Full Text

Title
Thermal tolerance of Echinolittorina species in Hong Kong:

implications for their vertical distributions
Advisor(s)
Williams, GA
Author(s)
Li, Hoi-ting, Kathy.; .
Citation
Issued Date
URL
Rights
2012
http://hdl.handle.net/10722/173874
The author retains all proprietary rights, (such as patent rights)

and the right to use in future works.
THERMAL TOLERANCE OF ECHINOLITTORINA SPECIES

IN HONG KONG:
IMPLICATIONS FOR THEIR VERTICAL DISTRIBUTIONS
by
Hoi Ting Kathy, LI

B. Sc. in Biology, The Hong Kong University of Science and Technology, Hong Kong
A thesis submitted in partial fulfillment of the requirement for

the Degree of Master of Philosophy
at The University of Hong Kong
August, 2012
Abstract of thesis entitled
THERMAL TOLERANCE OF ECHINOLITTORINA SPECIES

IN HONG KONG:
IMPLICATIONS FOR THEIR VERTICAL DISTRIBUTIONS
Submitted by Hoi Ting Kathy, LI
for the Degree of Master of Philosophy

at The University of Hong Kong
in August, 2012
Intertidal rocky shores represent an extremely stressful physical environment

dictated by the rise and fall of the tides.
One of the major environmental stresses
over this gradient is temperature, especially towards the upper reaches of the
shore where species spend long periods out of water exposed to hot, desiccating
conditions.
As a result, the thermal tolerance of intertidal species is often
positively correlated with their vertical distributions, and the physiological and
molecular limits that drive such patterns have been the subject of recent research.
Understanding these tolerance limits, from small (e.g. vertical distribution) to
large (e.g. latitudinal) spatial scales, may provide information to predict species
success under future climate change scenarios, and thus possible changes in
community structure.
Given their abundance in the high shore, and well resolved taxonomy and
phylogeography, the littorinids Echinolittorina malaccana, E. radiata and E.
vidua are excellent models to investigate the relationship between thermal

tolerance and spatial distribution patterns.
These littorinids are widely
distributed on Hong Kong shores and exhibit a distinct and consistent vertical
distribution that ranges from temperate to tropical regions along the western
Pacific coast.
Field surveys in summer and winter at two moderately exposed
shores (Stanley and South Bay, Hong Kong) showed that E. malaccana was
distributed highest on the shore, followed by E. radiata and E. vidua respectively,
and all the three species were found ~ 0.25m lower on the shore in summer than
winter.
Laboratory experiments, including determination of survival limits
(LT50), Arrhenius breakpoint temperature of heart rate (ABT of HR) and activities
of metabolic enzymes (MDH and LDH), were used to establish if the
physiological attributes of the three species were related to their distribution
patterns.
The LT50 of E. malaccana were the highest of the three species
(56.47oC), followed by E. radiata (55.5oC), and finally the lower shore species E.
vidua (53.7oC); while ABT of HR in E. malaccana (48.2oC) was also higher than
E. radiata (46.5oC) and E. vidua (46.6oC). The enzyme activities did not show
any clear patterns.
In terms of seasonal variation, LT50 and ABT of HR of all
three Echinolittorina species were higher in summer than winter, which showed
the potential for the littorinids to acclimate when environmental conditions
become more severe.
The present study provided a fundamental understanding of how physiological,

temperature tolerance may determine the spatial and temporal distribution patterns
of Echinolittorina species at a local scale where strong environmental gradients
vary vertically and also between seasons.
Information on the tolerance limits of
physiological traits such as LT50, heart rates and enzyme functioning may direct
further investigations to identify the underlying causes of the survival limits of

these species to temperature variation, and whether this tolerance is genetically or
environmentally determined, for example through acclimation.
Such studies will
provide insights into how a species' physiology may limit their present-day
distributions at multiple scales from local to biogeographical, but also enable
predictions of how species may respond to changing temperature regimes.
ACKNOWLEDGEMENTS
I would like to express my deepest gratitude to

Prof. Gray Williams, my supervisor, for his generous guidance and support,
especially during my endless thesis writing progress, as well as giving me the chance
to have lots of fun at Tsitsikamma and St. Petersburg, and BEER of course.
Dr. Stephen Cartwright, for his patience and guidance on detailed revisions on
this thesis.
Dr. Yunwei Dong, Dr. Wai-chuen Ng and Dr. Priscilla Leung, for teaching me
the techniques on enzyme works.
My field/lab helpers - June Leung (the all-the-time driver :]), Karen Villarta (the
prison breaker at Stanley), Terence Ng (the littorinids catcher), Stella Wong, Samuel
Wang, Sally Lau, Charmaine Yung, Adela Li, etc, for spending their time and
assisting me to find the little snails, either under hot & strong sun in summer or cold,
wet (by the strong wave action) & mysterious nights in winter.
All the students and staff at the Swire Institute of Marine Science (SWIMS), and
the School of Biological Sciences, The University of Hong Kong (SBS, HKU),
especially Cecily Law and Patrick Chan for their technical support.
My softball teammates at HKUST, PTIs from Mai Po, previous labmates at
CML, classmates from UST Biology, great friends from GHS, for their unlimited
supports and cheers, esp when I was desperate, and I want to say I love you all!.
My family, both real and host family from US, for letting me do whatever I want,
despite my willfulness.
Last, but most importantly, Miss Cecilia Chow, my Biology teacher at GHS, for
her generous sharing during classes that inspire my interest in the environmental field,
and lead to completion of this M.Phil. degree.
ii
CONTENTS
Declaration
Acknowledgements
ii
Contents
iii
..
CHAPTER 1 GENERAL INTRODUCTION

INTRODUCTION
Intertidal rocky shores as model systems to investigate

species distribution patterns
Temperature - a prominent factor controlling species
distribution patterns
Littorinids as a model for studies on thermal stress
Aims of study and project outline
The Hong Kong climate
Site descriptions
CHAPTER 2 SEASONAL VARIATION IN VERTICAL DISTRIBUTION OF

ECHINOLITTORINA SPECIES
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
iii
..
20
CHAPTER 3 THERMAL TOLERANCE OF Echinolittorina SPECIES IN

..
HONG KONG
INTRODUCTION
Thermal tolerance and species distribution on intertidal rocky

shores
How to determine thermal tolerance of species?
Thermal tolerance and vertical distribution of Echinolittorina
species on Hong Kong rocky shores
MATERIALS AND METHODS

Preliminary studies as quality control of experimental protocol
Seasonal variation in lethal temperature
Activities of malate dehydrogenase and lactate dehydrogenase
Data analysis
RESULTS
Seasonal variation in Arrhenius breakpoint temperature of
heart rate
DISCUSSION
Lethal temperature in relation to vertical distribution
Arrhenius breakpoint temperature of heart rate in relation to
vertical distribution
Activities
of
malate
dehydrogenase
and
dehydrogenase in relation to vertical distribution
iv
lactate
46
Seasonal variation of LT50 and ABT of HR
CHAPTER 4 GENERAL DISCUSSION
..
104
DISCUSSION
Vertical and seasonal distribution of Echinolittorina
species in Hong Kong
Thermal tolerance of Echinolittorina species and their
seasonal variations in Hong Kong
Present study: implications for future investegations
Conclusions
LIST OF REFERENCES
..
115
APPENDIX 1
..
138
Name used for Echinolittorina malaccana, E.

radiata and E. vidua in previous literature
..
APPENDIX 2 .1
139
Heart rate of Echinolittorina species vs

temperature (Winter)
..
APPENDIX 2 .2
Heart rate of Echinolittorina species vs

temperature (Summer)
140
CHAPTER 1:
GENERAL INTRODUCTION
1.1 INTRODUCTION
Intertidal rocky shores as model systems to investigate species
distribution patterns
Temperature - a prominent factor controlling species distribution
patterns
Site descriptions
Chapter 1: General introduction
1.1 INTRODUCTION
Determination of the causes of species distribution patterns is a complex matter in

all types of ecosystems, as it involves the interplay of physical factors (e.g.
temperature, salinity, water availability) and biological interactions (e.g. predation,
competition, facilitation) between species (see Brown, 1984; Krebs, 2009 for
reviews).
Given the complex nature of these interactions, ecologists are keen to
investigate whether individual factors can play a dominant role in determining

these distribution patterns; from large-scale latitudinal species' ranges to
small-scale microhabitat selection (see Begon et al., 1996 for review).
Identifying the factors which drive these patterns would facilitate greater
understanding of community dynamics within an ecosystem (e.g. in freshwater
systems: Welborn et al., 1996; intertidal systems: Underwood and Chapman, 1996;
also see Tilman, 1982 and Menge and Olson, 1990 for reviews).
Intertidal rocky shores as model systems to investigate species distribution

patterns
Intertidal rocky shores are a model system to assess the impacts of physical
factors on species' distribution patterns, as shores experience a very extreme and
dynamic physical environment gradient determined by tidal events (see Lewis,
1964; Stephenson and Stephenson, 1972; Menge and Branch, 2001; Little et al.,
2009 for reviews).
The changes brought by the rise and fall of the tides creates
alternating aquatic and terrestrial conditions which intertidal organisms have to

tolerate, and thus establishes a regularly fluctuating, severe, environmental
2
gradient along the vertical range of the shore (see Rafaelli and Hawkins, 1996;
Menge and Branch, 2001; Little et al., 2009 for reviews).
The unique situation
of severe changes in environmental conditions (e.g. temperature, water

availability) and biological interactions (e.g. predation, competition) over a short
vertical gradient (often less than 10m), therefore, makes intertidal rocky shores a
model system to investigate how these factors affect the distribution of species.
With variation in the degrees of physical stress and biological interactions along
the intertidal zone, organisms exhibit different ranges of tolerance to the
conditions they can experience along this vertical environmental gradient (see
Underwood, 1979 for review).
Species are unable to tolerate conditions along
the entire vertical gradient, and are assumed to have become adapted to perform
optimally to the conditions experienced at certain heights along the shore (see
Nybakken and Bertness, 2005 for review). As a result, species are found to have
their own, specific, vertical distribution patterns.
The upper limit of species'
distribution along the shore is generally regarded to be set by physical factors,

including temperature and desiccation (e.g. Lewis, 1964; Stephenson and
Stephenson, 1972; Wolcott, 1973; Schonbeck and Norton, 1978; Stillman and
Somero, 2001) whilst lower limits are considered to be driven by biological
interactions, such as competition and predation (e.g. Connell, 1961a; Paine, 1974;
Wethey, 1984).
These interactions are also mediated by changing environmental
conditions, for example, seasonal changes (e.g. Evans, 1948; Fraenkel, 1968);
physical disturbance (e.g. Boulding and Van Alstyne, 1993; Hutchinson and
Williams, 2003b; Sanpanich et al., 2006) and also the supply of juveniles to the
shore (Underwood and Denley, 1984; Underwood and Fairweather, 1989; Menge
3
and Branch, 2001).
Ultimately, knowing how species respond to these
interactions in relation to their vertical distributions will allow a better

understanding of the processes driving intertidal community structure (Menge and
Olson, 1990; Underwood and Chapman, 1996).
Temperature - a prominent factor controlling species distribution patterns
Environmental temperature is widely accepted as one of the most important

physical factors associated with both latitudinal and local distribution patterns of
species in terrestrial and marine ecosystems (see Huey and Kingsolver, 1989;
Prtner, 2002; Parmesan, 2006; Angilletta, 2009 for reviews).
The temperatures
species experience can affect their distributions by altering the fitness of

organisms through influencing their crucial life functions, from biochemical to
physiological levels, hence affecting species performance such as survival,
locomotion, growth and reproduction (see Hochachka and Somero, 2002; Prtner,
2002; Prtner et al., 2007; Angilletta, 2009 for reviews).
Since environmental
temperatures vary at a variety of spatial and temporal scales, from local to

geographic scales, studies have investigated the relationship between variation in
temperature tolerance and both small-scale, local species distributions (for
example between lizards along a 1000-m altitudinal gradient, Spellerberg, 1972;
and gastropods that occupy a narrow vertical range, often < 10m, Garrity, 1984)
as well as large-scale, latitudinal species distributions (e.g. in insects:
Addo-Bediako et al., 2000; ectotherms in both marine and terrestrial
environments, Sunday et al., 2010).
Understanding how temperature tolerance
plays a role in determining the different scales of species' spatial patterns, may
provide information to predict species success, for example, under future climate
change scenarios and hence potential changes in community structure (Somero,
2010).
On intertidal rocky shores, environmental temperatures fluctuate greatly due to

the alternation of tidal events (Lewis, 1964; Stephenson and Stephenson, 1972).
Due to the variation in species tolerance limits, previous studies have often found
clear positive correlations between thermal tolerance of intertidal species and their
vertical distributions (e.g. Stirling, 1982; McMahon, 2001; Stillman and Somero,
2000; also see Underwood, 1979 for review).
As well as showing changes
within the tidal cycle, environmental temperatures are often most extreme when
low tides occur during day time in hot seasons, or at low latitudes (Moore, 1972).
In Hong Kong, for example, during summer low tides in the afternoon, rock
surface temperatures can exceed 50oC (Williams, 1994b; Williams and Morritt,
1995; Williams et al, unpubl data).
Such variations in environmental
temperatures, therefore, require further investigation to identify if thermal

tolerance of intertidal species can change with time (e.g. seasonal or
laboratory-based acclimation, e.g. Stirling, 1982; Obermlle et al., 2011, also see
Table 3.13 in Chapter 3).
Determining a species thermal tolerance will allow an
understanding of the factors affecting a species vertical and seasonal distribution,

and also provide a scientific basis which may help predict species success under
long-term, global, environmental changes (Somero, 2010).
Littorinids (Gastropoda: Littorinidae), also known as periwinkles, are abundant

worldwide in different types of intertidal habitats, such as mangroves and rocky
shores, where many of these species dominate the upper intertidal zone (see
Lewis, 1964; Stephenson and Stephenson, 1972; McQuaid, 1996a,b; Reid, 2007
for reviews). Due to their abundance and ubiquitous distribution, the taxonomy
of littorinids has well been documented (e.g., the genus Littoraria, Reid, 1986;
Littorina, Reid, 1996 and Echinolittorina, Reid, 2007) and, therefore, this well
resolved
taxonomy
provides
researchers
with
accurate
and
confident
identifications of species and their phylogenetic relationships to aid comparative

studies.
From the ecological aspect, littorinids are dominant grazers on the high shore of
intertidal rocky shores (see Norton et al., 1990; McQuaid, 1996b for reviews).
The grazing activity of littorinids has been shown to reduce the biomass (e.g.
Castenholtz, 1961, 1964; Nicotri, 1977, Underwood, 1984, Mak and Williams,
1999) and alter the composition of the biofilm (e.g Lubchenco, 1978; Hunter and
Russell-Hunter, 1983, Vadas and Elner, 1992; Williams, 1994a; Stafford and
Davies, 2005).
Littorinids are, therefore, key players in shaping community
structure in the high shore (see Norton et al., 1990; McQuaid, 1996b).
Living on the high shore, littorinids can experience prolonged emersion periods (~
8 days) during neap tides (McMahon, 1990; Uglow and Williams, 2001), as well
as extreme environmental temperatures (> 50oC, Williams, 1994b; Williams and
Morritt, 1995; Marshall et al, 2010) and are, therefore, model organisms to study
the relationship between their thermal tolerance and vertical distributions.
Species living in such stressful environmental conditions are assumed to be living
close to their physiological limits (Somero, 2010) and this is thought to be
especially true for tropical species (Tewkesbury et al, 2011), as their potential to
acclimate to higher environmental temperatures is thought to be less as compared
to temperate species (see Somero, 2005; 2010 for reviews).
The majority of
previous studies have, however, been restricted to the eastern Pacific and Atlantic
coasts (e.g. Sandison, 1967; Hamby, 1975; Cannon and Hughes, 1992; Clarkes et
al., 2000b; Davenport and Davenport, 2005), and there have been few studies
along the western Pacific or on tropical shores (but see McMahon, 1990; Stirling,
1982).
It is therefore important to address this knowledge gap and to investigate
responses of species found in these understudied regions, so as to determine if

there are any general patterns related to species' distribution patterns and their
acclimation potentials worldwide (see Foster, 1990).
The genus Echinolittorina (formerly Nodilittorina, see Appendix 1 for former and
present scientific names), is the largest genus in the family Littorinidae (Reid,
2007). Containing 59 recognized species, close to half of the genus can be found
in the Indo-West Pacific region (Reid, 2007).
Like many other littorinids,
Echinolittorina malaccana, E. radiata and E. vidua (Figure 1.1) play an important

ecological role by controlling biofilm abundance on the high shore (Mak, 1996),
and are common along the Indo-West Pacific coast, with representatives of the
three species covering over 70o of latitude (Table 1.1). These three species are,
therefore, excellent representative candidates for determining the relationship
7
between thermal tolerance and their vertical distributions.
The three species are
abundant on rocky shores in Hong Kong and their ecology has been studied in
detail by Mak (1996) and Walters (2002). All three species inhabit the mid-high
shore, between 1.3-2.7m above C.D. (Mak, 1996), where they frequently
encounter extreme fluctuations in environmental conditions driven by tidal cycles
and weather conditions.
Given the fact that competition and predation pressure
on Echinolittorina species are assumed to be low in Hong Kong (see Stafford,

2002), hence the vertical distributions of these species are assumed to be related
to their tolerance to physical stresses (Stirling, 1982; Yipp et al., 1990).
The aims of this study were to measure: (i) variation in thermal tolerance between
the Echinolittorina species in Hong Kong, in relation to their local spatial and
temporal distributions; which would provide the scientific basis to direct further
investigations on larger-scale patterns (e.g. large scale latitudinal patterns and any
potential changes over temporal scales of years); and, (ii) seasonal variability of
the littorinids thermal tolerance, in order to determine the acclimation potential of
each species to higher environmental temperatures; ultimately to help predict and
identify relative species success as temperatures are proposed to increase in a
warming world.
To achieve these goals, this study is divided into two major parts as follows:
Chapter 2
Seasonal variation in vertical distribution of Echinolittorina

species
Since the vertical distributions of Echinolittorina malaccana, E. radiata and E.

vidua in Hong Kong have been previously investigated by Mak (1996) and
Walters (2002), in this chapter, therefore, the focus was to measure the seasonal
variation in the distribution and abundance of the Echinolittorina species. Three,
monthly seasonal surveys were conducted in both winter and summer on
moderately exposed rocky shores in South Bay and Stanley, Hong Kong Island.
This data helped to determine how the distribution and abundance of the
littorinids may vary between seasons, which may be related to their tolerances to
the seasonally variable physical environment in Hong Kong.
Chapter 3
Thermal tolerance of Echinolittorina species in Hong Kong
Using a variety of physiological parameters, including lethal temperature,

Arrhenius breakpoint temperature of heart rate and activities of the enzymes
malate and lactate dehydrogenases, this chapter examined variation in thermal
tolerance of Echinolittorina species.
With these information, thermal tolerance
of Echinolittorina species in relation to their vertical and seasonal distribution

could be demonstrated, and their potential to acclimate to high temperature could
also be identified.
Hong Kong (22o20 N and 114o10E), is situated on the southern coast of China,
south of the Tropic of Cancer and has a strong seasonal, monsoon climate
(Morton and Morton, 1983).
In general, between June to September, Hong
Kong experiences a hot, wet summer due to the dominant south-eastern monsoon.
In 2010 for example, the average maximum air temperature was > 30oC, average
relative humidity of ~ 82% (Hong Kong Observatory, see Figure 1.2).
In
contrast, the north-eastern monsoon during the winter brings cold, dry conditions
from December to March (Kaehler and Williams, 1996), for example during 2010,
the average maximum air temperature was < 20oC, average relative humidity of ~
70% (Hong Kong Observatory, Figure 1.2).
As well as differences in air
temperature and humidity, rainfall is higher in summer, with an average monthly

rainfall > 400mm in 2010, as compared with the winter average monthly rainfall
of < 50mm (Hong Kong Observatory, Figure 1.2).
The periods between these
two dominant monsoon seasons, i.e. April May and October November, are
considered transition periods when weather conditions are variable as the
monsoon influences change (Kaehler and Williams, 1996).
Sea surface temperatures also exhibit strong seasonal variation in Hong Kong.
In summer, the Hainan current from the South China Sea brings warm water
northwards along the south China coast (Morton and Morton, 1983), and sea
surface temperature at, for example, Waglan Island (south of Hong Kong Island)
is maintained at ~ 27oC (Hong Kong Observatory).
In winter, while the Taiwan
current from the East China Sea brings relatively cold water (19-23oC, Morton
10
and Morton, 1983) to the coast of Hong Kong, the Hainan current is replaced by
the warm Kuroshio current (26-29oC, Morton and Morton, 1983).
The mixing of
water from the Taiwan and Kuroshio current, therefore, contributes to a relatively
warmer sea surface temperature (monthly average=18.9oC, Hong Kong
Observatory) than air temperature in winter (monthly average=15.7oC, Hong
Kong Observatory) in Hong Kong.
In general wave action is more calm in
summer than winter (Apps and Chen 1973), with the exception of periods of
tropical depressions or typhoons when wave action and wind speeds are extreme
(Morton and Harper, 1995; Morton et al., 1996).
The daily tidal pattern in Hong Kong ranges between mixed, semi-diurnal, tides
during spring tides, and almost diurnal neap tides (Morton et al., 1996), with an
average tidal range of ~ 2.2m (Hutchinson and Williams, 2003a).
During spring
tides, the lowest of the low tides generally occurs during late night early
morning in winter, while during summer, the lowest of the low tides occurs in the
early afternoon and evening (Williams, 1994b). The seasonal variation in timing
of low tides, therefore, creates a strong seasonal contrast in environmental
temperatures that intertidal organisms experience (see Figure 1.2), with winters
being cool and dry and animals emersed during the night, whilst summers are hot
and wet and animals are emersed at the hottest time of the day when rock surface
temperatures can exceed 50oC (Williams, 1994b, Williams and Morritt, 1995;
Williams et al, unpubl data) .
11
Site descriptions
All field surveys and animal collections were conducted at South Bay (SB,
2213N, 11411E), Stanley (ST, 2212N, 11413E) and Tai Tau Chau (TTC,
2213N,11415E). All the three sites are located within the eastern, oceanic
waters of Hong Kong (Figure 1.3), to minimize the confounding effect of salinity
variation brought by the Pearl River Estuary which influences Hong Kong's
western waters. The three sites experience similar levels of wave exposure and
can be categorized as moderately exposed shores as defined by the distribution of
flora and fauna (Morton and Morton, 1983, Kaehler and Williams, 1996). While
South Bay is composed of a nearly vertical rock cliff (see Figure 1.4 a), and faces
south-west; Stanley and Tai Tau Chau are composed of more flattened rock
platforms (Figures 1.4 b & c), and face north and east respectively.
12
Table 1.1 Distribution of Echinolittorina species in the Indo-West Pacific Ocean

(after Reid, 2007)
Species
Latitudinal distribution
Latitudinal range
(North - south range)

E. malaccana
Zhejiang, China Indonesia
3015'N - 500'N
E. radiata
Hokkaido, Japan Nha Trang, Vietnam
4317N - 1214N
E. vidua
Miura, Japan Sydney, Australia
3508N - 3352S
13
E. malaccana
5 mm
E. radiata
5 mm
E. vidua
5 mm
Figure 1.1 The three common rocky shore Echinolittorina species in Hong Kong.
14
Air temperature
Air temperature (o C)
35
30
25
20
15
10
0
Rainfall
600
Total rainfall (mm)
500
400
300
200
JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
OCT
NOV
DEC
JAN
FEB
MAR
APR
MAY
JUN
JUL
AUG
SEP
100
2010
2011
Month
Figure 1.2 Monthly variation in: (upper graph) air temperature (
maximum;
= average;
monthly rainfall (
= average
= average minimum); (lower graph) total
) in Hong Kong within the study period (January,
2010-September, 2011) (data from Hong Kong Observatory). S = summer; W =

winter and areas shaded in grey represent the transition periods.
15
NEW TERRITORIES
KOWLOON
LANTAU
ISLAND
HONG KONG
ISLAND
Figure 1.3 Location of the three study sites: South Bay, Stanley and Tai Tau Chau in Hong Kong ( ).
16
Figure 1.4 a Photograph of study site at South Bay (SB) in summer.
17
Figure 1.4 b Photograph of study site at Stanley (ST) in winter.
18
Figure 1.4 c Photograph of study site at Tai Tau Chau (TTC) in summer.
19
CHAPTER 2:
SEASONAL VARIATION IN VERTICAL
DISTRIBUTION OF ECHINOLITTORINA
SPECIES
2.1 INTRODUCTION
21
2.2 MATERIALS AND METHODS
26
2.3 RESULTS
29
2.4 DISCUSSION
40
Chapter 2: Seasonal variation in vertical distribution of Echinolittorina species
2.1 INTRODUCTION
Intertidal ecologists have been trying to understand how physical factors and
biological interactions drive distribution patterns using sessile species (in
barnacles: Connell, 1961a,b; Wethey, 1984; mussels: Paine, 1974; algae:
Schonbeck and Norton 1978, 1980; Underwood, 1980; also see Underwood, 1979
for reviews). The lower limit of distribution of sessile species has been proposed
to be driven by biological interactions, such as competition and predation
(Connell, 1961a,b; Wethey, 1984; Paine, 1974; Schonbeck and Norton, 1980).
Connell (1961a), for example, showed that the rapid growth in the lower shore
Balanus balanoides was responsible for mortality of the upper shore barnacle,
Chthamalus stellatus, when they were found low on the shore, and proposed that
the lower limit of C. stellatus was determined by competition.
The upper limit
of species' distributions, on the other hand, is generally driven by physical factors,

such as temperature and desiccation stresses (Connell, 1961a; Schonbeck and
Norton, 1978; Underwood, 1980).
Three species of fucoid algae (Pelvetia
canaliculata, Fucus spiralis and Ascophyllum nodosum), for example, showed

clear tissue damage at their maximum height of distribution when they were
emersed for over a 20-day period (Schonbeck and Norton, 1978), illustrating the
important roles played by temperature and desiccation in determining the upper
limit of species' distributions. Sessile species are also unable to escape from
extreme environmental conditions during emersion, especially in summer.
During hot summer months, environmental conditions can exceed the survival
limits of sessile species, often resulting in high shore kills of these species (e.g.
Connell, 1961a,b; Hawkins and Hartnoll, 1985; Chan et al., 2006).
21
Unlike sessile species, mobile species have the advantage to avoid extreme
environmental conditions by moving to microhabitat refuges, such as crevices,
cracks and rock pools (Garrity, 1984; Williams and Morritt, 1995; Helmuth and
Hofmann, 2001), or migrating down shore to avoid hot summer conditions (Lewis,
1954; Branch, 1975; Liu, 1994; Williams and Morritt, 1995; Takada, 1996; Harper
and Williams, 2001; see Underwood, 1979 for review).
Given the ability of
mobile species to use behavioural mechanisms to avoid extreme environments,

mobile species, therefore, may have less risk of mortality as compared to sessile
species. The upper limit of mobile species distribution is, however, in a similar
manner to sessile species, largely determined by physical factors (for example, in
limpets: Wolcott, 1973; gastropods: Britton and McMahon, 1990; McMahon,
1990; 2001; porcelain crabs: Stillman and Somero, 2001; see Underwood, 1979
for review).
These studies generally showed that the physiological tolerance of
mobile species to physical stresses was positively correlated to their

corresponding shore heights.
The behavioural and physiological strategies
exhibited by mobile intertidal species cannot be placed into context without a

basic understanding of the vertical distribution of a species (Underwood et al.,
2000).
Understanding the vertical level that a species inhabits allows the
description or quantification of the physical environment that they will experience

and the ranges of environmental conditions that they will have to tolerate. To fully
appreciate the ecology of intertidal species, therefore, requires an initial
assessment of their vertical distribution patterns (Underwood et al., 2000).
Generally categorized as high shore gastropods, littorinids are abundant at the
22
upper edge of intertidal habitats (e.g. rocky shores and mangroves) throughout the
world (see Lewis, 1964; Stephenson and Stephenson, 1972; McQuaid, 1996a,b;
Reid, 2007 for reviews).
Littorinids are commonly restricted to areas above the
high water mark and, therefore, experience prolonged periods of emersion, which
can last longer than 8 days during neap tides (McMahon, 2001; Uglow and
Williams, 2001).
Throughout the prolonged emersion period, littorinids
frequently experience extreme environmental conditions (McMahon, 1990;

Williams, 1994b; McQuaid, 1996b; Uglow and Williams, 2001).
To survive the
harsh conditions on the high shore, littorinids are well-adapted to this environment
through morphological (Vermeij, 1973; McQuaid and Scherman, 1988; Lee and
Lim, 2009), behavioural (e.g. Vermeij, 1971; McQuaid and Scherman, 1988;
Muoz et al., 2005), and physiological (e.g. Evans, 1948; Stirling, 1982;
McMahon, 2001) strategies.
Morphologically, many species have light-coloured
and nodulated shells, which help reflect heat (Vermeij, 1973; McQuaid and
Scherman, 1988; Lee and Lim, 2009).
In terms of behavioural aspects, most
littorinids withdraw their foot, and attach themselves via a mucus thread when the
substratum reaches a high temperature (Vermeij, 1971; McQuaid and Scherman,
1988). This 'standing' behaviour reduces their direct contact with the substratum
and, therefore, minimizes heat uptake through conduction from the rock surface
(Marshall et al., 2010).
Such strategies, therefore, allow littorinids to dominate
the physically harsh environment high on intertidal shores.
Littorinids, similar to other intertidal gastropods (limpets Liu, 1994; Williams and
Morritt, 1995; top shells: Takada, 1996; various intertidal gastropods: Harper and
23
Williams, 2000), also show seasonal variation in their vertical distribution, which
has been suggested to minimize physical stresses (Lambert and Farley, 1968;
Hannaford Ellis, 1985; Ohgaki, 1988b; Mak, 1996).
These studies, in general,
observed that adult individuals are distributed lower on shores during hot periods
of the year (i.e. summer) than during cooler, winter periods.
Such migrations
may, however, be related to other factors, such as wave exposure, food availability
and reproduction (Lambert and Farley, 1968; Williams and Ellis, 1975). For
example, Ohgaki (1988b; 1989; Ito et al., 2002) suggested that the downshore
migration of Echinolittorina radiata in Japan was related to their spawning
pattern.
Mak (1996) observed a similar pattern in Hong Kong, which he
suggested may be related to seasonal variation in abundance of microalgae.

While explanations for the cause of migration patterns remain controversial,
determination of such patterns may be important in understanding the physical
environment species face, and the forces which drive these migration patterns.
Echinolittorina malaccana, E. radiata and E. vidua are three common littorinds

that are widely distributed on Hong Kong rocky shores (Ohgaki, 1985, Williams,
1994; Mak 1996; Walters, 2002). Given that these Echinolittorina species live
close to the upper limit of the intertidal zone, they show distinct and consistent
vertical distribution patterns over shores of varying wave exposure (Ohgaki, 1985;
Mak, 1996), as well as seasonal vertical migration (Mak, 1996).
On all three
shores of varying wave exposure that Mak (1996) studied, E. malaccana inhabited
the highest level of the intertidal zone (mean height = 2.2-2.7 m C.D.), followed
by E. radiata (1.8-2.5 m C.D.) and E. vidua (1.3-2.0 m C.D.).
24
From his surveys,
Mak also found that all three Echinolittorina species were distributed at lower
shore levels in summer as compared to winter (Mak, 1996).
There are several
explanations or theories/hypotheses which can be applied to explain the

distribution patterns of Hong Kong Echinolittorina species.
Understanding both
the spatial and temporal distribution of the littorinids, however, is an essential

starting point to help generate hypotheses about how a specific factor may play a
role in determining the observed distributions (Underwood et al., 2000).
In this study, as well as determining the species' vertical distributions (which have
been previously described in earlier studies, Mak, 1996), variation in the seasonal
distribution and abundance of E. malaccana, E. radiata and E. vidua in Hong
Kong were also quantified.
Seasonal surveys were conducted in winter and
summer months on moderately exposed rocky shores at South Bay and Stanley,
Hong Kong (see Chapter 1).
These surveys aimed to establish the physical
environment these snails experienced to inform further investigations on the

relative physiological tolerances of these species to physical factors, such as
temperature, and how these may be related to the spatial and temporal
distributions of Echinolittorina species in Hong Kong (see Chapter 2).
25

Field surveys were conducted to determine the vertical distributions of
Echinolittorina malaccana, E. radiata and E. vidua in winter and summer.
Surveys were carried out on three, randomly selected, replicate days in winter
(Dec 2010-Feb, 2011) and summer (Jun-Aug, 2011, as defined by Kaehler and
Williams, 1996) at the two study sites (refer to Chapter 1).
Average monthly air
temperatures were between 13.7-20.2oC during winter, and 27.1-29.5oC in

summer (Hong Kong Observatory, see Chapter 1).
Surveys were conducted
during low water on spring tides (tidal height 1.20m above Chart Datum, C.D.),
when the littorinids were emersed and inactive (Mak, 1996).
All surveys were
conducted during dry days, to minimize potential disturbance to the littorinids

distributions due to dislodgement by rain (Ohgaki, 1988a).
The survey method was adopted from Mak (1996), who also conducted surveys
on Echinolittorina species in Hong Kong.
At each site, a horizontal, 10m stretch
of shore was vertically divided into 25cm height intervals from 1.5-3.25m above
C.D., which is the normal vertical range for Echinolittorina species (Mak, 1996).
At each height, 10, 25x25cm quadrats were randomly placed, and the total
number of each Echinolittorina species in each quadrat was recorded.
The
vertical distribution for each Echinolittorina species was presented in terms of

mean number and mean percentage of individuals at different tidal heights from
the three replicate days during each season, as individual day to day differences
were not of interest in this study.
The percentage of individuals at different tidal
heights was calculated as follows:

26
The overall mean height of each species was calculated following Harper and
Williams (2001):
The sample area covered at each height was 6250cm2 [25 x 25cm (area of each
quadrat) x 10 quadrats], whereas the total area covered on each sampling day was
50000cm2 [Total area= 6250cm2 (area of each height) x 8 heights].
Due to inherent local variability (e.g. wave exposure, topography, inclination), the
vertical distribution of a species would be expected to show some degree of
variation between different shores (Mak, 1996).
As the present study aimed to
obtain a species-level view of overall distribution patterns of the three

Echinolittorina species, between shore variation was not analyzed, and a two-way
Analysis of Variance (ANOVA) was used to test for any possible variation in
vertical distribution and total abundance of Echinolittorina species between
seasons at South Bay and Stanley separately, using WinGMAv5 (EICC, The
University of Sydney), with species (sp) [three levels: E. malaccana, E. radiata
and E. vidua] and seasons (se) [two levels: Summer and Winter] treated as fixed
27
factors.
Cochrans test was used to check for homogeneity of variances
(Cochran, 1951) and data were transformed, where possible, if they were
heterogeneous.
Analyses were still conducted when data failed tests for
homogeneity of variances after transformation, as ANOVA is considered to be

robust to such heterogeneity, provided the error degrees of freedom are relatively
large (Underwood, 1997).
Where variances were homogenous, to minimize the
chance of Type I errors, p-values were adjusted to a more conservative level (p <
0.01, Underwood, 1981).
Significant difference(s) for fixed factors were further
analysed by post-hoc Student-Newman-Keuls (SNK) tests to identify potential

differences in treatment means.
28
2.3 RESULTS
The distribution of Echinolittorina malaccana, E. radiata and E. vidua showed
consistent vertical patterns at both South Bay and Stanley.
In general, E.
malaccana occupied the highest position on both shores (mean height S.D. =
2.76m above C.D. 0.24), which overlapped with E. radiata which was found
slightly lower on the shore (2.46m above C.D. 0.29), and finally E. vidua which
overlapped E. radiata, but was found lowest on the shore (1.96m above C.D.
0.23, see Figures 2.1 & 2.2).
The vertical distribution ranges for the three
species between the two shores were similar, although the average mean height
for all species in Stanley, was ~ 0.25m lower than at South Bay (Figure 2.3).
The abundance of Echinolittorina species also showed a consistent pattern at the

two shores (Figures 2.4 & 2.5, Tables 2.2 & 2.3).
E. malaccana was the most
abundant among the three species (total abundance from all surveys: 2,151
individuals for South Bay; 2,853 for Stanley), whilst E. radiata was ~ 50% less
abundant on both shores (total abundance: 1,071 for South Bay; 1,512 for Stanley),
and E. vidua was the least abundant (total abundance: 377 for South Bay; 221 for
Stanley, see Figures 2.4 & 2.5).
Both the vertical distribution and abundance of the individual species varied
between seasons (Tables 2.1-2.3).
The vertical distributions of E. malaccana, E.
radiata and E. vidua were generally lower by at least 0.25m during the summer
than in the winter months at both sites (Figures 2.1 & 2.2), with the exception of E.
vidua at Stanley which showed a similar distribution between seasons.
29
The
mean height of all species was significantly lower in summer than winter at both
sites (Figure 2.3, Table 2.1). The total abundance of Echinolittorina species,
although not significantly different within species (except for E. malaccana at
South Bay, see Tables 2.2 & 2.3) was, in general, less in summer than winter at
both sites (Figure 2.6, Table 2.2). Both E. radiata and E. vidua were ~ 25% less
abundant in summer than winter at South Bay, and more than 50% less abundant
at Stanley.
This temporal pattern was also observed for E. malaccana at Stanley,
which was also ~ 25% less abundant during summer as compared with winter.
In contrast, however, E. malaccana were significantly more abundant during
summer than winter at South Bay by ~ 40%.
To conclude, consistent patterns were observed for both the vertical distribution
and abundance of Echinolittorina species at South Bay and Stanley.
Echinolittorina malaccana occupied the highest tidal level and was the most
abundant species, followed by E. radiata which dominated relatively lower tidal
levels and was less abundant, and finally E. vidua, which was found the lowest on
the shore and was the least abundant among the three species.
In terms of
temporal variation, all three species were less abundant and found at lower shore
levels in summer than winter, with the exception of E. malaccana at South Bay.
30
South
Bay
225
E. malaccana
200
175
150
125
100
75
Mean no. of individuals
50
25
0
200
E. radiata
175
150
125
100
75
50
25
0
75
E. vidua
50
25
25
3.
00
3.
75
2.
50
2.
25
2.
00
2.
75
1.
1.
50
Height above C.D. (m)

Figure 2.1 Vertical distribution of Echinolittorina species at South Bay. Mean
number of individuals of Echinolittorina malaccana, E. radiata and E. vidua,
sampled on three separate days in both winter (
summer (
, Jun-Aug, 2011) (mean S.D., n = 3).

31
, Dec, 2010-Feb, 2011) and
Mean no. of individuals
Stanley
275
250
225
200
175
150
125
100
75
50
25
0
175
150
E. malaccana
E. radiata
125
100
75
50
25
0
100
E. vidua
75
50
25
25
3.
00
3.
75
2.
50
2.
25
2.
00
2.
75
1.
1.
50

Figure 2.2 Vertical distribution of Echinolittorina species at Stanley. Mean
number of individuals of Echinolittorina malaccana, E. radiata and E. vidua,
, Dec, 2010-Feb, 2011) and
summer (
, Jun-Aug, 2011) (mean S.D., n = 3).
32
3.5
South Bay
3.0
2.5
Mean height (m)
2.0
1.5
1.0
0.5
0.0
3.5
Stanley
3.0
2.5
2.0
1.5
1.0
0.5
0.0
E. malaccana E. radiata
E. vidua
Species
Figure 2.3 Seasonal variation in mean vertical height of Echinolittorina species
at South Bay and Stanley. Mean of mean vertical height of Echinolittorina
malaccana, E. radiata and E. vidua, sampled on three separate days in both
winter (
, Dec, 2010-Feb, 2011) and summer (
S.D., n = 3).
33
, Jun-Aug, 2011) (mean m
South
Bay
100
E. malaccana
nwinter = 869
nsummer = 1282
E. radiata
nwinter = 584
nsummer = 487
E. vidua
nwinter = 221
nsummer = 156
75
50
Mean % of individuals
25
0
100
75
50
25
0
100
75
50
25
25
3.
00
3.
75
2.
50
2.
25
2.
00
2.
75
1.
1.
50

Figure 2.4 Relative vertical distribution of Echinolittorina species at South Bay.
Mean % of individuals of Echinolittorina malaccana, E. radiata and E. vidua,
, Dec, 2010-Feb, 2011) and
summer (
, Jun-Aug, 2011) (mean % S.D., n = 3).
34
Stanley
100
E. malaccana
nwinter = 1668
nsummer = 1185
E. radiata
nwinter = 1127
nsummer = 385
E. vidua
nwinter = 171
nsummer = 50
75
50
Mean % of individuals
25
0
100
75
50
25
0
100
75
50
25
25
3.
00
3.
75
2.
50
2.
25
2.
00
2.
75
1.
1.
50

Figure 2.5 Relative vertical distribution of Echinolittorina species at Stanley.
Mean % of individuals of Echinolittorina malaccana, E. radiata and E. vidua,
, Dec, 2010-Feb, 2011) and
summer (
, Jun-Aug, 2011) (mean % S.D., n = 3).
35
Total abundance / 50000 cm2
500
450
400
350
300
250
200
150
100
50
0
South Bay
Stanley
700
600
500
400
300
200
100
0
E. vidua
Figure 2.6 Seasonal variation in total abundance of Echinolittorina species at

South Bay and Stanley. Mean total abundance (per 50000 cm2) of Echinolittorina
malaccana, E. radiata and E. vidua, sampled on three separate days in in both
winter (
, Dec, 2010-Feb, 2011) and summer (
individual S.D., n = 3).
36
, Jun-Aug, 2011) (mean
Table 2.1 Two-way ANOVA to compare variation in mean height of Echinolittorina species (Sp) between seasons (Se) at South Bay and
Stanley. Variances were homogenous (Cochran's test: C = 0.4370, p > 0.05 for South Bay; C = 0.5712, p > 0.05 for Stanley). Significant
differences are indicated by asterisk(s): * (<0.05); ** (<0.01). Post-hoc Student-Newman-Keuls tests (mean S.D.) were conducted for
multiple comparisons of means.
South Bay
Stanley
Source
df
SS
MS
Sp
Se
Sp x Se
2
1
2
1.738
0.708
0.007
0.869
0.708
0.004
28.88
23.53
0.12
<0.001** Residual
<0.001** Residual
0.887
Residual
Residual
12
0.361
SNK tests
Sp
Se
E. malaccana
2.84 (0.19)
Winter
2.70 (0.21)
Source
df
SS
MS
Sp
Se
Sp x Se
2
1
2
2.250
0.289
0.007
1.125
0.289
0.004
39.39
10.11
0.13
<0.001** Residual
0.008**
Residual
0.881
Residual
0.030
Residual
12
0.343
0.029
>
SNK tests
Sp
>
E. radiata
2.57 (0.21)
Summer
2.30 (0.23)
Fvs
>
E. vidua
2.09 (0.17)
Se
37
E. malaccana
2.68 (0.14)
Winter
2.42 (0.23)
>
>
E. radiata
2.35 (0.19)
Summer
2.16 (0.14)
Fvs
>
E. vidua
1.83 (0.10)
Table 2.2 Two-way ANOVA to compare variation in total abundance of

Echinolittorina species (Sp) between seasons (Se) at South Bay. Variances were
homogenous (Cochran's test: C = 0.4039, p > 0.05). Significant differences are
indicated by asterisk(s): * (<0.05); ** (<0.01). Post-hoc Student-Newman-Keuls
tests (mean S.E.) were conducted for multiple comparisons of means.
Source
df SS
Sp
Se
Sp x Se
Residual
1 3500.1
2 27200.4
12 41403.3
MS
Fvs
266395.1 133197.6 38.60 <0.001** Residual

3500.1
13600.2
3450.3
SNK tests
Se (Sp)
E. malaccana
Winter
<
289.67 (91.45)
E. radiata
Sp (Se)
F
1.01
3.94
0.334
0.048*
Residual
Residual
Summer
427.33 (69.25)
Winter
=
194.67 (85.34)
E. vidua
Winter
=
73.67 (23.76)
Summer
E. malaccana >
427.33 (69.25)
Winter
Summer
162.33 (9.28)
>
E. vidua
52.00 (17.44)
E. malaccana =
289.67 (91.45)
E. radiata
>
194.67 (85.34)
E. vidua
73.67 (23.76)
Summer
52.00 (17.44)
E. radiata
162.33 (9.28)
38
Table 2.3 Two-way ANOVA to compare variation in total abundance of

Echinolittorina species (Sp) between seasons (Se) at Stanley. Variances were
homogenous (Cochran's test: C = 0.5521, p > 0.05). Significant differences are
indicated by asterisk(s): * (<0.05); ** (<0.01). Post-hoc Student-Newman-Keuls
tests (mean S.D.) were conducted for multiple comparisons of means.
MS
Source
df SS
Sp
Se
Sp x Se
Residual
1 100650.9 100650.9 11.92 0.005**

2 32431.4 16215.7 1.92 0.189
12 101300.0 8441.7
Fvs
577354.8 288677.4 34.20 <0.001** Residual
SNK tests
Sp
E. malaccana
>
475.50 (104.93)
Se
Winter
>
329.56 (133.47)
Residual
Residual
E. radiata
> E. vidua
252.00 (109.48)
36.83 (21.01)
Summer
180.00 (108.69)
39
2.4 DISCUSSION
In Hong Kong, clear vertical zonation patterns of three Echinolittorina species
were consistently observed on two moderately exposed rocky shores.
E.
malaccana was distributed highest on the shore, and its range overlapped with E.
radiata, which was dominant slightly lower on the shore, while E. vidua was
found lowest on the shore.
The vertical zonation pattern of the three
Echinolittorina species matches previous observations on Hong Kong shores

(Ohgaki, 1985; Cleland and McMahon, 1990; Dudgeon and Yipp, 1990; Williams,
1994; Mak, 1996; Walters, 2002). Cleland and McMahon (1990), for example,
also showed that the vertical distribution of E. malaccana and E. radiata
overlapped, and E. radiata were also found ~ 0.5m lower on the shore than E.
malaccana.
A similar pattern was also observed for E. malaccana and E. radiata
in Singapore (Lee and Lim, 2009), while in Tanabe Bay (Japan) and the Gulf of
Thailand, E. vidua was found below E. radiata and E. malaccana respectively and
overlapped with the vertical distribution of barnacles, Tetraclita spp. (Habe, 1958;
Tsuchiya and Lirdwitayapasit, 1986; also see Reid, 2007 for review).
Authors working in Hong Kong have proposed that Echinolittorina species

experience reduced biological interactions, with low predation risk (Mak, 1996;
Stafford, 2002; Li, personal observation) and competition pressures (Dudgeon and
Yipp, 1990) by living high on the shore. These potential benefits may be offset
by the high physical stresses which the littorinids will experience living in the
high-shore.
Species specific variation in tolerance to thermal and desiccation
stresses may positively match with the vertical distributions of the three
40
Echinolittorina species.
Laboratory experiments, for example, suggest that E.
malaccana are more tolerant to desiccation stress than E. radiata in Hong Kong,
given that the LT (lethal time) 50 in air was 34 days more for E. malaccana than E.
radiata (Yipp et al., 1986), although such exposure times are highly unrealistic.
Similarly, thermal tolerance, in terms of heat coma and lethal temperature, were
higher in E. malaccana than E. radiata according to studies conducted in Hong
Kong by Stirling (1982) and McMahon (2001).
These data, although not able to
fully explain the zonation patterns of Echinolittorina species, do illustrate the

importance of physical factors in relation to species' distribution, and hence
implications for community structure and ecological processes (Menge and Olson,
1990; Underwood and Chapman, 1996).
As was recorded by Mak (1996), Echinolittorina malaccana, E. radiata and E.

vidua, showed seasonal variation in their vertical distribution, with all three
species generally being distributed higher on the shore during winter than summer.
This phenomenon has been interpreted to enhance the reproductive success of
littorinids (Kojima, 1959; Hannaford Ellis, 1985; Ohgaki, 1988a; Mak, 1996), as
downward migration of the littorinids generally coincides with their spawning
periods.
Moving downshore, therefore, was suggested to increase the chance for
the littorinids to release their eggs in water by increasing their duration of

immersion. The immersion period can, for example, be up to 2 hours longer at
1.5m C.D. than 1.75m during summer neap tides on Hong Kong shores (Ng,
2007). Most of these studies, however, failed to provide any evidence of the
actual benefits of an increased duration to release eggs when littorinids were
41
immersed in support of this theory.
Other littorinid species, such as Littorina
unifasciata and L. cincta, for example, do not migrate downwards during their
spawning period (Pilkington, 1971), therefore, the downward movement of
littorinids in summer may not be fully explained by spawning migrations.
The
downward migration of littorinids may also possibly be related to food availability

(Mak, 1996; Ito et al., 2002), or perhaps seasonal variation in tidal height. The
lowest tides are generally lower in the summer than winter (Kaehler and Williams,
1996) which may account for the lower distribution of littorinids at this time of
year (Ohgaki, 1989).
The littorinids may also migrate downshore to avoid
extreme physical stress by minimizing emersion duration (Lambert and Farley,

1968; Williams and Ellis, 1975), as described for other intertidal species in Hong
Kong (Harper and Williams, 2001).
Seasonal variation in the abundance of Echinolittorina species was also detected

at Stanley.
The lower abundances recorded during summer months matches with
Mak (1996)s monthly surveys at three shores of varying exposures in Hong Kong
(Big Wave Bay, Cape d Aguilar and South Bay).
Similar patterns of seasonal
abundance were also observed for E. radiata and E. vidua at South Bay, although
these were not statistically significant.
A decrease in abundance of littorinids
during the summer months may be related to mortality associated with conditions
at this time of year; such as heat stress (Williams, 1994b; Mak, 1996).
Dislodgement of littorinids caused by heavy rainfall has also been proposed to
affect populations in both temperate Japan (Ohgaki, 1988b) and Hong Kong due
to tropical storms in summer (Mak, 1996).
42
Authors have suggested that a decrease in abundance of intertidal species often

occurs following tropical storms (Kohn, 1980; Boulding and Van Alstyne, 1993;
Mak, 1996; Hutchinson and Williams, 2003b; Sanpanich et al., 2006). During
tropical storms, intertidal gastropods may move to refuges, such as cracks and
crevices, to avoid dislodgement by strong wave action, and if they fail to find
these refuges they may be washed away resulting in mortality (Kohn, 1980).
In
2011, the first tropical storm (Sarika) arrived on 9th June after the first summer
survey was conducted, and rainfall was not heavy during the storm period
(accumulated rainfall 7th 11th June < 20 mm, Hong Kong Observatory).
This
storm did not seem to have any observable effect on littorinid abundance as the
abundance of E. malaccana and E. radiata was observed to have increased, or
was similar, in the second summer survey.
E.vidua, did, however, decline in
abundance between the first and second survey, and this continued between the
second and third survey, although there were no further storms.
Mortality due to
tropical storms is, therefore, unlikely to explain the reduction in littorinid

abundance in the present study, as also noted by Walters working on
Echinolittorina in Hong Kong (2002).
Despite the potential effect of tropical storms, heavy rainfall (i.e. > 46mm per day,
Ohgaki, 1988b), may also contribute towards the decrease in abundance of
Echinolittorina species in summer. Ohgaki (1988b) showed that heavy rainfall
would increase detachment of mucus threads in E. radiata, thus dislodging the
littorinids. As the first heavy rainfall (total rainfall per day: 69.8.mm, Hong
43
Kong Observatory) was reported on 22nd May, 2011, this effect may explain the
lower abundance observed in, for example, E. malaccana in the first field survey
in both South Bay (~ 30%) and Stanley (~ 80%) as compared with the second
survey. Heavy rainfall, may, therefore, have been responsible for the decline in
littorinid abundance in summer, although no direct evidence is available to link
this with snail mortality, since no tagging experiments have been conducted on
Echinolittorina species to determine their survival in relation to rainfall (see Mak,
1996).
The major link to the reduction in abundance of the three Echinolittorina species
during summer in Hong Kong may be associated with the stressful physical
environment experienced on rocky shores during this time.
In summer the
lowest tides occur during the afternoon, when rock surface temperatures may
exceed 50oC (see previous references).
Such extreme high temperature
conditions during summer can cause 50% mortality of mobile species on Hong
Kong shores (Williams et al., unpubl data).
Large mortality events are
commonly observed, for example, in the limpet, Cellana toreuma (Firth and
Williams, 2009) and barnacle, Tetraclita japonica (Chan et al., 2006), when
physical stresses exceed species tolerance limits (Williams and Morritt, 1995;
Dong and Williams, 2011) and attempting to reduce this stress by migrating
downshore may explain the seasonal variation in the littorinids vertical
distribution (Williams and Morritt, 1995; Harper and Williams, 2001).
Little is
known, however, about possible seasonal variation in littorinids tolerance to

physical stresses in Hong Kong.
While the reduction in littorinid abundance in
44
summer may be caused by mortality, the increased abundance in winter was

suggested to be a result of recruitment between October to January (Mak, 1996).
The contrary lower abundance of E. malaccana in winter than summer at South
Bay is, however, difficult to explain and was not recorded by Mak (1996),
suggesting this may be an anomaly.
This anomaly may also be related to the
recruitment patterns of E. malaccana at South Bay (Menge and Branch, 1999), for
example, variation in sporadic recruitment and recruitment failure.
In conclusion, Echinolittorina malaccana, E. radiata and E. vidua, as in many

previous studies, showed a consistent vertical pattern on rocky shores in Hong
Kong.
In general, Echinolittorina species were found lower down the shore, in
reduced numbers in summer than winter. While the downward migration in

summer may be a response to enhance reproductive success, the decline in
abundance may be related to mortality/ dislodgement of littorinids during heavy
rainfall, however there is little direct evidence to support these explanations.
As
these Echinolittorina species will experience relatively low negative biological

interactions, such as predation and competition (Stafford, 2002), living in the high
shore on Hong Kong shores; it seems logical to assume that physical factors, e.g.
temperature and water availability, may play important roles in determining
spatial and temporal variation in their vertical distribution and abundance.
As a
result, a series of laboratory based assays are required to try and establish whether
there is a correlation between their physical tolerances and spatial and temporal
patterns, and these are reported in the next chapter.
45
CHAPTER 3:
THERMAL TOLERANCE OF
ECHINOLITTORINA SPECIES IN HONG KONG
3.1 INTRODUCTION
48
Thermal tolerance and species distribution on intertidal rocky

shores
Thermal tolerance and vertical distribution of Echinolittorina
species on Hong Kong rocky shores
58

Seasonal variation in Arrhenius breakpoint temperature of heart
rate
Data analysis
3.3 RESULTS
Seasonal variation in Arrhenius breakpoint temperature of heart
rate
72
3.4 DISCUSSION
84

Arrhenius breakpoint temperature of heart rate in relation to
Activities of malate dehydrogenase and lactate dehydrogenase in
relation to vertical distribution
Chapter 3: Thermal tolerance of Echinolittorina species in Hong Kong
3.1. INTRODUCTION
Thermal tolerance and species distribution on intertidal rocky shores
The intertidal zone is an extreme and dynamic environmental gradient, where

species can experience severe environmental stress over a short vertical distance
(< 10m, see Chapter 1).
This is especially true of intertidal rocky shores, as the
rock surface is a two-dimensional habitat which offers little protection against

physical stresses, which are generally suggested to set the upper limit of species
distribution (e.g. Connell, 1961a; Newell, 1970; Moore, 1972; Wolcott, 1973; also
see Underwood, 1979 for review).
The linkage between thermal tolerance and
vertical distribution of species has been widely studied, with comparative studies
of genera distributed at different shore levels being relatively common.
For
example, McMahon (2001) investigated heat coma temperature (see below for
definition) for 60 species of gastropods collected from various types of intertidal
habitats from different geographic areas, and found a strong positive relationship
between thermal tolerance and corresponding shore height.
A similar trend, with
thermal tolerance being correlated with tidal height, was also observed by Stirling
(various gastropods, 1982) and Davenport and Davenport (various gastropods,
barnacles & bivalves, 2005).
Although these studies compared thermal tolerance
across genera to illustrate this linkage, such comparisons may be confounded by

the role phylogenetic relationships play in determining the distribution of species
(see Somero, 2002).
Somero (2010), therefore, suggested studies which focus
on comparative physiology of congeneric species instead of across different

genera to overcome this problem.
Using this approach, Stillman and Somero
(2000) showed that upper shore porcelain crabs in the genus Petrolisthes, tended
48
to have greater thermal tolerance than lower shore species in the same genera.
This pattern was also supported by Tomanek and Somero (1999) and Somero
(2002) for the top shells Chlorostoma (previously Tegula) and littorinids in the
genus Littorina respectively.
Temporal variation in environmental temperatures on intertidal rocky shores, for

example between seasons, also poses the question of whether the intertidal species
have the ability to acclimate to the changes in environmental temperatures, thus
altering their thermal tolerance. Such potential impacts on thermal tolerance
have been investigated either by seasonal field-based (e.g. Evans, 1948; Fraenkel,
1968; Stirling, 1982) and/or laboratory-based acclimation experiments (e.g.
Newell, 1971; Wolcott, 1973; McMahon et al., 1995; Tomanek and Somero, 1999;
Stillman and Somero, 2000; Stenseng et al., 2005; Hopkin et al., 2006).
Fraenkel (1968), for example, tested thermal tolerance of Littorina littorea
between seasons, and found that thermal tolerance was greater in summer than
winter; whilst Stillman and Somero (2000) acclimated three porcelain crab
species in the laboratory at 8oC and 18oC, and reported that thermal tolerance of
warmer acclimated crabs was greater than those acclimated to cooler
temperatures.
Species that have less potential to acclimate for greater thermal tolerance may
exhibit other strategies to avoid or minimize this stress such as seeking refuge in
benign microhabitats (Garrity, 1984; McQuaid and Scherman, 1988; Helmuth and
Hoffman, 2002), orientating their body positions (Muoz et al., 2005) or even
migrating downshore (Williams and Morritt, 1995; Harper and Williams, 2001) to
49
minimize exposure to stressful conditions (also see Chapter 2).
However, in
terms of longer temporal scales, for example, under climate change scenarios
where environmental temperature is predicted to be elevated by 2-6oC by 2100
(McCarthy, 2001), knowing the acclimation potential of intertidal species would
allow us to identify which species are more likely to survive or become locally
extinct, and therefore, possible impacts on community structures may also be
understood (Somero, 2002, 2010).
Thermal tolerance generally refers to the survival limit of species under a given
set of thermal conditions (see Angilletta, 2009 for review).
Traditional
approaches to determine species' thermal tolerances in intertidal ecology are to

measure species lethal (LT50) or heat coma temperatures (HCT) (e.g. Evans, 1948;
Newell, 1958, Southward, 1958; Fraenkel, 1960; Sandison, 1967; Stirling, 1982;
Urban, 1994; McMahon, 2001; Davenport and Davenport, 2005).
LT50 is
defined as the temperature at which 50% mortality of individuals of a species is

observed within the tested population, where mortality is determined as when
individuals fail to give any response to external stimulus and fail to recover after
return to ambient conditions (Evans, 1948; Fraenkel, 1960).
HCT is the
temperature at which 50% of individuals in the population start to lose their

normal neural function (i.e. enter a coma), and under such conditions, an
individual ceases locomotion and fails to attach to the substratum (Evans, 1948;
Southward, 1958; Stirling, 1982; McMahon, 2001).
50
Most studies have measured LT50 and/or HCT of various intertidal species while
immersed in water (see Table 3.1), in order to minimize the confounding effect of
desiccation, and generally showed a positive correlation between thermal
tolerance of species and their vertical distribution (see Underwood 1979 for
review).
Most species, however, and especially those which live on the upper
shore such as littorinids, do not experience extreme temperatures while they are
immersed but when they are emersed (Marshall et al., 2010), and may respond
differently when stressed under unnatural conditions.
For example, Sandison
(1976) measured HCT and LT50 for four Littorina species and Thais lapillus in air
and water and, in general, found both HCT and LT50 to be higher when
determined in air than water.
It is, therefore, important to select an
environmentally realistic heating condition when determining species thermal

tolerances.
When determining species thermal tolerance, although much of the past focus has
investigated survival limits, the tolerance of physiological systems from the
biochemical (e.g. protein synthesis, enzyme stability) to organ (e.g. heart function,
respiratory response) levels will provide important insights in relation to species'
survival limits, and thus their distributions (see Somero, 2002 for review).
Using four congeneric species of porcelain crabs (Petrolisthes), that live at
different shore levels, Stillman (2003) found that the Arrhenius breakpoint
temperature of heart rate (ABT of HR, see below for definition) was positively
correlated with the species LT50, and hence their vertical distributions. Similarly,
at the biochemical level, the stability and function of the metabolic enzyme,
malate dehydrogenase, in five species of Lottia was found to be correlated with
51
the respective species vertical and geographic distributions (Dong and Somero,
2009).
Heart rate, at the organ-level, has traditionally been used as a physiological

indicator of intertidal species response to thermal conditions (e.g. Pickens, 1965;
Trueman, 1967; Jones, 1970; Marshall and McQuaid, 1992; Santini et al., 1999;
Williams et al., 2005, 2010; Marshall et al., 2010; Dong and Williams, 2011; also
see Underwood, 1979; Somero, 2010 for reviews), as heart rate is known to be a
thermally sensitive measure of metabolic performance in many marine
invertebrates (see Hochachka and Somero, 2002).
As well as measuring species
metabolic performance, heart rate has shown to be positively related to oxygen

consumption of an organism (Marshall and McQuaid, 1992; see Prtner, 2001;
2010 for reviews).
This implies that once heart function of an individual starts
dropping drastically at high temperatures, individuals switch to a less-energy

producing anaerobic metabolism, which ultimately will result in mortality (see
Prtner, 2001 for review).
It can be illustrated in the case of Stillman's work on
porcelain crabs, where the temperature breakpoint of heart rate (see below for
definition) was shown to be positively correlated to an individuals' lethal
temperature (reviewed in Somero, 2010).
Heart rate can, therefore, be used as a
sensitive, sub-lethal, indicator to determine thermal tolerance of intertidal species.
To determine thermal tolerance using heart rate, the Arrhenius breakpoint

temperature (ABT of HR) of heart rate (defined as the temperature at which heart
rate of an individual drops dramatically, Stillman and Somero, 1996), is
commonly determined to assess the initiation of breakdown of heart function
52
(Tomanek and Somero, 1999; Stillman, 2003; Stenseng et al., 2005; Braby and
Somero, 2006; Marshall et al., 2010, 2011; Dong and Williams, 2011).
Similar
to survival limits, the ABT of HR for congeneric intertidal species has also been
found to be positively correlated to their vertical distributions (porcelain crabs:
Stillman and Somero, 1996; top shells: Stenseng et al., 2005; limpets: Dong and
Williams, 2011).
Enzyme function is also known to be thermally labile, with function breaking

down at temperatures when enzymes denature (see Hochachka and Somero, 2002).
Therefore, to study thermal tolerance limits in marine invertebrates at the
biochemical level, the activities of a suite of metabolic enzymes have been widely
determined (e.g. Sokolova and Prtner, 2001, 2003; Dahlhoff et al., 2002; Lee and
Lim, 2009; see Prtner, 2001, 2002; Hochachka and Somero, 2002; Somero, 2002,
2010; Dahlhoff, 2004 for reviews).
Malate dehydrogenase (MDH) and lactate
dehydrogenase (LDH) are common metabolic enzymes used to investigate

thermal tolerance of species, as their kinetics and stability to temperature have
been found to vary between several congeneric species (e.g. Dahlhoff and Somero,
1993; Fields et al., 2002, 2006; Dong and Somero, 2009).
Malate dehydrogenase (MDH) in general, contains two isoforms, mitochondrial

malate dehydrogenase (mMDH) and cytosolic malate dehydrogenase (cMDH)
and serves several functions involved in aerobic metabolism (see Goward and
Nicholls, 1994; Mathews et al., 2000 for reviews).
Aerobic metabolism refers to
the cellular energy production mechanism that oxidizes glucose through

glycolysis, the Krebs cycle and the electron transport chain, under the presence of
53
oxygen, which serves as the final electron acceptor in the electron transport chain
to synthesize adenosine triphosphate (ATP) as a temporary energy store (see
Mathews et al., 2000; Hochachka and Somero, 2002 for reviews).
LDH, on the other hand, is a terminal enzyme in anaerobic glycolysis, catalyzing

the conversion of pyruvate into lactate (see Mathews et al., 2000).
It is often
used to monitor the anaerobic capacity of species (e.g. Childress and Somero,
1979; Wu and Lam, 1997; Rinke and Lee, 2009; Ombres et al., 2011; see
Hochachka and Somero, 2002; Dahlhoff, 2004 for reviews), which is particularly
critical at high temperatures when oxygen transportation is limited due to a
reduction in heart function (see Prtner, 2001; Hochachka and Somero, 2002 for
review).
Both enzymes have been shown to demonstrate adaptive variation with
temperature in several marine invertebrates (abalone: Dahlhoff and Somero, 1993;

mussels: Fields et al., 2006; littorinids: Sokolova and Prtner, 2001; limpets:
Dong and Somero, 2009), which has been correlated with species biogeographic
and vertical distributions (e.g. Dahlhoff and Somero, 1993).
Most studies to determine thermal tolerance of intertidal species in relation to

their vertical distribution have, however, been conducted on the eastern Pacific
coast, therefore, more research needs to be conducted on the western Pacific and
especially the tropics to determine if there are any general patterns which emerge
with relation to species' responses to thermal stress (see Foster, 1990).
54
Thermal tolerance and vertical distribution of Echinolittorina species on Hong

Kong rocky shores
Littorinids are, among the intertidal gastropods, the most tolerant family to high
temperature in different intertidal habitats from temperate to tropical regions
(Evans, 1948; Newell, 1958, Sandison, 1967; Stirling, 1982; McMahon, 2001;
Davenport and Davenport, 2005).
Within this family, the high-shore
Echinolittorina species are the most thermally tolerant species on Hong Kong
shores (Stirling, 1982; McMahon, 2001).
Given the well-studied taxonomy and
ecology of this genus (Reid, 2007; Mak, 1996), Echinolittorina is an excellent

genus to study how their thermal tolerance, measured at different organization
levels, can be related to species' distribution patterns.
In Hong Kong, the three high-shore Echinolittorina species (E. malaccana, E.

radiata and E. vidua), as well as showing distinct vertical distribution patterns
(see Chapter 2), also exhibit strong seasonal variation in their vertical distribution
(see Mak, 1996; Chapter 2).
All three Echinolittorina species are found higher
on the shore during winter, and move down-shore in summer, with their average
tidal heights varying by ~ 0.5m between seasons (see Chapter 2; Mak, 1996).
Many studies have related this phenomenon to spawning behaviour of littorinids
(Hannaford Ellis, 1985; Ohgaki, 1988b), however, the cause and benefits of this
migration behaviour remain unclear.
Migrating down-shore may be a strategy
for these littorinids to avoid thermal stress at higher shore levels during the hot,
wet summer, where rock temperature can reach close to 58C (Williams et al.,
unpubl data) and exceed their optimal range, or even be close to their survival
55
limits (Marshall et al., 2011). Determining variation in the littorinids thermal

tolerance between season, may also allow an understanding of their potential to
acclimate due to the seasonal differences in environmental temperatures (see
Chapter 1).
This study, as well as investigating the thermal tolerance of the three

Echinolittorina species in relation to their vertical distribution, also aimed to test
if there was any seasonal variation in thermal tolerance of these littorinids, which
may be linked to their seasonal migration patterns, as well their potential to
acclimate to higher temperatures.
Thermal tolerances in the littorinids were
assessed through lethal temperature (LT50), Arrhenius breakpoint temperature of

heart rate (ABT of HR) and activities of malate dehydrogenase and lactate
dehydrogenase.
Using such an integrated approach at the organismal,
physiological and biochemical levels, was intended to provide more detailed

insights into the factors which drive survival limits in these species.
56
Table 3.1 Studies on thermal tolerance of intertidal species in relation to different factors, heating condition, and parameters used to determine
survival limits.
Aim of study
Species
(no. of species tested)
(Factor(s) in relation to thermal tolerance)
Heating
Thermal
Condition
Tolerance
Water
Air
Reference
HCT
LT50
11
Evans, 1948
Environmental temperatures and geographical distribution Molluscs and barnacles
15
Southward, 1958
Illustration of temperature adaptation
Cod, littorinid and hermit crab
Fraenkel, 1960
Geographical distributions
Dog whelks and littorinids
Sandison, 1967
Season, vertical and geographical distribution
Gastropods
22
Stirling, 1982
Vertical distributions
Gastropods
El Nino effect
Bivalves
10
Shore height and geographical distribution
Littorinids
Clarke et al, 2000b
Habitats types, vertical and geographical distribution
Gastropods
60
McMahon, 2001
Shore height, wave exposure, geographical distance
Gastropods, bivalves & barnacles 10
Vertical distributions
Littorinids
Vertical distribution
Molluscs
57
Cleland and McMahon, 1990

Urban, 1994
Davenport and Davenport, 2005
Lee and Lim, 2009
3.2. MATERIALS AND METHODS
To assure better quality of data and to minimize the possible confounding effects
that might contribute to significant differences between the assessments of
thermal tolerance, the effect on littorinid size, sample size, duration of short-term
acclimation, and time for recovery were tested through LT50 measurements using
E. malaccana (see Table 3.2) to aid development of standard protocols. All
experiments from the preliminary studies were conducted based on Stirling (1984)
with modifications of heating condition and rate of heating.
Potential
differences in LT50 with littorinid size, sample size, short-term acclimation and
recovery time were tested using Students t-tests.
Preliminary studies showed that significant differences were only detected

between different sized littorinids, but there was no difference in LT50 with sample
size, duration of short-term acclimation and recovery time, thus a standard
protocol was developed according to these results. Firstly, given the size range
of 7-8mm is more commonly used for studies on Echinolittorina species than the
smaller range of 6-7mm (e.g. Lee and Lim, 2009; Marshall et al., 2010), therefore,
7-8mm littorinids were collected for the experiments.
Secondly, to avoid
over-collection of littorinids that might affect populations at the collection site, a

sample size of 5 individuals per replicate (see below for details) was chosen.
Thirdly, to facilitate experimental work, littorinids were not acclimated before the
experiment.
Lastly, 12 hours recovery was chosen as opposed to 24 hours,

58
which was commonly used in previous studies (e.g. Fraenkel, 1968; Stirling,
1982), since there was no significant difference in LT50 assessments between the
two recovery times.
Apart from the factors tested, spatial variation in LT50 of the three Echinolittorina
species in Hong Kong was also tested to assure generality of the data collected for
sites in Hong Kong (Figure 3.1, Table 3.3).
To test for possible variation
between sites, a nested Analysis of Variance (ANOVA) was employed, with sites
(si) nested within species (Sp) [three levels: E. malaccana (Tai Miu Wan and
Stanley beach), E. radiata (Tai Miu Wan and Tai Tau Chau) and E. vidua (Stanley
prison and South Bay)].
The LT50 of the three Echinolittorina species showed
significant differences with species but no effect of sites (Figure 3.1, Table 3.3),
which leads to the assumption that there is little spatial variation in thermal
tolerance, determined using LT50, of Echinolittorina species on moderately
exposed shores of Hong Kong
59
Table 3.2 Mean LT50 (C S.D., n = 3) for preliminary studies on littorinid size,
sample size, acclimation and recovery time used for protocol standardization
using E. malaccana. Significant difference was detected by Students t-test and
indicated by asterisk(s): * (< 0.05).
Mean
S.D.
6-7mm
55.92
0.07
0.001*
7-8mm
57.22
0.31
10 individuals
56.68
0.23
5 individuals
56.99
0.02
0 day
56.35
0.50
7 days
56.91
0.16
Recovery Time 13 hrs
56.68
0.23
24 hrs
56.68
0.09
Factor
Size
Sample Size
Acclimation
60
0.072
0.072
0.482
58
LT50 (C)
57
56
55
54
53
52
0
E. malaccana
E. radiata
E. vidua
Species
Figure 3.1 Spatial variation of LT50 in Echinolittorina species Mean LT50 (C S.D., n = 6) of Echinolittorina malaccana (
= Tai Miu Wan;
= Stanley Beach), E. radiata (
= Tai Tau Chau;
= Shek Mei Tau) and E. vidua (
= Stanley Prison;
= South Bay) measured
in September, 2010.
61
Table 3.3 Nested ANOVA to compare variation of LT50 over sites (Si) nested
within Echinolittorina species (Sp): Echinolittorina malaccana (Tai Miu Wan;
Stanley Beach), E. radiata (Tai Tau Chau; Shek Mei Tau) and E. vidua (Stanley
Prison; South Bay). Variances were not homogenous (Cochran's test: C = 0.6047;
p < 0.01); therefore, p-value for significant differences was adjusted to 0.01.
Significant differences are indicated by asterisk(s): * (<0.01); ** (<0.001).
Source
df
SS
MS
Fvs
Sp
37.1931
18,5965
70.06
0.003*
Si (Sp)
Si (Sp)
Residual
3
20
0.7963
7.8324
0.2654
0.2611
1.02
0.399
Residual
62
Echinolittorina species were collected from South Bay and Stanley (see Chapter 1
for site description) on two randomly selected, replicate, days in each season,
during non-rainy, low water spring tide as applied for the field surveys.
Littorinids were collected from the height at which each species was most
abundant, i.e. their average mean height S.D. as determined from the field
survey (Chapter 2, E. malaccana: 2.76m 0.24; E. radiata: 2.46m 0.29; E.
vidua: 1.96m 0.23).
To minimize variation that might be related to size (Clarke
et al., 2000a; also see Table 3.1), individuals with shell length of 7-8mm
(measured from the apex to the base of the columella, see Figure 3.2 modified
from Mak, 1996), were collected for all three species.
All littorinids were
collected ~ 10m outside the population sampling sites, to ensure collection would
not impact the vertical distribution study.
At each site, ~ 60-100 individuals of
each species were taken dependant on availability of individuals of the required

size range. Animals were kept moist in unsealed plastic bags that were placed
inside an insulated box, and were immediately transported to the Swire Institute of
Marine Science (Cape dAguilar, Hong Kong) within 70 minutes of collection.
Littorinids were rehydrated in Petri dishes for two hours in natural seawater
collected from the respective site.
63
Shell length
Figure 3.2 Shell length of E. radiata (Mak, 1996) for measurement of littorinids.
The lethal temperature (LT50) of Echinolittorina species was determined for each
of the two days within summer and winter, using modifications of the method of
Stirling (1982).
Only active littorinids, as defined by having their foot attached
to the substratum when immersed, were selected and blotted dry.
Five
individuals were placed in labelled, cylindrical screw-top plastic vials (diameter

2.7cm and height 5.7cm) in air, with caps loosely fitted to allow air exchange.
Vials were randomly positioned in a grid and immersed in a Grant GP200
programmable water bath at 25C.
The temperatures of the water bath (internal
thermometer) and inside a test vial, in a position where the littorinids were located
(K-type thermocouples, C 0.05, Omega, recorded using digital thermometers,
Lutron TM-903A, Taiwan), were continuously monitored throughout the
experiment.
Temperature differences between the water bath and the vial were <
0.7C during the experiment.
Temperature was initially increased at a rate of 5C / 10 minutes until 50C, when

the heating rate was reduced to 1C / 10 minutes, and the duration of heating after
64
50C varied between species as preliminary trials showed E. vidua to be less

tolerant than E. malaccana and E. radiata ( Duration of exposure = (E.
malaccana & E. radiata) (5 x 10) + (8 x 10) = 130 minutes; (E. vidua) (5 x 10) +
(5 x 10) = 100 minutes).
When temperature inside the test vial reached 52, 53,
54 and 55C (for E. vidua) or 53, 54, 55, 56, 57 and 58C (for E. malaccana and
E. radiata), three randomly selected vials (= three replicates), were removed from
the water bath at each temperature interval, and cooled at ambient laboratory
temperature (in summer: ~ 23-26C; in winter: ~ 16-19C) for 5 minutes.
For
each vial, littorinids were then removed and gently placed into a labelled Petri
dish (8.8 cm diameter) with a film of natural seawater (~ 2-3mm in depth)
collected from the site at ambient temperatures for a 12-hour recovery period (see
Table 3.2).
After 12 hours, individuals that had their foot extended and attached
to the substratum, or responded to gentle pricking by a blunt forceps were

recorded as alive (Evans, 1948), while the remaining littorinids were assumed to
be dead.
LT50 of littorinids, which represents 50% mortality in the tested population, was
determined according to the following protocol. First, the number of dead snails
in each vial was converted to a proportion, i.e. percentage of mortality.
Then, to
obtain three LT50 values from each set of experiments, the percentage mortality of
individual vials at each temperature point were randomly assigned to obtain three
sets of equal temperature groups, each with one value for the temperatures: 52, 53,
54 and 55C (for E. vidua) or 53, 54, 55, 56, 57 and 58C (for E. malaccana and
E. radiata).
Finally, three LT50 values were calculated by fitting the proportion
65
of mortality data of each of the three temperature groups to a sigmoidal

log(dose)-response function (variable slope) using GraphPad Prism version 5.00
for Windows (GraphPad Software, San Diego California USA) for each of the two
days within each season.
Seasonal variation in Arrhenius breakpoint temperature of heart rate
Variation of heart rate in Echinolittorina species with temperature was monitored

according to Marshall and McQuaid (2011) with modification of the rate of
heating, while determination of Arrhenius breakpoint temperature of heart rate
(ABT of HR) was based on the method used by Stillman and Somero (1996).
Echinolittorina species were collected from Tai Tau Chau (see Chapter 1 for site
description) between February March 2011 and June August 2011 during low
tide (tidal height 1.2m above CD). Approximately 20 individuals of each
species were collected following the criteria described above and transported to
the Swire Institute of Marine Science under the same procedures for the LT50
experiment (see above), and rehydration of snails was carried out within a period
of 24 hours.
Littorinids were placed in a 30C oven (Binder FD115) for 30 minutes to stabilize
heart rate, in order to facilitate heart rate measurements (DJ Marshall; pers
comm.).
Preliminary studies showed no mortality after drying E. malaccana in
the oven for 4 days (Marshall and McQuaid, 2011).
Littorinids were then fitted
with infrared sensors (Vishay Semiconductors, CNY70), secured with Blu-tack
66
(Bostik), and placed in plastic vials (see LT50 experiment).
Signals were
amplified and filtered using a custom-made pre-amplifier (a modified version of

Chelazzi et al., 1999, and used by Marshall and McQuaid, 2011 in E. malaccana),
and data were digitally logged using a data logger (PowerLab 8/30, ADI
Instruments, Figure 3.3).
Littorinids were heated in a programmable water bath
(Grant GP200) at a rate of 0.5C/ minute (initial heating rate for LT50 experiment),
as a constant heating rate is commonly used when determining ABT of HR in
intertidal species (e.g. Stenseng et al., 2005; Marshall et al., 2010), between
30-55C after an initial 10 minutes at 30C (i.e. total duration = 60 minutes), and
heart rate was logged every minute and expressed as beats per minute (bpm).
Arrhenius breakpoint temperature (ABT) of heart rate, which is defined as the

temperature that shows a sharp discontinuity in the Arrhenius plot, indicates the
initial breakdown of heart function of the snail (Stillman and Somero, 1996).
Plots of heart rate versus temperature were constructed with heart rate (beat per
minute, bpm) versus 1000/temperature (Kelvin scale).
By fitting the two best-fit
regression lines before and after the distinctive change in heart rate, the Arrhenius
breakpoint temperature could be determined by back-calculating the intersection
point of the two regression lines and converting this point on the x axis to the
Celsius scale (see Stillman & Somero, 1996; Stenseng et al., 2005).
Echinolittorina species were collected from Tai Tau Chau on 15th July, 2011
during low tide (tidal height 1.2m above C.D.).
67
Approximately 120
individuals of each species were collected following the criteria described above
and transported to the Swire Institute of Marine Science and prepared for heating
under the same procedures carried out in the LT50 experiment (see above).
Temperature was increased at a rate of 5C/ 10 minutes from 30C to 55C (i.e.
total duration = 60 minutes).
When temperature reached 30, 35, 40, 45, 50 and
55C, three randomly selected vials, each containing five individuals, were
removed from the water bath at each temperature interval.
Littorinids were then
immediately placed in 2 ml eppendorf tubes and frozen in liquid nitrogen, and

were stored in 80C freezer (Sanyo, Japan) prior to enzyme measurements.
For sample preparation, foot muscle tissue, with the operculum removed, was
dissected from 5 individuals (approx. 0.2 g of tissue) in each vial and pooled.
The samples were pooled to minimize possible variations in enzyme activities
between individuals treated at the same temperature, which may be for example
caused by variation in food availability (Yang and Somero, 1993). The tissue
was then homogenised in 500 l homogenization buffer (50 mmol l-1 potassium
phosphate, pH 6.8 at 20C; 1 mmol l-1 DL-Dithiothreitol) on ice with a
rotor-stator
homogenizer
(IKA
Works,
Staufen,
Germany).
After
homogenisation, the homogenate was centrifuged at 12,500 rpm at 4C for 10

minutes (Eppendorf centrifuge 5810 R, Eppendorf-Netheler-Hinz, Germany), and
the supernatant was stored at 80C for determination of enzyme activities and
protein quantification.
68
Activities
of
malate
dehydrogenase
(MDH,
E.C.1.1.1.37)
and
lactate
dehydrogenase (LDH, E.C.1.1.1.27) were measured according to Yang & Somero

(1993) with modifications in concentration of chemicals and expression unit for
enzyme activity, i.e. (mol substrate converted to product min1) per gram protein.
To monitor enzyme activities, the homogenate was thawed on ice at room
temperature, and diluted by a factor of 25 (determined from preliminary trials)
using homogenisation buffer for MDH assay, while no dilution was made for
LDH. MDH was then assayed in 200mmol l-1 imidazole-HCl (pH 7.0 at 20C),
150 mol l-1 NADH and 0.2mmol l-1 oxaloacetate; while LDH was assayed in 200
mmol l-1 imidazole-HCl (pH 7.5 at 20C), 150mol l-1 NADH and 4mmol l-1
pyruvate. The activities of MDH and LDH were assayed at 30C 0.05 for all
samples in 96-well microplates (Serological plates SLP-000-09C, Jet Biofil), and
changes in absorbance at 340nm measured using a SpectraMax M2
spectrophotometer (Molecular Devices) equipped with a temperature-controlled
micro-plate reader.
The protein content of each sample was quantified using the
procedures adopted in the Bio-Rad Bradford Protein Assay (Bio-Rad , United

States). The homogenate was diluted by a factor of 25 times (determined from
preliminary trials) using homogenisation buffer, and was assayed in Protein Dye
Reagent Concentrate (Bio-Rad , United States) using the 96-well microplate at
30C
0.05.
The absorbance was measured at 595 nm using the
spectrophotometer as described, and protein content was obtained by comparing

the value to a standard curve, which was constructed using bovine serum albumin
standards at concentrations of 12.5, 25, 50, 100, 200 and 400ng/l. Enzyme
assays and protein quantifications were done in duplicate for all samples, and the
69
mean value was taken for statistical analyses.
Data analysis
To test for any possible variation in lethal temperature of Echinolittorina species

(n = 3 from each set of experiment x 2 sampling days = 6) between seasons, a
two-way Analysis of Variance (ANOVA) was employed using WinGMAv5 (EICC,
The University of Sydney), with species (Sp) [three levels: E. malaccana, E.
radiata and E. vidua] and seasons (Se) [two levels: summer & winter] treated as
fixed factors.
The two replicate days were pooled to increase statistical power,
as within season variation was not of interest in this comparison. Cochrans test
was used to check for homogeneity of variances (Cochran, 1951). Data were
transformed, if they were heterogeneous, however, where data failed tests for
homogeneity of variances after transformation, analyses were still conducted as
ANOVA is considered to be robust to such heterogeneity provided there is a large
error degrees of freedom (Underwood, 1997).
To be conservative in
interpretation of such ANOVA results p-values for significant differences

(originally p < 0.05) were adjusted to a lower value (p < 0.01, Underwood, 1981).
If any significant difference(s) was detected, post-hoc Student-Newman-Keuls
(SNK) tests were performed to identify the difference by multiple comparisons of
treatment means.
Potential difference(s) in Arrhenius breakpoint temperature of heart rate between

seasons and activities of malate dehydrogenase and lactate dehydrogenase among
70
the Echinolittorina species were analysed similarly using WinGMAv5 (EICC, The
University of Sydney).
For Arrhenius breakpoint temperature of heart rate (n =
14), a two-way orthogonal ANOVA was performed, with species (Sp) [three levels:
E. malaccana, E. radiata and E. vidua] and seasons (Se) [two levels: summer &
winter] as fixed factors; while for activities of malate dehydrogenase and lactate
dehydrogenase (n = 3), a two-way orthogonal ANOVA was performed, with
species (Sp) [three levels:
E. malaccana, E. radiata and E. vidua] and
temperature (Te) [temperature: 30, 35, 40, 45, 50 and 55C] as fixed factors.
Cochrans test was used to check for homogeneity of variances (Cochran, 1951)
and data were treated as above where variances were heterogeneous.
significant
difference(s)
for
fixed
factors
was
detected,
If any
post-hoc
Student-Newman-Keuls (SNK) tests were performed to identify differences by

multiple comparisons of treatment means.
71
3.3 RESULTS
In the analysis of lethal temperature (LT50) of Echinolittorina species, an

interaction was found between species and season (Figure 3.3, Tables 3.4 & 3.5).
E. malaccana, which occupies the highest location on the shore (Chapter 2), in
general, had the highest LT50, followed by E. radiata and E. vidua in both seasons
at South Bay (Table 3.4) and Stanley (Table 3.5).
Similar patterns of LT50 were
observed between summer and winter, however, the LT50 for each Echinolittorina
species was higher in summer than winter, and the response was greatest for E.
radiata, (1.15C, South Bay and 1.53C Stanley), followed by E. malaccana
(0.74C, South Bay and 1.14C Stanley), with E. vidua having the smallest
difference (0.57C South Bay and 0.5C Stanley).
72
58
South Bay
57
56
55
54
LT50 (C)
53
52
0
58
Stanley
57
56
55
54
53
52
0
E. vidua
Species
Figure 3.3 Temporal variation of LT50 in Echinolittorina species Mean LT50 (C
S.D., n = 6) of Echinolittorina malaccana, E. radiata and E. vidua measured
during winter (Dec, 2010 - Feb, 2011) (
at South Bay and Stanley.
) and summer (Jun - Jul, 2011) (
73
Table 3.4 Two-way ANOVA to compare variation of LT50 in Echinolittorina

species (Sp) between seasons (Se) at South Bay. Variances were not homogenous
(Cochran's test: C = 0.7387; p < 0.01); therefore, p-value for significant difference
was adjusted to 0.01 instead of 0.05. Significant differences are indicated by
asterisk(s): * (<0.01); ** (<0.001). Post-hoc Student-Newman-Keuls tests (mean
S.D.) were conducted for multiple comparisons of means.
Source
df
SS
MS
Sp
6.00
6.00
167.24
<0.001** Residual
Se
Sp x Se
Residual
1
2
30
49.14
0.54
1.08
24.57
0.27
0.04
684.55
7.49
<0.001** Residual
0.002*
Residual
SNK tests
Se (Sp)
Sp (Se)
E. malaccana
Summer
56.93 (0.07)
E. radiata
Fvs
>
Winter
56.19 (0.12)
Summer
56.06 (0.07)
E. vidua
Summer
54.01 (0.02)
Summer
E. malaccana
56.93 (0.07)
Winter
>
Winter
54.91 (0.03)
>
Winter
53.44 (0.28)
>
E. radiata
>
56.06 (0.07)
E. vidua
54.01 (0.02)
E. malaccana
56.19 (0.12)
>
E. radiata
>
54.91 (0.03)
E. vidua
53.44 (0.28)
74
Table 3.5 Two-way ANOVA to compare variation of LT50 in Echinolittorina

species (Sp) between seasons (Se) at Stanley. Variance were not homogenous
asterisk(s): * (<0.01); ** (<0.001). Post-hoc Student-Newman-Keuls tests (mean
S.D.) were conducted for multiple comparisons of means.
Source
df
SS
MS
Sp
10.14
10.14
70.42
<0.001** Residual
Se
Sp x Se
Residual
1
2
30
42.97
1.67
4.32
21.48
0.84
0.14
149.14
5.81
<0.001** Residual
0.007*
Residual
SNK tests
Se (Sp)
Sp (Se)
E. malaccana
Summer
56.95 (0.09)
E. radiata
Fvs
>
Winter
55.81 (0.45)
Summer
56.28 (0.33)
E. vidua
Summer
54.01 (0.02)
Summer
E. malaccana
56.95 (0.09)
Winter
>
Winter
54.75 (0.24)
>
Winter
53.51 (0.24)
>
E. radiata
>
56.28 (0.33)
E. vidua
54.01 (0.02)
E. malaccana
55.81 (0.45)
>
E. radiata
>
54.75 (0.24)
E. vidua
53.51 (0.24)
75
Seasonal variation in Arrhenius breakpoint temperature of heart rate
The Arrhenius breakpoint temperature (ABT) of heart rate (HR) showed an

interaction between Echinolittorina species and seasons (Figure 3.4, Table 3.6).
During summer, the mean ABT of HR for E. malaccana was the highest, followed
by E. radiata, and E. vidua.
In winter, however, the pattern of ABT differed
from summer, with E. malaccana having the highest mean ABT, followed by E.
vidua, and E. radiata. The ABT for E. radiata and E. vidua were generally
greater in summer than winter (Table 3.6), whereas the ABT for E. malaccana
was similar between summer and winter.
76
50
49
ABT (C)
48
47
46
45
44
43
42
0
E. vidua
Species
Figure 3.4 Temporal variation in Arrhenius breakpoint temperature (ABT) of
heart rate (HR) in Echinolittorina species Mean ABT of HR (C S.D., n = 14)
for Echinolittorina malaccana, E. radiata and E. vidua measured during winter
(March, 2011) (
Hong Kong.
) and summer (June - July, 2011) (
77
) at Tai Tau Chau,
Table 3.6 Two-way ANOVA to compare variation of ABT of HR in

Echinolittorina species (Sp) between seasons (Se) at Tai Tau Chau. Variances
were not homogenous (Cochran's test: C = 0.3433; p < 0.05); therefore, p-value
for significant difference was adjusted to 0.01 instead of 0.05. Significant
differences are indicated by asterisk(s): * (<0.01); ** (<0.001). Post-hoc
Student-Newman-Keuls tests (mean S.D.) were conducted for multiple
comparisons of means.
Source
df
SS
MS
Sp
Se
Sp x Se
Residual
2
1
2
78
54.43
108.42
41.51
172.85
27.22
108.42
20.76
2.22
12.28
48.92
9.37
<0.001** Residual
<0.001** Residual
<0.001** Residual
SNK tests
Se (Sp)
Sp (Se)
E. malaccana
Summer
48.76 (1.05)
E. radiata
Summer
48.58 (1.09)
E. vidua
Summer
47.36 (1.83)
Summer
E. malaccana
48.76 (1.05)
Winter
E. malaccana
47.71 (1.53)
Fvs
Winter
47.71 (1.53)
>
Winter
44.34 (2.13)
>
Winter
45.84 (0.86)
>
E. radiata
>
48.58 (1.09)
E. vidua
47.36 (1.83)
>
E. vidua
>
45.84 (2.13)
E. radiata
44.34 (0.86)
78
Activities of both malate dehydrogenase (MDH) and lactate dehydrogenase (LDH)

varied with temperature, however, only MDH was shown to vary between species
(Figures 3.5 & 3.6, Tables 3.7 & 3.8). MDH activity was highest for E. vidua,
followed by E. malaccana, and then E. radiata (Figure 3.5, Table 3.7).
While
LDH activity showed no difference among the species, activity of this enzyme
was generally higher for E. malaccana than E. vidua and E. radiata (Figure 3.6,
Table 3.8).
Both activities of MDH and LDH were highest in the three littorinid
species when temperatures reached 55oC (Figures 3.5 & 3.6, Tables 3.7 & 3.8).
79
MDH Activity
(x 103 I.U. per g protein)
80
60
40
20
0
30
35
40
45
50
55
Temperature (C)
Figure 3.5 Activities of malate dehydrogenase (MDH) for Echinolittorina species along a thermal gradient. Mean MDH activity (x 103 I.U. per
g protein S.D., n = 3, with each replicate pool with 5 individuals) at 30, 35, 40, 45, 50 and 55C for Echinolittorina malaccana, E. radiata and
E. vidua collected on 15th July, 2011 at Tai Tau Chau, Hong Kong. Bars filled in white (
in dark grey (
) = E. vidua.
80
) = E. malaccana, in grey (
) = E. radiata, and
Table 3.7 Two-way ANOVA to compare variation in malate dehydrogenase

(MDH) activities of Echinolittorina species (Sp) collected from Tai Tau Chau
sampled at different temperatures (Te). Variances were not homogenous
asterisk(s): * (<0.01); ** (<0.001). Post-hoc Student-Newman-Keuls tests
(meanS.D.) were conducted for multiple comparisons of means.
Source
df
SS
Sp
Te
Sp x Te
Residual
2
5
10
36
105600731.28 7800365.64 18.37

903956115.45 180791223.09 6.06
231327791.26 23132779.13 0.78
1073764820.57 29826800.57
SNK tests
Sp
Te
MS
<0.001** Residual
<0.001** Residual
0.651
Residual
E. vidua
60.2 x 103
(2989.66)
>
E. malaccana
54.1 x 103
(2947.55)
>
E. radiata
49.2 x 103
(2007.81)
30C
55.1 x 103
(3406.42)
45C
50.8 x 103
(3718.04)
35C
53.4 x 103
(2532.34)
50C
50.8 x 103
(2694.53)
40C
52.9 x 103
(4378.68)
55C
63.2 x 103
(6461.85)
81
Fvs
<
LDH Activity
(I.U. per g protein)
125
100
75
50
25
0
30
35
40
45
50
55
Temperature (C)
Figure 3.6 Activities of lactate dehydrogenase (LDH) for Echinolittorina species along a thermal gradient. Mean LDH activity (I.U. per g
protein S.D., n = 3, with each replicate pooled with 5 individuals) at 30, 35, 40, 45, 50 and 55C for Echinolittorina malaccana, E. radiata and
E. vidua collected on 15th July, 2011 at Tai Tau Chau, Hong Kong. Bars filled in white (
in dark grey (
) = E. vidua.
82
) = E. malaccana, in grey (
) = E. radiata, and
Table 3.8 Two-way ANOVA to compare variation in lactate dehydrogenase (LDH)

activities of Echinolittorina species (Sp) collected from Tai Tau Chau sampled at
different temperatures (Te). Variances was homogenous (Cochran's test: C =
0.2304; p > 0.05) after transformed to natural logarithm (Ln). Significant
differences are indicated by asterisk(s): * (<0.05); ** (<0.01). Post-hoc
Student-Newman-Keuls tests (mean S.D.) were conducted for multiple
comparisons of means.
Source
df
SS
MS
Fvs
Sp
Te
Sp x Te
Residual
2
5
10
36
0.069
0.226
0.065
0.414
0.035
0.451
0.007
0.01 2
3.00
3.92
0.57
0.062
0.006**
0. 828
Residual
Residual
Residual
SNK tests
Te
30C
4.38 (0.05)
45C
4.36 (0.03)
=
=
35C
4.36 (0.05)
50C
4.34 (0.07)
83
=
<
40C
4.35 (0.05)
55C
4.53 (0.09)
3.4 DISCUSSION
Littorinids inhabiting the high shore (reviewed by Lewis, 1972; see Chapter 1 for
definition) of intertidal habitats have been well documented to have a stronger
ability to tolerate extreme high temperatures than other gastropod taxa (Evans,
1948; Southward, 1958; Fraenkel, 1966; Stirling, 1982; McMahon and Britton,
1985; Cleland and McMahon, 1990; McMahon, 1990, 2001; Britton, 1992).
McMahon (2001), for example, categorized 60 species of gastropods from various
intertidal habitats, including rocky shores, salt marshes and mangroves, into two
groups, the littorinids and nonlittorinids, and showed that the mean HCT for
littorinids was significantly higher than the nonlittorinids by ~ 2.5C, and that this
group generally went into coma only when temperatures reached > 40C.
This is
also true for littorinids in Hong Kong (see Table 3.9), and in this study, lethal
temperatures determined for E. malaccana, E. radiata and E.vidua, in general,
exceeded 53C. This lethal temperature is slightly below the maximum rock
surface temperatures recorded at +2 m above CD (~ 57.6C) in summer at Cape
dAguilar, Hong Kong (Williams et al., unpubl i-button data).
Echinolittorina malaccana, E. radiata and E. vidua in Hong Kong have a strong

tolerance to high temperatures and showed a consistent pattern in their tolerance
which matched their vertical distributions at South Bay and Stanley (see Chapter 2;
Williams, 1994a; Mak, 1996).
E. malaccana, which occupies the highest level
84
of the high shore, has the highest lethal temperatures in both summer and winter,
followed by the lower-level E. radiata and E. vidua, respectively.
This pattern
matches with the HCT determined by Stirling (1982) and McMahon (2001, see
Table 3.9), who conducted similar thermal stress experiments, but in water, on E.
malaccana and E. radiata during summer in Hong Kong.
In terms of LT50,
Stirling (1984) showed no difference between E. malaccana and E. radiata during

summer in Hong Kong.
Stirling's results are similar to those obtained in the
present study, with the LT50 of E. malaccana being only ~ 0.5oC higher than that
of E. radiata (see Table 3.9). The LT50 for E. malaccana and E. vidua, however,
demonstrated a greater difference, with the relatively higher shore E. malaccana
LT50s being ~ 2oC greater than E. vidua in the present study, as was also recorded
by Lee and Lim in Singapore (2009, see Table 3.10).
Based on the survival
limits determined, therefore, thermal tolerance of Echinolittorina species in Hong

Kong generally increases with increasing shore levels.
Despite the close concordance of patterns of thermal tolerance of Echinolittorina

species with previous results, there is likely to be some degree of variation
introduced by the methodologies used, such as heating conditions and rates
(Terblanche et al., 2011; also see Table 3.10), which makes absolute comparison
with other established work difficult (Clarke et al., 2000a).
Many studies (see
Table 3.1), to minimize the effect of desiccation, have determined survival limits
while organisms were immersed in seawater.
There are only a few studies
(Sandison, 1976; Davenport and Davenport, 2005, 2007; present study) that have
measured limits whilst animals are emersed, which is the normal condition that
85
animals will experience thermal stress. During summer low tide periods, for
example, rock surface temperatures in Hong Kong rocky shores can exceed 50oC
(Williams and Morritt, 1995; Williams, unpubl data).
Intertidal animals living at
the high shore are more likely to experience such extreme thermal conditions, and
can spend more than 85% of their time emersed between successive spring tides
(McMahon, 2001; Uglow and Williams, 2001; Lee and Boulding, 2010).
Due to
variations in the environmental medium and thermal conditions, animals may,

therefore, exhibit different respiratory rates in air and water. Sandison (1967),
for example, showed a greater oxygen uptake in air than water in Littorina species
and Thais lapillus.
Based on this finding, Lee and Boulding (2010) pointed out that HCT determined
in water is positively correlated to that in air, therefore, implying that heating
condition is not critically important in determining species survival limits, and
allowing direct comparison with previous studies.
Lee and Boulding, however,
did not supply any evidence to support this view (2010).
Despite being
positively correlated, realistically, different heating conditions may still contribute

to variation in recorded LT50 values (Sandison, 1967; Davenport and Davenport,
2007).
The lethal temperature of Littorina littoralis, L. littorea and Thais
lapillus were, for example, found to be 1-3C higher in air than water, while no
difference was found in L. neritoides and L. saxatilis (Sandison, 1967). Similar
to Littorina littoralis, L. littorea and Thais lapillus, in the present study, the LT50
of E. malaccana was significantly lower in water than air and a similar, but not
significant, trend was also observed in E. radiata (Table 3.11). This implies that
86
when comparing the LT50 as a measure of thermal stress in Echinolittorina species,

heating conditions have to be noted, as these may confound potential differences
or similarities between species.
Apart from heating conditions, differences in heating rates may also result in
variation in measured species survival limits. Stirling (1982), for example, used
two different approaches (i.e. 6C/hr and constant C/hr) to determine the LT50 of
intertidal gastropods in Dar es Salaam, Tanzania, and showed that constant
heating at a fixed temperature led to lower LT50 values than an increasing rate.
Similarly the above results also apply to the limpets Patella vulgata, P. depressa
and P. athletica (Evans, 1948), where survival limits were found to be lower at a
slower heating rate. Conclusions made from the above studies also hold true for
E. radiata, where the LT50 was positively related to heating rate, although this was
not true in E. malaccana (see Table 3.11). When comparing survival limits in
Echinolittorina species, therefore, heating rate should also be considered as it may
potentially influence the results obtained (Terblanche et al., 2011).
In general, regardless of differences in methodology, such as heating rate, sizes of

littorinids, heating conditions (see Table 3.10) that make absolute comparison
with other research difficult (Clarke et al., 2000a), the pattern of survival limits to
high temperatures in the three Echinolittorina species remains consistent, with E.
malaccana having greater survival limits than E. radiata, and followed by E.
vidua, i.e., the LT50 of Echinolittorina species increases with higher shore heights.
The positive relationship between survival limits and vertical distribution of the
87
Echinolittorina species also agrees with most previous work on intertidal species,
either on non-congeneric (Broekhuysen,1940; Evans, 1948; Stirling, 1982;
Cleland and McMahon, 1990; Clarke et al., 2000b; McMahon, 2001; Davenport
and Davenport, 2005; Lee and Lim, 2009), or on congeneric species (in littorinids:
Markel, 1971; top shells: Tomanek and Somero, 1999; porcelain crabs: Stillman
and Somero, 2000; littorinids: Somero, 2002).
88
Table 3.9 Heat coma temperature of common gastropod species recorded from previous studies (Stirling, 1982; McMahon, 2001)
collected at different shore heights (Cleland and McMahon 1990), on Hong Kong rocky shores; and LT50 of E. malaccana and E.
radiata determined in present study.
Shore
Species
height
Echinolittorina malaccana
Echinolittorina radiata
Planaxis sulcatus
Monodonta labio
Morula musiva
Lunella coronate
Heat coma temperature (oC)
LT50 (oC)
Stirling, 1982
McMahon, 2001 Present study
48.5
46
50.5
43
44.5
43.5
46.3
44.8
40.8
37.4
39.6
38.8
89
56.95
56.17
-
Table 3.10 Variation in experimental approaches (collection site, heating rate, size, heating condition and thermal tolerance (HCT
and/or LT50) to determine lethal temperatures of E. malaccana, E. radiata and E. vidua from previous studies.
Species
Collection Site
Heating Rate
Size
Heating
Thermal tolerance
(mm)
Condition
HCT/LT (C S.D.)
Reference
50
Echinolittorina
Japan
Constant C/hr
Air
LT
50
48.5
Fraenkel, 1966
malaccana
Hong Kong
1C/10 mins
Water
HCT
48.5
Stirling, 1982
LT
56.5
50
Hong Kong
1C/5 mins
Water
HCT
46.5 0.75
Cleland & McMahon, 1986
Hong Kong
1C/5 mins
Water
HCT
46.31 0.71
McMahon, 2001
Singapore
Constant C/hr
7-11
Water
LT
50
50.4
Lee & Lim, 2009
Hong Kong
5C/10 mins;
7-8
Air
LT
50
56.95 0.11
Present study
Water
HCT
46.0
Stirling, 1982
LT
56.5
1C/10 mins
Echinolittorina
Hong Kong
1C/10 mins
radiata
50
Hong Kong
1C/5 mins
Water
HCT
44.8 1.46
Cleland & McMahon, 1986
Hong Kong
1C/5 mins
Water
HCT
44.8 1.46
McMahon, 2001
Hong Kong
5C/10 mins;
7-8
Air
LT
50
56.17 0.33
Present study
50
48.1
Lee & Lim, 2009
50
54.01 0.03
Present study
1C/10 mins
Echinolittorina
Singapore
Constant C/hr
7-11
Water
LT
vidua
Hong Kong
5C/10 mins;
7-8
Air
LT
1C/10 mins
90
Table 3.11 Lethal temperature (LT50) of (a) E. malaccana and (b) E. radiata
under different heating conditions and heating rates. Students t-tests were
used to compare variation in LT50 between heating conditions; while one-way
ANOVA was used to compare LT50 difference(s) in heating rates.
Significant differences are indicated by asterisk(s): * (< 0.05).
(a) E. malaccana
Factor
Condition
Rate
Mean S.D. n
Air
56.99 0.02 3
Water
55.99 0.06 3
5C /10 min
56.89 0.13 3
1C /5 min
56.95 0.02 3
5C /10 min (25-50C);

1C / 10 min (50-58C)
56.99 0.02 3
p
<0.001*
0.299
(b) E. radiata
Mean S.D. n
Air
56.59 0.49 3
0.077
Water
55.95 0.06 3
5C /10 min
56.98 0.04 3
1C /5 min
56.05 0.02 3
5C /10 min (25-50C);

1C / 10 min (50-58C)
56.59 0.49 3
Factor
Condition
Rate
91
0.02*
Arrhenius breakpoint temperature of heart rate in relation to vertical

distribution
In Echinolittorina species in Hong Kong, the pattern of ABT of HR is similar to

their LT50, and was positively related to species' vertical distributions.
The
relatively higher species, E. malaccana, in general, has the highest ABT of HR,
than the two relatively lower species, E. radiata and E. vidua.
This result
matched with studies on congeneric intertidal species (e.g. porcelain crabs, top
shells, limpets) that also showed that the ABT of HR for species was positively
correlated to their corresponding shore heights (Stillman and Somero, 1996;
Stenseng et al., 2005; Dong and Williams, 2011).
When comparing the ABT of
HR in congeneric top shells (Tegula = genus Chlorostoma), Stenseng and

colleagues (2005), for example, found that the ABT of HR in the relatively higher
shore Tegula funebralis was > 6oC higher than the lower shore Tegula brunnea
and Tegula monteryi.
In general, therefore, congeneric species inhabiting higher
shore heights tend to have higher ABT of HRs than lower shore species.
The ABT of HR refers to the temperature that heart rate of an individual drops
dramatically, and is used to indicate the initiation of loss of the ability for efficient
oxygen transportation of an individual (Stillman and Somero, 1996).
With a
higher ABT of HR, therefore, E. malaccana can maintain efficient oxygen

transportation at a higher temperature than E. radiata and E. vidua.
This
matches the higher LT50 determined for E. malaccana than the other two species.
A weak positive correlation between ABT of HR and LT50 for Echinolittorina
92
species (see Figure 3.7), however, matches with the trend shown in four
congeneric porcelain crabs Petrolisthes (summarized by Somero, 2010), which
suggests a general positive relationship between the two parameters.
Both ABT
of HR and LT50 of Echinolittorina species, therefore, play an important role in

determining the vertical distribution of the species (see Somero, 2010 for review).
Although there have been no comparative studies on ABT of HR of congeneric

littorinid species, the heart rate of E. malaccana has recently been studied
(Marshall and McQuaid, 2011; Marshall et al., 2010, 2011). After a slower
heating rate (i.e. 1oC per 5 minutes) the ABT of HR for E. malaccana in Brunei
Darussalam (see Marshall and McQuaid, 2011; Marshall et al., 2011) was 1-3oC
higher than recorded in the present study. Such variation in ABT of HR may be
due to latitudinal differences, as shown in previous studies on intertidal
gastropods (e.g. McMahon, 2001; Stillman, 2002; Kuo and Sanford, 2009) where
thermal tolerance of species increases with decreasing latitude. However, such a
conclusion can only be drawn when the experimental protocols used are similar,
in order to reduce the confounding effects of experimental factors, such as animal
sizes, heating conditions and rates (se LT50 section).
To conclude, ABT of HR in Echinolittorina species in Hong Kong, like many

other intertidal species from other parts of the world, was found to be positively
correlated to their vertical heights.
The positive correlation between ABT of HR
for Echinolittorina species and LT50 may, therefore, help explain that thermal
tolerance of the littorinids heart is linked to their survival limits under thermal
93
stresses.
Activities of malate dehydrogenase and lactate dehydrogenase in relation to
In the present study, the two isoforms of MDH were not isolated for the enzyme
assays, thus, the activity measured refers to the overall activities of the two
isoforms.
It has been reported that the overall activities of the isoforms are
positively related to oxygen consumption (in mussels, Mytilus californianus and

dogwhelks, Nucella ostrina Dahlhoff et al., 2002), and so this enzyme has been
used as an indicator for aerobic metabolism (e.g. Childress and Somero, 1979;
Yang and Somero, 1993; Thuesen and Childress, 1994; Dahlhoff et al., 2002;
Ombres et al., 2011).
The in vivo MDH and LDH activities of the Echinolittorina species, in general,
did not show any clear trend with increasing temperature, except for the relatively
higher activities detected when temperatures reached 55C.
This lack of
variation with temperature implies that the amount of active MDH and LDH in
the littorinids remained relatively constant as temperatures increased, even beyond
the ABT of HR, which is assumed to mark the start of the breakdown in aerobic
metabolism (Stillman and Somero, 1996; Stenseng et al., 2005; Marshall et al.,
2010, 2011; Dong and Williams, 2011).
Enzyme activity is generally expected to decrease with increasing temperature

once temperatures exceed the enzymes' optimal temperature, as enzymes are
94
thermally liable and denature at extreme temperatures (see Mathews et al., 2000;
Hochachka and Somero, 2002; Angilletta, 2009).
Sokolova and Prtner (2001),
determined the in vivo activities of two metabolic enzymes, citrate synthase (CS)
and NADP-dependent isocitrate dehydrogenase (NADP-IDH), in Littorina
saxatillis and L. obtusata, and found that activity levels decreased after heating
the littorinids from 20-25C to 45C at a constant rate of 1C per 5 minutes.
The
contrasting results obtained in this study, where there was little change in enzyme
activity with increasing temperatures, could possibly be explained by MDH and
LDH being thermally stable over the high temperatures experienced by
Echinolittorina species.
This hypothesis, however, would need to be confirmed
using in vitro methods (e.g. Sokolova and Prtner, 2003; Lee and Lim, 2009) by
measuring the activities of MDH and LDH along the temperature gradient using
cell extracts.
When heating the littorinids in vivo, individuals may respond by
continuously inducing production of MDH and LDH to maintain the activity

levels of these enzymes.
The in vitro method would, therefore, help to establish
whether the stable activity levels of the enzymes along the temperature gradient
was due to the stability of enzyme structure, and not a result of increased enzyme
production rates.
When comparing activity among species, it was found that E. vidua, which
inhabits the lowest shore level, had the highest MDH activity, and this implies this
species has the greatest aerobic capacity, followed by the two upper shore species,
E. malaccana and E. radiata respectively. The higher enzyme activity of MDH
in E. vidua than E. malaccana matches the pattern seen in a study on the activity
95
of glutamate oxaloacetate transaminase (GOT, which is responsible for ammonia

detoxification and nitrogen excretion), between the two species (Lee and Lim,
2009).
This finding does not, however, support the hypothesis that E. vidua has
a greater aerobic capacity than E. malaccana as GOT is not involved in energy

metabolism, but does indicate that E. vidua may have a higher metabolic rate than
E. malaccana. This appears to be true, as the basal heart rate for E. vidua, in
general, is slightly higher than that of E. malaccana (see Appendix 2.1 and 2.2).
Enzyme activity is commonly used as an indicator in determination of metabolic

rate (see Dahlhoff, 2004 for review), as it has been reported to be positively
correlated with metabolic rate in pelagic fishes (Childress and Somero, 1979;
Sullivan and Somero, 1983; Torres and Somero, 1988a,b; Yang and Somero,
1993).
If this is the case, the lower metabolic rate of E. malaccana may result
from their enhanced capability for metabolic depression (Marshall et al., 2011).
In some cases, E. malaccana is known to depress metabolism, and their heart rate
at 40C has been shown to be lower than at 30C during constant heating
(Marshall and McQuaid, 2011; Marshall et al., 2011). Possibly due to different
heating approaches, metabolic depression was not observed in the Echinolittorina
species in the present study (see Appendix 2) as well as the study by Matumba
and colleagues (unpubl data), who also worked on E. malaccana and E. vidua.
Yet, both studies showed that the heart rate of E. malaccana was relatively
independent of temperature, i.e. the HR did not show much increase as
temperature increased as compared with E. vidua.
This phenomenon seen in the
heart rate for E. malaccana may due to metabolic depression, which may also
96
apply to E. radiata which showed a lower level of MDH activity than E. vidua.
Such physiological mechanisms may, therefore, help E. malaccana to conserve
energy to protect themselves from thermal stress (e.g. to be able to produce heat
shock proteins, Marshall and McQuaid, 2011; Marshall et al., 2011).
The activity of LDH between the species was slightly different from MDH,
although these differences were not significant.
Activity was highest in E.
malaccana, while E. radiata and E. vidua showed similar LDH activities.
High
LDH activity means that, despite having the ability to undergo metabolic
depression, the high shore E. malaccana also has the greatest anaerobic capacity
when compared with E. radiata and E. vidua.
High shore species have, in
general, been proposed to have a greater capacity for anaerobic metabolism than
lower shore species (Simpfendrfer et al., 1995; Sokolova and Prtner, 2001;
Rinke and Lee, 2009).
Sokolova and Prtner (2001), for example, compared the
anaerobic capacity of two Littorina species, through the ratio of pyruvate kinase
(PK) and phosphoenolpyruvate carboxykinase (PEPCK). The higher shore L.
saxatilis had a lower PK/PEPCK activity ratio than the lower shore L. obtusata,
and Sokolova and Prtner suggested that L. saxatilis had a greater anaerobic
capacity than L. obtusata.
Similarly, in the present study, although using a
different anaerobic enzyme, the higher shore species tend to have greater
anaerobic capacity than the lower species.
Therefore, even when rock surface
temperature reaches close to their ABT of HR, when oxygen transportation is

limited, the higher shore species are still able to produce energy through anaerobic
metabolism, in order to maintain their basal needs for survival.
97
Seasonal impacts on thermal tolerance, including survival limits and heart rate, in
intertidal species, especially gastropods, have been previously studied (for heart
rate: Segal, 1956; survival limit: see Table 3.13).
These studies have shown that
thermal tolerance was higher when animals were collected at higher air
temperature environments during summer than winter.
Evans (1948), for
example, determined the seasonal HCT in the limpets Patella vulgata, P. depressa
and P. athletica, and found that their HCTs in summer were ~ 0.7-1.6oC higher
than those recorded in winter. Such observations, in general, are also true for the
LT50 and ABT of HR in the three Echinolittorina species in Hong Kong, where the
LT50 was ~ 0.5-1.5oC higher in summer, and ABR of HR was ~ 0.1-4.2oC higher
than in winter.
Seasonal migration patterns are a common phenomena observed in littorinids

which, apart from being linked with reproductive success (e.g Kojima, 1959;
Fretter and Graham, 1962; Hannaford Ellis, 1985; Ohgaki, 1988b; Mak, 1996),
have also been suggested to be related to environmental factors (e.g. Lambert and
Farley, 1968; Williams and Ellis, 1975; Harper and Williams, 2001), such as
temperature.
Lambert and Farley (1968) and Williams and Ellis (1975), for
example, suggested that the downward migration observed in Littorina littorea

during winter allowed it to escape from cold conditions high on the shore.
The
three Echinolittorina species in Hong Kong exhibit seasonal migration, moving

down shore in summer (Mak, 1996; see Chapter 2). Data on thermal tolerance
suggest that this behaviour may not be driven by exposure to high temperatures in
98
summer, as the littorinids are more tolerant to high temperature in summer than
winter. This behaviour may, however, be to enhance their reproductive success
and increase duration of feeding (Ohgaki, 1988a; Mak, 1996; Ito et al., 1998).
To enhance littorinids reproductive success, for example, seasonal migration of
Echinolittorina species in Hong Kong was found to coincide with their spawning
period (Mak, 1996).
As the low tides are lower in Hong Kong in summer than
winter (Kaehler and Williams, 1996), the seasonal migration may, therefore, help
facilitate the littorinids chance to release their eggs whilst immersed, as has been
observed for E. radiata in Shirahama, Japan, (Ohgaki, 1988a).
The cause of the seasonal variation in thermal tolerance of the Echinolittorina

species in Hong Kong was not investigated in the present study.
It may, however,
be linked to the littorinids ability to acquire acclimation-induced heat shock

responses, as shown in other intertidal species (see Feder and Hofmann, 1999;
Dahlhoff, 2004; Tomanek, 2010; Somero, 2010 for reviews), which help the
species to survive in extreme environmental conditions during the hot summer.
Heat shock response, as defined by Tomanek (2010), is the production of a group
of heat shock proteins (hsp) that help stabilize denatured proteins as well as to
refold denatured proteins to correct suboptimal conformations produced under
stressful environmental conditions, such as high temperatures.
Seasonal or
laboratory-based acclimation to higher temperature can coincide with an increase

in levels of hsps in many intertidal species (e.g., topshells, Chlorostoma: Tomanek
and Somero, 1999; Tomanek, 2002; 2008; mussels, Mytilus: Ioannou et al., 2009;
multiple species: Barua and Heckathron, 2004).
99
Both hsp 38 and 70, for
example, showed increases when top shells Tegula brunnea (=genus Chlorostoma)
and T. funebralis were acclimated separately at 13 and 18oC (Tomanek and
Somero, 1999), which implied that the top shells are more readily protected from
extreme high thermal conditions when acclimated at higher temperatures.
Physiological systems may, therefore, be protected by increased levels of hsps,
which convey a greater ability to survive thermal stresses. This can also be seen
by the fact that the ABT of HR in T. brunnea and T. funebralis were higher when
the snails were acclimated at higher temperatures (Stenseng et al., 2005), thus,
enhancing an individuals survival limits.
The higher ABT of HR recorded when
organisms were acclimated to higher temperatures was also demonstrated by

Stillman (2000) where the LT50 of porcelain crabs Petrolisthes cinctipes, P.
eriomerus, and P. manimaculis increased after they had been acclimated at a
higher
temperature.
Acclimation-induced
heat
shock
responses
of
Echinolittorina species were, although, not investigated in the present study,

investigated by Marshall and colleagues (2011) who found that concentration of
hsp 70 was doubled in E. malaccana on reaching high temperatures, i.e. 50oC
after an initial stage of metabolic depression.
This result implies E. malaccanas
ability to induce heat-shock response even under a short heating period, and may
reflect acclimation-induced heat shock responses which may explain their higher
thermal tolerance in summer than winter.
In summary, the LT50 and ABT of HR of Echinolittorina malaccana, E. radiata

and E. vidua in Hong Kong displayed a distinct pattern in relation to their vertical
distribution. Variations in the activities of MDH and LDH for the littorinids,
100
however, were less clear and require further in vitro experiments to confirm the
present results.
In addition, activities of more enzymes are suggested to be
examined to acquire a better understanding of the aerobic and anaerobic scope of

energy metabolism of the Echinolittorina species in relation to their distribution
patterns. Seasonal variation of LT50 and ABT of HR for Echinolittorina species
were also detected in the present study, which, in general, were higher in summer
than winter.
These variations are unlikely to contribute to the seasonal
down-shore migration pattern commonly observed in these littorinids, which is

probably related to other factors, such as spawning and food availability. The
cause of the variation may possibly be brought by the ability of the littorinids to
acquire acclimation-induced heat shock responses, which enhance the thermal
tolerance of physiological systems of individuals, and thus their survival, although
there is currently little evidence for this, and this is an area for further
investigation.
101
Table 3.13 Thermal tolerance of marine invertebrates in different seasons (winter and
summer). Thermal tolerances were determined by heat coma temperature (HCT) and
lethal temperature (LT) using various protocols (see Table 3.11).
Thermal
Mean (oC)
Tolerance
Winter
Summer
HCT
40.3
42.0
P. depressa
41.0
42.6
P. athletica
39.3
41.0
34-35
35-36
A. scutumn
34-35
35-36
A. persona
37-38
38-39
A. digitalis
38-39
39-40
A. scabra
40-41
41-42
LT
39.0
40-41
Fraenkel, 1960; 1968
HCT
32.6
37.6
Hamby, 1975
28.0
27.0
Clarke et al., 2000b
36.0
38.0
Cannon and Hughes, 1992
37.0
39.0
HCT
27.0
30.0
HCT
27.5
31.0
L. obusata
28.5
32.5
L. fabalis
29.0
31.0
56.0
56.9
E. radiata
54.3
56.2
E. vidua
53.5
54.0
50.0
50.5
51.5
52.5
20.4
24.9
Carcinus maenas
31.9
34.1
Hyas araneus
24.5
26.1
Liocarcinus depurator
27.7
28.0
Necora puber
26.4
29.5
Species
Limpets
Patella vulgata
Acmaea pelta
Littorinids Littorina littorea
L. neglecta
LT
LT
L. saxatilis
L. arcana
Echinolittorina. malaccana
Top shells Nerita plicata
LT
LT
N. textilis
Crabs
Cancer pagurus
LT
102
Reference
Evans, 1948
Wolcott, 1973
Present study
Stirling, 1982
Hopkin et al., 2006
52
Summer
50
E. malaccana
48
E. radiata
E. vidua
46
y = 0.4948 x + 20.67
r 2 = 0.9804
p = 0.0893
44
ABT of HR (oC)
42
52
Winter
50
E. malaccana
48
E. vidua
46
y = 0.6939 x + 7.959
r 2 = 0.2693
p = 0.6526
E. radiata
44
42
52
Overall
50
E. malaccana
48
E. radiata
46
E. vidua
44
y = 0.5171 x + 18.54
r 2 = 0.5268
p = 0.4829
42
52
54
56
58
LT50 (oC)
Figure. 3.7 Regressions between ABT of HR and LT50 for E. malaccana, E. radiata
and E. vidua (from top to bottom: summer, winter and overall) in Hong Kong. Each
dot () represents one Echinolittorina species (n = 3).
103
CHAPTER 4:
GENERAL DISCUSSION
4.1 DISCUSSION
Vertical and seasonal distribution of Echinolittorina species
in Hong Kong
Thermal tolerance of Echinolittorina species and their
seasonal variation in Hong Kong
Present study: implications for future investigations
Conclusions
105
Chapter 4: General discussion
4.1 DISCUSSION
Vertical and seasonal distribution of Echinolittorina species in Hong Kong
On two moderately exposed shores, i.e. Stanley and South Bay, in Hong Kong,
Echinolittorina malaccana, E. radiata and E. vidua, showed a consistent vertical
pattern and were located between 1.75 to 3.25m above C.D. (Chapter 2).
E.
malaccana was found highest on the high shore (mean height ~2.76m above C.D.),
and overlapped with E. radiata which dominated ~ 0.3m lower down the shore
(~2.46m above C.D.) than E. malaccana, and E. vidua which was found the
lowest (~1.96m above C.D.) among the three species, living at the edge between
high and mid shore.
Through field-manipulation experiments, Yipp and
Dudgeon (1990) showed that vertical distributions of E.malaccana and E. radiata

did not vary with the existence of the other species, therefore suggesting that
competition between the species may not be the major factor determining the
differences in their vertical distributions.
Predation is also unlikely to drive the
vertical distribution of Echinolittorina species on Hong Kong shores (Mak, 1996;

Stafford, 2002; Walters, 2002; Li, personal observation) as the density of their
potential predators, for example, the Pacific reef egret Egretta sacra, is not high
enough to affect their distribution.
With limited competition and predation
pressure observed in Hong Kong (Stafford, 2002), it can be assumed that their
vertical distribution is, therefore, related to their tolerance to physical stresses
(Stirling, 1982; Yipp et al., 1990).
Each of the three Echinolittorina species was also found lower on the shore by ~
105
0.25-0.5m in summer than winter.
The seasonal variation in the littorinids
distribution have been commonly proposed to enhance their reproductive success

by increasing their chance to spawn while immersed (e.g. Kojima, 1959;
Hannaford Ellis, 1985; Ohgaki, 1988a).
However, this phenomena, as observed
in other Echinolittorina species, has also been suggested to be related to a

seasonal shift in food availability (e.g. Ito et al., 2002), seasonal variation in wave
action (e.g. Ohgaki, 1989), as well as physical stresses (e.g. Lambert and Farley,
1968).
In terms of abundance, Echinolittorina species were, in general, less abundant in

summer than winter on both shores.
The lower abundance in summer was
possibly related to mortality due to thermal stress as suggested for other intertidal
species in Hong Kong (e.g. limpets: Firth and Williams, 2009; barnacles: Chan et
al., 2006) or dislodgement of littorinids during heavy rainfall (Ohgaki, 1988b;
Mak, 1996).
Loss of snails due to tropical storms, as described in previous
studies (e.g. Kohn, 1980; Boulding and Van Alstyne, 1993), with the littorinids
being washed away by strong wave action is, however, thought to be unlikely in
the present study as there were few storms in 2010.
Mortality may also be
increased in summer due to a decrease in food availability, as the biofilm is known

to be less abundant in summer months (Nagarkar and Williams, 1999).
decrease in food supply, in tandem with increased physical stress, may lead to
increased mortality rates and hence reduced numbers of littorinids.
In winter,
however, the increase in abundance of Echinolittorina species was associated with

the recruitment period of the littorinids (Mak, 1996), with the exception of E.
malaccana, which was less abundant in winter than summer in South Bay, a factor
106
which is difficult to explain.
Since these Echinolittorina species experience relatively low negative biological

interactions, such as predation and competition, on Hong Kong shores (Mak, 1996;
Walters, 2002; Stafford, 2002; Li, personal observation), it seems logical to
assume that physical factors, e.g. temperature and water availability, may play
important roles in determining spatial and temporal variation in their vertical
distribution and abundance.
The physiological tolerances of the littorinids to
temperature were, therefore, determined through laboratory based experiments

(Chapter 3) to try and establish possible correlations with their spatial and
temporal distribution in directing further investigations to understand larger-scale
spatial patterns as well as to predict species success under long-term, climate
change.
Thermal tolerance of Echinolittorina species and their seasonal variation in

Hong Kong
Thermal tolerance of Echinolittorina species was assessed using an integrated

approach at the organismic, physiological and biochemical level by lethal
temperature (LT50), Arrhenius breakpoint temperature of heart rate (ABT of HR)
and activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH).
The LT50 and ABT of HR of E. malaccana, E. radiata and E. vidua, as in other

congeneric intertidal species (e.g. in top shells: Tomanek and Somero, 1999;
porcelain crabs: Stillman and Somero, 2000), displayed a distinct pattern in
107
relation to their vertical distribution (Chapter 3).
In summer, for example, E.
malaccana which lived highest on the shore, had the greatest LT50 value (56.95oC,
see Table 3.10, Chapter 3) close to 0.8oC higher than the slightly lower E. radiata
(56.17oC), while the lowest shore E. vidua had the lowest LT50 (54.01oC). The
LT50 pattern of Echinolittorina species in Hong Kong also matched with previous
studies on similar species in Hong Kong (e.g. Stirling, 1982) and Singapore (Lee
and Lim, 2009), regardless of differences in methodology used to calculate the
value (see Table 3.11) that make absolute comparison difficult (Clarke et al.,
2000a). Similar to LT50 that showed a positive correlation with their vertical
distribution, the ABT of HR was also higher in E. malaccana (48.2oC) than the
two lower shore E. radiata and E. vidua (46.5 and 46.6oC respectively) (see Table
3.6, Chapter 3), which implied that E. malaccana can maintain efficient oxygen
transportation for aerobic metabolism under higher environmental temperatures
than E. radiata and E. vidua.
The weak, but positive relationship between ABT
of HR and LT50, (Figure 3.7, Chapter 3) suggest that thermal tolerance at the
physiological level may be linked to their survival limits under thermal stress, as
reviewed by Somero (2010) in other intertidal species (e.g. top shells Tegula (=
genus Chlorostoma) and porcelain crabs, Petrolisthes).
The activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH)

did not show a clear pattern in relation to the vertical distribution patterns of the
Echinolittorina species in Hong Kong, which was in contrast to the clear patterns
seen for LT50 and ABT of HR.
Activity of MDH in E. vidua, on the contrary,
was higher than the two upper shore species E. malaccana and E. radiata at all
sampling temperatures (Chapter 3). This result cannot confidently confirm that,
108
in general, E. vidua has the greatest aerobic capacity than the other two species,
however, the metabolic rate of E. vidua has been suggested to be higher than E.
malaccana as illustrated by the higher activity of glutamate oxaloacetate
transaminase (GOT, Lee and Lim, 2009) and differences in basal heart rates (see
Appendices 2.1 & 2.2) in E. vidua and E. malaccana.
The higher metabolic rate
in E. vidua, however, does not give a direct explanation to explain their thermal
tolerance limits.
In contrast to MDH, LDH activity was similar between the
three species littorinids, although E. malaccana had a slightly greater activity than
E. radiata and E. vidua (Chapter 3).
This implies the possibility that E.
malaccana has a higher anaerobic capacity than the other two species, therefore,
when environmental temperatures reach close to their ABT of HR, it would still
be able to produce energy through anaerobic metabolism to maintain basal needs,
which may attribute to their greater thermal tolerance limits.
However, as suggested (Chapter 3), in vitro methods should be conducted to

confirm the current results on activity of MDH and LDH, for example, activities
of CS and NADP-IDH in L. saxatilis and L. obtusata were measured by both in
vivo and in vitro methods by Sokolova and Prtner (2001), and both methods
showed decreases in activities of the enzymes.
Further, detailed examination on
enzyme function, for example, by determining enzyme kinetics and stability and
by measuring resistance to denaturing at high temperatures (e.g. Dahlhoff and
Somero, 1993; Fields et al., 2006; Dong and Somero, 2009) is recommended, as
such approaches may also help in understanding the causes of the vertical
distribution pattern of these species at the biochemical level.
As in these studies,
Dong and Somero (2009) for example, used congeneric limpets, Lottia species, to
109
show that enzyme resistance to denaturing of cMDH between species increased

with increasing shore levels.
Such differences could be attributed to the structure
of cMDH, as for L. digitalis (lower shore) and L. austrodigitalis (higher shore), as

an extra hydrogen bond exists in the cMDH of L. austrodigitalis, and therefore,
contributes to a more stable conformation for enzymatic reactions. Activities of
more metabolic enzymes that are involved in aerobic and anaerobic metabolism,
such as citrate synthase, pyruvate kinase, phosphoenolpyruvate carboxykinase,
NADP-dependent isocitrate dehydrogenase, should also be tested (e.g. Childress
and Somero, 1979; Sokolova and Prtner, 2001; Rinke and Lee, 2009; Ombres et
al., 2011), in order to acquire a more general picture of how their activities
contribute to an individuals overall response to thermal stress.
Thermal tolerance, in terms of LT50 and ABT of HR, was also found to vary
seasonally for the Echinolittorina species (Chapter 3), in which both LT50 and
ABT of HR for individual species were higher in summer than winter by ~
0.5-1.5oC and ~ 1-4oC respectively (see Chapter 3).
A higher thermal tolerance
in summer than winter has also been recorded in other intertidal species, such as
limpets, top shells and crabs (see Chapter 3, Table 3.13).
This seasonal variation
may not well be linked to the seasonal distribution patterns of the Echinolittorina
species (Chapter 2), however, the variation in thermal tolerance between seasons
showed that Echinolittorina species have the potential to acclimate to higher
environmental temperatures, which may lead to a higher survival limit to
increasing temperatures (as has been noted for other species, Stillman and Somero,
2002; Stenseng et al., 2005).
The cause of this seasonal variation, although not
investigated in the present study, like many other intertidal species (e.g. Tomanek
110
and Somero, 1999; Ioannou et al., 2009), may be due to the littorinids acquiring
acclimation-induced heat shock responses to increase the production of heat shock
proteins, which may enhance the thermal tolerance of the physiological systems of
individuals, and thus their survival.
There is currently little evidence for this,
and this, therefore, requires further investigation.
One exception is the work by
Marshall and colleagues (2011) who showed that at higher temperatures, after a
period of metabolic depression, E. malaccana metabolism increased to induce the
production of heat shock proteins, so these Echinolittorina species do possess the
ability to produce these heat shock responses.
Present study: implications for future investigations
The present study has demonstrated a positive relationship between the vertical
distributions of Echinolittorina species with their thermal tolerance on two
moderately exposed shores in Hong Kong. Such results may, therefore, be able
to direct future investigations on larger-scale spatial patterns (e.g. between local
shores, latitudes).
Latitudinal species distributions, for example, have been
hypothesized to give similar trends, in which, thermal tolerance of intertidal

species tends to increase with decreasing latitude.
Although such trends between
thermal tolerance and latitude have been shown in previous studies (e.g.
McMahon, 2001; Stillman, 2002), they are often, however, not linear with latitude
in some intertidal species (in littorinids: Clarke et al., 2001; Lee and Boulding,
2010; dog whelks: Sorte et al., 2011), with thermal tolerance of species sometimes
being found to be greater at a higher latitude.
Such unique places along the
latitudinal gradient were termed hot spots by Helmuth and colleagues (2002), as
111
they found that environmental temperatures experienced by the mussel, Mytilus

californianus, were unexpectedly higher in some higher latitude sites than at
places at lower latitudes due to local variation in the timing of low tides at these
hot spots.
Such observations, therefore, lead to the question of whether the
patterns of thermal responses seen in the Echinolittorina species from Hong Kong
shores can be applied over wider latitudinal scales.
Any abnormality observed in
the patterns may imply that hot spots exist, not only at a latitudinal scale, but
also at a local (within Hong Kong) spatial scale.
If such phenomenon could be
observed locally, it might help to understand the possible environmental

conditions which may cause these hot spots other than being due to variation in
the timing of low tides.
Based on the preliminary studies, for example, on
spatial variation of LT50, the littorinids showed little spatial variation between
different shores in Hong Kong (see Chapter 3, Figure 3.1). This may not hold
true over larger spatial gradients, if thermal tolerance is solely determined by
genetics, as E. malaccana is thought to have a number of clades distributed over
various latitudes, and there is some question as to whether E. vidua also varies
genetically along its latitudinal distribution range (see Reid, 2007 for review).
To give possible clues to answer this question, potential factors, for example,
genetic variation (Kuo and Sanford, 2009), response to wave exposure (Davenport
and Davenport, 2005), and local environmental factors such as the timing of low
tides (Helmuth et al., 2002), all of which may drive variations in thermal tolerance
of Echinolittorina species, should be considered and studied.
The fundamental goal of this study was to understand thermal tolerance of

Echinolittorina species in relation to their vertical and seasonal distribution. A
112
secondary purpose was to provide basic information on species distribution limits

and acclimation potential of these species.
Such information will allow
predictions of the littorinids response to changing environmental temperatures

such as under future climate change scenarios (see Somero, 2002, 2010 for
reviews).
In his reviews, Somero suggested that warm-adapted species with
narrow distribution ranges, with little genetic variation or capacity to acclimate

would be most vulnerable towards climate warming; while species with wide
distribution range and with both genetic variability and phenotypical plasticity
would be less vulnerable. Understanding which species will be winners or
losers, he suggested, would enable prediction of future changes in species
distribution patterns and hence community structure.
In terms of acclimation ability, from the present study, E. radiata had the greatest
ability (Chapter 3) and are, therefore, more likely to be able to cope with
environmental changes than E. malaccana and E. vidua.
With the little
information available, however, the present study alone cannot help conclude
which of the three Echinolittorina species may be winners or losers (sensu
Somero, 2010).
Further investigations and detailed examination of the littorinids
phylogeny related to their thermal tolerance, as well as their phenotypic potentials

over a longer time scale are therefore required to predict species success under
climate change scenarios.
113
Conclusions
The positive correlation between thermal tolerance of Echinolittorina species and

their vertical distribution on two moderately exposed shores in Hong Kong has
illustrated that temperature, as a physical factor, may play an important role in
determining the vertical distribution of these species.
Thermal tolerance of
Echinolittorina species was also found to vary seasonally in the present study,
suggesting their potential to acclimate to higher environmental temperatures.
These results provide a basic understanding on small-scale vertical and seasonal
patterns in thermal tolerance of Echinolittorina species which, linked to their
larger-scale spatial distribution patterns, may help predict which species will be
successful under long-term changes in the environment.
114
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APPENDIX 1
NAME USED FOR ECHINOLITTORINA MALACCANA, E. RADIATA AND E. VIDUA
IN PREVIOUS LITERATURE
Scientific name
Current
Echinolittorina malaccana
Reference
Past
Nodilittorina pyramidalis
Nodilittorina trochoides
Echinolittorina radiata
Nodilittorina exigua
Nodilittorina millegrana
Nodilittorina radiata
Echinolittorina vidua
Nodilittorina millegrana
Nodilittorina vidua
138
Fraenkel, 1966
Stirling, 1982
Ohgaki, 1985
Yipp and Dudgeon, 1990
Yipp et al., 1990
Cleland and McMahon, 1990
McMahon, 2001
Williams, 1994a
Mak, 1996
Walters, 2002
Ohgaki, 1985, 1988a,b, 1989
Yipp and Dudgeon, 1990
Yipp et al., 1990
Ohgaki, 1988, 1989
McMahon, 2001
Stirling, 1982
Williams, 1994a
Mak, 1996
Walters, 2002
Ohgaki, 1985
Williams, 1994a
Mak, 1996
Walters, 2002
APPENDIX 2.1
HEART RATE OF ECHINOLITTORINA SPECIES (n=14 per species, illustrated by
different symbols) VS TEMPERATURE (WINTER)
350
300
WINTER
E. malaccana
For all species

n=14
250
200
150
100
50
0
Heart rate (bpm)
350
300
E. radiata
250
200
150
100
50
0
350
300
E. vidua
250
200
150
100
50
0
20
30
40
50
Temperature (oC)
139
60
APPENDIX 2.2
HEART RATE OF ECHINOLITTORINA SPECIES (n=14 per species, illustrated by
different symbols) VS TEMPERATURE (SUMMER)
SUMMER
350
300
E. malaccana
For all species

n=14
250
200
150
100
50
0
Heart rate (bpm)
350
300
E. radiata
250
200
150
100
50
0
350
300
E. vidua
250
200
150
100
50
0
20
30
40
50
Temperature (oC)
140
60

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Title

Thermal tolerance of Echinolittorina species in Hong Kong:

Li, Hoi-ting, Kathy.; .

The author retains all proprietary rights, (such as patent rights)

THERMAL TOLERANCE OF ECHINOLITTORINA SPECIES

Hoi Ting Kathy, LI

A thesis submitted in partial fulfillment of the requirement for

Abstract of thesis entitled

THERMAL TOLERANCE OF ECHINOLITTORINA SPECIES

Submitted by Hoi Ting Kathy, LI

for the Degree of Master of Philosophy

Intertidal rocky shores represent an extremely stressful physical environment

One of the major environmental stresses

As a result, the thermal tolerance of intertidal species is often

vidua are excellent models to investigate the relationship between thermal

These littorinids are widely

Field surveys in summer and winter at two moderately exposed

Laboratory experiments, including determination of survival limits

The LT50 of E. malaccana were the highest of the three species

In terms of seasonal variation, LT50 and ABT of HR of all

The present study provided a fundamental understanding of how physiological,

Information on the tolerance limits of

further investigations to identify the underlying causes of the survival limits of

Such studies will

I would like to express my deepest gratitude to

CHAPTER 1 GENERAL INTRODUCTION

Intertidal rocky shores as model systems to investigate

CHAPTER 2 SEASONAL VARIATION IN VERTICAL DISTRIBUTION OF

CHAPTER 3 THERMAL TOLERANCE OF Echinolittorina SPECIES IN

Thermal tolerance and species distribution on intertidal rocky

MATERIALS AND METHODS

dehydrogenase in relation to vertical distribution

Seasonal variation of LT50 and ABT of HR

CHAPTER 4 GENERAL DISCUSSION

Name used for Echinolittorina malaccana, E.

Heart rate of Echinolittorina species vs

Heart rate of Echinolittorina species vs

Chapter 1: General introduction

Determination of the causes of species distribution patterns is a complex matter in

Given the complex nature of these interactions, ecologists are keen to

investigate whether individual factors can play a dominant role in determining

Intertidal rocky shores as model systems to investigate species distribution

alternating aquatic and terrestrial conditions which intertidal organisms have to

Chapter 1: General introduction

The unique situation

of severe changes in environmental conditions (e.g. temperature, water

Species are unable to tolerate conditions along

The upper limit of species'

distribution along the shore is generally regarded to be set by physical factors,

These interactions are also mediated by changing environmental

Chapter 1: General introduction

and Branch, 2001).

Ultimately, knowing how species respond to these

interactions in relation to their vertical distributions will allow a better

Temperature - a prominent factor controlling species distribution patterns

Environmental temperature is widely accepted as one of the most important

species experience can affect their distributions by altering the fitness of

temperatures vary at a variety of spatial and temporal scales, from local to

Understanding how temperature tolerance

Chapter 1: General introduction

On intertidal rocky shores, environmental temperatures fluctuate greatly due to

As well as showing changes

Such variations in environmental