Sixty adult barnacles of each species with 4– Water temperature 0.28
6 mm (for C. malayensis) and 10–12 mm (for A. amphitrite) basal Water temperature, salinity, dissolved oxygen 0.17
rostral-carinal diameter were collected from Tanyong Po. These Water temperature, salinity, pH 0.16
sizes were determined to be small enough to represent young Water temperature, salinity, dissolved oxygen, pH 0.16
and vulnerable individuals but large enough to be handled in Water temperature, dissolved oxygen, 0.15
the experiment. Specimens were acclimated in aerated seawater Salinity, dissolved oxygen 0.15
for 48 h prior to the experiment. Each species of barnacle Water temperature, dissolved oxygen, pH 0.14
was maintained in two treatments: continuous submersion and Dissolved oxygen, salinity, pH 0.14
control for 14 days. In the continuous submersion treatment,
barnacles were continuously immersed in seawater. In the control
treatment, barnacles were removed from the water for 4 h and
immersed again in seawater for 8 h. This procedure was repeated
two times a day to simulate the inundation period of the Effects of Temperature and Salinity on
semidiurnal tide on the Andaman coast (a total of 8 h of emersion Barnacle Survival
and 16 h of immersion per day). Each treatment had three Three hundred and sixty adult barnacles of C. malayensis and
replicates. For each replicate, 10 barnacles were kept in a 500 ml A. amphitrite were collected and acclimated as in section Effect
plastic container filled with continuously aerated 32◦ /oo seawater of Continuous Submergence on Barnacle Survival. Barnacles
prepared from artificial seawater (Aquarise©). Barnacles were fed were maintained in combinations of four different salinities
with an ad libitum diet of Chaetoceros sp. (concentration: 3 × (0, 33, 50, and 60◦ /oo ) and three temperature levels (30, 40,
105 cell/ml) daily. Lamps (120 W Phillips Par 38 Flood) were and 50◦ C), with three replications per treatment. For each
suspended ∼30 cm above the containers and turned on for 12 h replication, 10 barnacles were kept in a 500 ml plastic container
(06:00 to 18:00) per day. Heating from the lamps created an filled with continuously aerated solution prepared from artificial
ambient temperature similar to the average shore temperature seawater. Containers were placed in a thermostatic heating
(∼30◦ C). Seawater was changed every 2 days and the number of water bath and the solution was heated from 27◦ C (ambient)
surviving barnacles was scored daily. Surviving barnacles were at ∼0.3◦ C min−1 until the target temperature was reached.
checked by stimulating their operculum using a wooden stick Barnacles were exposed to the treatments for 5 h, after which
(Fraser and Chan, 2019). the number of surviving barnacles was counted. Salinity was
The percentages of barnacles that survived were compared checked every 15 mins to ensure that the designated salinity
between the continuous submersion and control treatments on levels were maintained. Distilled water was added in containers
different days by two-way ANOVA. Treatment (two levels) and in which salinity had increased due to the heating. Barnacles were
Day (15 levels) were treated as fixed factors. Treatment was not fed.
crossed with Day. Tukey’s test was performed on significant terms Two-way ANOVA was performed to test the effect of
for post hoc multiple comparisons. temperature and salinity on the percentage of C. malayensis

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Buasakaew et al. Rarity of Barnacles in Rock Pools

and A. amphitrite survival separately. Data was sqrt(x+1) RESULTS

transformed. Temperature (three levels) and salinity (four
levels) were treated as fixed factors. Temperature was Physico-Chemical Environment of Rock
crossed with salinity. A Student–Newman–Keuls (SNK) Pools and Barnacle Abundance
test was performed on significant terms for post hoc A total of 76 rock pools was measured for surface area, depth,
multiple comparisons. water temperature, salinity, pH and dissolved oxygen, and the

FIGURE 3 | Diurnal variation in water temperature, salinity, pH, and dissolved oxygen in six intertidal pools and inshore seawater. Blue regions indicate pools that were
immersed in high tides. White regions indicate pools that were isolated from the sea during low tides. Raining clouds indicate that rain occurred during sampling.

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Buasakaew et al. Rarity of Barnacles in Rock Pools

FIGURE 4 | Percentage cover of barnacles in rock pools and on open rock surfaces adjacent to pool. (A) Percentage cover of barnacles (mean ± SE) pooled from all
shores. (B,C) Percentage cover on five shores of C. malayensis and A. amphitrite, respectively. Icon: * = significant differences between habitats suggested by
pairwise tests.

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Buasakaew et al. Rarity of Barnacles in Rock Pools

corresponding abundances of C. malayensis and A. amphitrite. TABLE 3 | Permutational multivariate ANOVA (PERMANOVA) for the effects of
There was no significant correlation detected between the size habitat (rock pools and adjacent open rock surfaces), species and shore on
barnacle abundance.
dimension of pools (surface area × depth) and any physico-
chemical parameters measured (Figure 2). Water temperature Source of variation df MS Pseudo-F p (perm)
from all the pools measured ranged from an approximate
temperature of 27–37◦ C. Salinity ranged from ∼5–46◦ /oo , pH Pooled across shores

ranged from ∼6–9.7 and dissolved oxygen concentration ranged Habitat (Ha) 1 29674 86.67 <0.05*
from ∼0.9–11 mg L−1 (Figure 2 and Table 1). Species (Sp) 1 23284 68.01 <0.05*
Based on the Bio-Env multivariate analysis in the BEST (Bio- Ha × Sp 1 29085 84.96 <0.05*
Env) routine, the correlation coefficients from a combination of Residual 300 342.37
different subsets of physico-chemical parameters to the barnacle Total 303

abundance ranged from 0.14 to 0.30 (Table 2). The subset, C. malayensis

including water temperature and salinity, yielded the highest Habitat (Ha) 1 55229 33.71 <0.05*

correlation coefficient (0.30) to the barnacle abundance (Table 2). Shore (Sh) 4 1983.10 4.12 <0.05*

Having all the physico-chemical parameters in the analysis Ha × Sh 4 1714.50 3.56 <0.05*

yielded a correlation coefficient of 0.16, lower than the subset Residual 142 480.97
containing water temperature and salinity (Table 2). Total 151
A. amphitrite

Diurnal Variation in Physico-Chemical Habitat (Ha) 1 180.63 0.16 0.77

Shore (Sh) 2 801.15 5.15
Parameters in Rock Pools <0.05*
Ha × Sh 2 1213.40 7.81 <0.05*
Water temperature, salinity, pH, and dissolved oxygen followed
Residual 94 155.46
a diurnal pattern of variation. Pools were emersed and immersed
Total 99
two times daily. When the pools were immersed in seawater
during high tides, all their physico-chemical parameters were Bold values and asterisks indicate significant effects.
similar to those of inshore seawater. When the pools were isolated
during the daytime low tide, water temperature, salinity, pH, and between the open rock surfaces and pools (Table 3). In general,
dissolved oxygen started to increase gradually, peaking (water A. amphitrite had a lower abundance than C. malayensis on open
temperature at 33◦ C, salinity at 38◦ /oo , pH at 9.0 and dissolved rock surfaces (Figure 4 and Table 3).
oxygen at 15 mg L−1 ) in the late afternoon (at ∼ 15:00) and
then decreasing. When the pools were immersed again after the Barnacle Recruitment in Rock Pools and
first low tide, the physico-chemical parameters became similar to on Emergent Rock
those of inshore seawater again. The second low tides happened Barnacle recruitment was observed in October 2019 and from
in the very early morning (03:00 to 07:00). During this period, June to September 2020, when recruits could be seen on open
when the pools were isolated, the water temperature, dissolved rock surfaces (Figure 5). There was a significant difference in the
oxygen, and pH decreased and reached its lowest values (water density of recruits between the rock pools and adjacent open rock
temperature at ∼27–28◦ C, pH at ∼7.2 and dissolved oxygen at surfaces (Figure 5A and Table 4). Recruits were only recorded in
∼2 mg L−1 ) at around 05:00–06:00. In contrast to other physio- pools at Had Sai Yao in June 2020 and at Tanyong Po in August
chemical parameters, salinity continued to increase during the 2020. No recruitment was found in pools in any other month on
early morning low tide. On the second conservative day of either shore (Figure 5). In Tanyong Po, the density of recruits was
measurement, it rained during the afternoon low tides when the more than three times higher on adjacent open rock surfaces than
pools were emersed. Water temperature dropped (∼27◦ C) and inside pools (Figure 5A).
salinity plummeted to as low as 5◦ /oo , compared with 33◦ /oo for
the inshore seawater (Figure 3). The pH and dissolved oxygen Submersion Experiment
followed previous trends of increase, peaking at around 17:00 and Survivorship for C. malayensis differed between treatments
dropping again during high tides (Figure 3). (Figure 6A and Table 5). After 2 weeks, all C. malayensis survived
well in the control treatment (> 90% survival) but had a survival
Abundance of Barnacles in Rock Pools and of 80% under continuous submersion (Figure 6A). Survival
on Emergent Rock of A. amphitrite was not different between the treatments
Chthamalus malayensis had a higher abundance on the adjacent (Figure 6B, Table 5).
open rock surfaces than in pools on all shores (Figures 4A,B
and Table 3). The abundance of A. amphitrite on adjacent open Effect of Temperature and Salinity on
rock surfaces and in pools varied among shores (Figure 4C and Barnacle Survival
Table 3). In Tanyong Po, A. amphitrite was more abundant on The C. malayensis and A. amphitrite had similar survival patterns
open rock surfaces than in pools, and this pattern was reversed in at different temperatures and salinities (Figure 7). Based on
Pleum Sook (Figure 4C). Based on the pooled data from across the two-way ANOVA, the interaction between temperature ×
all shores, there were no significant differences in A. amphitrite salinity in barnacle survivals was significant. At 50◦ C, none of the

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Buasakaew et al. Rarity of Barnacles in Rock Pools

FIGURE 5 | Density of recruits (mean ± SE) in rock pools and on emergent rock from October 2019 to September 2020. Samplings were not carried out from March
2020 to May 2020 due to logistical difficulties during COVID-19 lockdown. (A) and (B) Tanyong Po and Had Sai Yao, respectively. Icon: *significant differences
between habitats as suggested by pairwise tests.

barnacles survived (0% survival), regardless of the salinity level. TABLE 4 | PERMANOVA on the effects of habitat, shore, and month on the
When barnacles were maintained at 40◦ C, ∼40–50% survived at recruitment of barnacles.

0◦ /oo , whereas survival was up to 90% under 33, 50 and 60◦ /oo Source of variation df MS Pseudo-F p (perm)
salinities, with no significant difference among these top three
salinities (SNK test; p <0.05). At 30◦ C, there was no significant Habitat (Ha) 1 456.19 1.42 <0.05*
difference in survival among salinity treatments, and >90% of all Shore (Sh) 1 318.16 24.16 <0.05*
barnacles survived (Figure 7 and Table 6). Month (Mo) 8 72.67 1.49 0.20
Ha × Sh 1 321.34 24.40 <0.05*
DISCUSSION Ha × Mo 8 71.10 1.42 0.21
Sh × Mo 8 48.63 3.69 <0.05*
Physico-Chemical Environment in Indian Ha × Sh × Mo 8 50.15 3.81 <0.05*
Ocean Rock Pools Residual 508 13.17
The present study is the first to describe the physico-chemical Total 543
environment of rock pools and its diurnal variation in the Indian
*indicate p <0.05.
Ocean. Water temperature, salinity, pH, and dissolved oxygen Bold values and asterisks indicate significant effects.
levels fluctuate across diurnal cycles, with one maximum and
minimum period per day during the two low tides periods. The
diurnal pattern of variations in physico-chemical parameters in of the Pacific region (Chan, 2000). Both regions have two periods
the rock pools of the present study is similar to the patterns of pool isolations from low tides, and the physico-chemical
addressed in the subtropical Hong Kong, in the South China Sea parameters of both peaked in the later afternoon. The range

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Buasakaew et al. Rarity of Barnacles in Rock Pools

FIGURE 6 | Percentage of survival (mean ± SE) of barnacles in submersion experiment. (A) C. malayensis and (B) A. amphitrite.

TABLE 5 | Two-way ANOVA on the effects of treatment and day on the pattern addressed in Balanus balanoides in the temperate Atlantic
percentage of surviving barnacles in the submergence experiment. (Singletary and Shadlou, 1983) that barnacles were more intense
Source of variation df MS F P
on rock surfaces than inside rock pools. High shore intertidal
species often inhabit open rock surfaces instead of inside rock
C. malayensis pools. The high shore limpet, Cellana grata, for example, are
Day (D) 14 27.30 0.51 0.92 common on open rock surfaces and at rock pool interface
Treatment (T) 1 2667.78 50.02 <0.001* (adjacent surface to pools) but never get submerged in rock
D×T 14 27.30 0.51 0.92 pools. The limpets at pool interface had relatively lower heat
Residual 60 53.33 stress when compared to open rock surfaces. The lower shore
Total 89 barnacle, Amphibalanus amphitrite, in contrast, had overall
A. amphitrite similar abundances (5–10%) in pools and also on the rock
Day (D) 14 71.11 1.68 0.08 surfaces. The vertical distribution of A. amphitrite extends from
Treatment (T) 1 17.78 0.42 0.52 lower intertidal to subtidal region. A. amphitrite often has intense
D×T 14 17.78 0.42 0.96 settlement on shores and subtidal man-made objects (Desai and
Residual 60 42.22
Anil, 2005). A. amphitrite is also one of the marine fouling
Total 89
species due to its intense settlement and high tolerance to
environmental stresses (Desai and Anil, 2005). A. amphitrite
*indicate p < 0.05. had higher abundance in rock pools when compared with C.
Bold values and asterisks indicate significant effects.
malayensis indicates this barnacle survives well inside the rock
pools environment.

of variations in water temperature, salinity, pH, and dissolved

oxygen is similar between Thailand and Hong Kong (Chan, Factors Affecting Barnacle Survival in
2000). Similar to the present study, the water temperature, Rock Pools
salinity, pH and dissolved oxygen concentration in the NW coast In the present study, sparse recruitment of barnacles was
of Brittany in the temperate region of the Atlantic had one recorded in some of the rock pools, suggesting that barnacle
maximum (later afternoon) and one minimum (early morning). larvae can settle in the rock pools and grow into recruits. This
However, rock pools in the Atlantic coast of France have a single does not support the first hypothesis, that barnacles are rare in
low tide isolation per day. This isolation can last up to 10 h, pools because of a lack of recruitment. However, the density
much longer than the 6- to 7-h low tide isolation in the present of recruits is much lower in pools than on adjacent open rock
study. Water temperature in the present study is higher than the surfaces. This suggests that some factors may affect the post-
summer water temperature in the Britany rock pools, but the settlement and post-recruitment mortality of barnacles recruited
range of salinity variation is similar between the present study inside of rock pools. Rock pools in the Andaman Sea of Thailand
and the Atlantic rock pools. are often inhabited by various species of gastropods including
the Monodonta and Nerita snails (Sanpanich and Duangdee,
Distribution of Barnacles in Rock Pools 2013). Bulldozing effect of the settled cyprids by these mobile
and Adjacent Open Rock Surfaces molluscan grazers can affect the success of the settlement (Chan
In the present study, it was found that Chthamalus malayensis and Williams, 2003). In the temperate Atlantic, the occurrence
was more abundant (>40%) on the adjacent rock surfaces of some algae and biofilms might affect the settlement and post-
than inside rock pools (<5%). This pattern is similar to the settlement mortality of barnacles (Singletary and Shadlou, 1983).

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Buasakaew et al. Rarity of Barnacles in Rock Pools

FIGURE 7 | Percentage of barnacles, namely, (A) C. malayensis and (B) A. amphitrite (mean ± SE), that survived under different temperature and salinity treatments.
Different small letters = significant difference between temperature within each salinity treatment. Note survival of barnacles is 0 in 50◦ C.

TABLE 6 | Two-way ANOVA on the effects of Temperature and Salinity on the had greater mortality (∼40–50%) only in the low salinity
percentage of survival of two species of barnacles. treatment (0◦ /oo ), but not in the high salinity treatments. For
Source of variation Df MS F P
the 30◦ C treatment, both barnacle species had high survivals
in all salinity treatments. When barnacles were exposed to low
C. malayensis salinity water, their opercular valves were closed to stop external
Temperature (T) 2 289.67 325.23 <0.001* water from getting inside the shell. As a result, mantle water
Salinity (Sa) 3 3.68 16.85 <0.001* and haemolymph can have higher osmolality than the external
T × Sa 6 2.72 12.48 <0.001* environment (Chan et al., 2001). However, at high temperatures,
Residual 24 0.22 barnacles can enter a coma state and lose the ability to close
Total 35 their opercular valves. As a result, low salinity water can
A. amphitrite enter the mantle and affect the osmolality of the hemolymph,
Temperature (T) 2 300.53 224.66 <0.001* reducing survival. This suggests that the combination of high
Salinity (Sa) 3 1.83 31.77 <0.001* water temperature and low salinity (after rain) can lead to
T × Sa 6 1.31 22.74 <0.001* high barnacle mortality in rock pools. In natural rock pools,
Residual 24 0.06
salinity can be as low as almost 0◦ /oo during the heavy rain
Total 35
monsoon season on the Andaman coast (May to October),
and intense solar radiation shortly after rains can heat up
*indicate p < 0.05. pool water. This condition can cause mortality in barnacles
Bold values and asterisks indicate significant effects.
inside pools. This is probably a common phenomenon on
tropical shores. During the wet season in Hong Kong, the limpet
Algae are more abundant in rock pools than on open rock Cellana grata experienced heavy rainfall and subsequently, heat
(Nylund and Pavia, 2003; Othmani et al., 2016). stresses had faster heart beat rates and lower haemolymph and
Submergence experiments indicated that both C. malayensis mantle osmolalities when compared with limpets that were
and A. amphitrite can have at least 80% lower survival under not stressed. Such patterns suggest that heat and raining can
submergence conditions compared to simulated tidal conditions both impose different levels of stress and protein responses,
(control) although barnacles suffer slight mortality under but their combination had an interactive effect on the limpets’
submerged conditions. Continuously submerged conditions in metabolism and survival (Williams et al., 2011). The brittle star,
pools do not appear to be the major cause of barnacle mortality Ophiopholis mirabilis, is common in the temperate intertidal
in rock pools. Compared to the high shore intertidal limpets, region of the West Pacific. The combination of temperature
Cellana grata, which are common on rock surfaces but not and salinity can affect its metabolism, ammonia excretion, and
found in rock pools, continuous submergence under laboratory tissue moisture. Reduced salinity conditions in combination
conditions can result in 70% mortality after 96 h (Gray, 2010). with temperature variations can result in mortality due to
This may explain continuous submergence can be a factor for the the tissue moisture beyond their regulatory capacity (Fang
absences of C. grata inside rockpools. et al., 2015). In the cold Baltic Sea, the barnacle Balanus
Based on the multivariate analysis of rock pool physico- improvisus had increased mortality in low salinity (5 psu)
chemical environment and barnacle abundance, water and low temperature (12◦ C) because low water temperature
temperature and salinity are the main factors controlling narrowed the barnacle’s tolerance toward low salinity (Nasrolahi
barnacle distribution. In the laboratory temperature and et al., 2013). Salinity and water temperature can thus interplay
salinity experiment, barnacles did not survive in the high together and impose interactive stress toward intertidal species
water temperature (50◦ C) environment. At 40◦ C, barnacles and affect their mortality.

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Buasakaew et al. Rarity of Barnacles in Rock Pools

However, the present study has not tested the effect of AUTHOR CONTRIBUTIONS
dissolved oxygen on barnacle survival. On the Atlantic coast of
the USA, pools may not be able to support high densities of KW conceived the idea and designed the study. NB analyzed the
settlers because such a condition would decrease the dissolved data and wrote the first draft of the manuscript. BC involved in
oxygen needed to support such densities of barnacles, thus experiment design, manuscript writing and provided advice. All
resulting in hypoxia and high barnacle mortality (Singletary authors read and commented on the manuscript.
and Shadlou, 1983). In the present study, some of the rock
pools had very low dissolved oxygen (1–3 mg. L−1 ). In such FUNDING
an environment, barnacles, if recruited in those pools, probably
would not survive. This study was supported by the Faculty of Science Research
In conclusion, barnacles are rare in tropical Indian Ocean Fund, Prince of Songkla University (Contract No. 1-2562-02-
rock pools. This is a result of the interplay of continuous 006) and an Academia Sinica research grant.
submergence, high water temperature, and low salinity causing
barnacle mortality. Future research should investigate biological ACKNOWLEDGMENTS
factors like predation (density of whelks in rock pools) or the
grazing of molluscs, which bulldoze settlers (Chan and Williams, We thank Lutfee Hayeewachi, Suphatsara Sangphuek,
2003). Vanyarat Kongsap, Apisara Nakpan, Phutita Wongwaiyut,
Nattacha Khantasimachalerm, Kanyanat Kaewutai, Thitiya
DATA AVAILABILITY STATEMENT Choosun, Arisa Somprom, and Suntaree Karnchananiyom
for their help with fieldwork. We also thank Thachanon
The original contributions generated for the study are included Sinso for his help in setting up the experiment
in the article/supplementary material, further inquiries can be and Noah Last (third draft editing) for his English
directed to the corresponding authors. language editing.

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larval settlement of the barnacle Balanus improvisus. Mar. Biol. 143, 875–882. absence of any commercial or financial relationships that could be construed as a
doi: 10.1007/s00227-003-1093-9 potential conflict of interest.
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Settlement inhibition of marine biofilm bacteria and barnacle larvae by Publisher’s Note: All claims expressed in this article are solely those of the authors
compounds isolated from the Mediterranean brown alga Taonia atomaria. J. and do not necessarily represent those of their affiliated organizations, or those of
Appl. Phycol. 28, 1975–1986. doi: 10.1007/s10811-015-0668-4
the publisher, the editors and the reviewers. Any product that may be evaluated in
Pochai, A., Kingtong, S., Sukparangsi, W., and Khachonpisitsak, S. (2017). The
this article, or claim that may be made by its manufacturer, is not guaranteed or
diversity of acorn barnacles (Cirripedia, Balanomorpha) across Thailand’s
coasts: the Andaman Sea and the Gulf of Thailand. Zoosyst. Evol. 93, 13–34. endorsed by the publisher.
doi: 10.3897/zse.93.10769
Sanpanich, K., and Duangdee, T. (2013). The biodiversity of marine Copyright © 2021 Buasakaew, Chan and Wangkulangkul. This is an open-access
gastropods of Thailand in the late decade. Malaysian J. Sci. 32, 47–64. article distributed under the terms of the Creative Commons Attribution License (CC
doi: 10.22452/mjs.vol32no3.5 BY). The use, distribution or reproduction in other forums is permitted, provided
Seabra, R., Wethey, D. S., Santos, A. M., and Lima, F. P. (2011). Side matters: the original author(s) and the copyright owner(s) are credited and that the original
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