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TitleThermal tolerance of Echinolittorina species in Hong Kong:implications for their vertical distributions

Advisor(s) Williams, GA

Author(s) Li, Hoi-ting, Kathy.; “§sý.

Citation

Issued Date 2012

URL http://hdl.handle.net/10722/173874

RightsThe author retains all proprietary rights, (such as patent rights)and the right to use in future works.

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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

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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 .

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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

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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.

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ii

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.

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iii

CONTENTS

Declaration ………………………………………………….…… i

Acknowledgements …………………………………………………….… ii

Contents ………………………………………………………. iii

CHAPTER 1 GENERAL INTRODUCTION ………………………..… 1

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 ………………………..… 20

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

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iv

CHAPTER 3 THERMAL TOLERANCE OF Echinolittorina SPECIES IN

HONG KONG ………………………..… 46

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 lethal temperature

Seasonal variation in Arrhenius breakpoint temperature of

heart rate

Activities of malate dehydrogenase and lactate dehydrogenase

DISCUSSION

Lethal temperature in relation to vertical distribution

Arrhenius breakpoint temperature of heart rate in relation to

vertical distribution

Activities of malate dehydrogenase and lactate

dehydrogenase in relation to vertical distribution

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v

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 Echinoli ttorina malaccana , E .

radiata and E . vidua in previous literature

APPENDIX 2 .1 ………………………..… 139

Heart rate of Echinolittorina species vs

temperature (Winter)

APPENDIX 2 .2 ………………………..… 140

Heart rate of Echinolittorina species vs

temperature (Summer)

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CHAPTER 1:

GENERAL INTRODUCTION

1.1

INTRODUCTION 2

I ntertidal r ocky shores as model systems to investigate species ’

distri bution patterns

Temperature - a prominent factor controlling species’ distri bution

patterns

L ittor inids as a model for studies on thermal stress

Aims of study and project outli ne

The Hong Kong cl imate

Site descriptions

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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).

I nter tidal r ocky shor es as model systems to investigate species ’ distri bution

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

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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

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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;

Pörtner, 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; Pörtner,

2002; Pörtner 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

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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; Obermülle 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).

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Littor ini ds as a model f or studies on thermal stress

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

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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

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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).

Aims of study and project outli ne

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.

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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.

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The Hong Kong cl imate

Hong Kong (22o20’ N and 114o10’E), 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

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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) .

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Site descri ptions

All field surveys and animal collections were conducted at South Bay (SB,

22°13’N, 114°11’E), Stanley (ST, 22°12’N, 114°13’E) and Tai Tau Chau (TTC,

22°13’N,114°15’E). 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.

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Table 1.1 Distribution of Echinolittorina species in the Indo-West Pacific Ocean

(after Reid, 2007)

Species Latitudinal distribution

(North - south range)

Latitudinal range

E. malaccana Zhejiang, China – Indonesia 30°15'N - 5°00'N

E. radiata Hokkaido, Japan – Nha Trang, Vietnam 43°17’N - 12°14’N

E. vidua Miura, Japan – Sydney, Australia 35°08’N - 33°52’S

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E . malaccana

E . radiata

E . vidua

Figure 1.1 The three common rocky shore Echinolittorina species in Hong Kong.

5 mm

5 mm

5 mm

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10

15

20

25

30

35

0

100

200

300

400

500

600

W S W

A i r t e m p e r a t u r e ( o C )

T o t a l r a i n f a

l l ( m m )

S

2010 2011

J A N

F E B

M A R

A P R

M A Y

J U N J U L A U G

S E P

O C T

N O V

D E C

J A N

F E B

M A R

A P R

M A Y

J U N J U L A U G

S E P

Month

0

Air temperature

Rainfall

Figure 1.2 Monthly variation in: (upper graph) air temperature ( = average

maximum; = average; = average minimum); (lower graph) total

monthly rainfall ( ) 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.

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16

Figure 1.3 Location of the three study sites: South Bay, Stanley and Tai Tau Chau in Hong Kong ( ).

NEW TERRITORIES

KOWLOON

HONG KONGISLAND

LANTAUISLAND

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17

Figure 1.4 a Photograph of study site at South Bay (SB) in summer.

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18

Figure 1.4 b Photograph of study site at Stanley (ST) in winter.

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19

Figure 1.4 c Photograph of study site at Tai Tau Chau (TTC) in summer.

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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

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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).

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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

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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;

Muñoz 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

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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.). From his surveys,

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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).

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2.2 MATERIALS AND METHODS

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:

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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

50000cm

2

[Total area= 6250cm

2

(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

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factors. Cochran’s 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.

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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. The

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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.

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0

25

50

75

100

125

150

175

200

225

250

275

E . malaccana

0

25

50

75

100

125

150

175

E . radiata

1. 5 0

1. 7 5

2. 0 0

2. 2 5

2. 5 0

2. 7 5

3. 0 0

3. 2 5

0

25

50

75

100

E . vidua

Height above C.D. (m)

M e a n

n o . o f i n d i v i d u

a l s

Figure 2.2 Vertical distribution of Echinolittorina species at Stanley. Mean

number of individuals of Echinolittorina malaccana, E . radiata and E . vidua,

sampled on three separate days in both winter ( , Dec, 2010-Feb, 2011) and

summer ( , Jun-Aug, 2011) (mean ± S.D., n = 3).

Stanley

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M e a n h e i g h t ( m )

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

E. malaccana E. radiata E. vidua

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Species

South Bay

Stanley

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 ( , Jun-Aug, 2011) (mean m

± S.D., n = 3).

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0

25

50

75

100

E . malaccananwinter = 869

nsummer = 1282

0

25

50

75

100

E . radiata

nwinter = 584

nsummer = 487

1. 5 0

1. 7 5

2. 0 0

2. 2 5

2. 5 0

2. 7 5

3. 0 0

3. 2 5

0

25

50

75

100

E . viduanwinter = 221

nsummer = 156


M e a n % o

f i n d i v i d u a l s

Figure 2.4 Relative vertical distribution of Echinolittorina species at South Bay.

Mean % of individuals of Echinolittorina malaccana, E . radiata and E . vidua,


summer ( , Jun-Aug, 2011) (mean % ± S.D., n = 3).

South

Bay

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0

25

50

75

100

E . malaccananwinter = 1668

nsummer = 1185

0

25

50

75

100

E . radiata

nwinter = 1127

nsummer = 385

1. 5 0

1. 7 5

2. 0 0

2. 2 5

2. 5 0

2. 7 5

3. 0 0

3. 2 5

0

25

50

75

100 E . vidua

nwinter = 171

nsummer = 50


M e a n % o

f i n d i v i d u a l s

Figure 2.5 Relative vertical distribution of Echinolittorina species at Stanley.

Mean % of individuals of Echinolittorina malaccana, E . radiata and E . vidua,


summer ( , Jun-Aug, 2011) (mean % ± S.D., n = 3).

Stanley

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0

50

100

150

200

250

300

350

400

450

500


0

100

200

300

400

500

600

700

T o t a l a b

u n d a n c e / 5 0 0 0 0 c m

2

South Bay

Stanley

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 ( , Jun-Aug, 2011) (mean

individual ± S.D., n = 3).

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37

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.

Source df SS MS F p Fvs Source df SS MS F p Fvs Sp 2 1.738 0.869 28.88 <0.001** Residual Sp 2 2.250 1.125 39.39 <0.001** Residual

Se 1 0.708 0.708 23.53 <0.001** Residual Se 1 0.289 0.289 10.11 0.008** Residual

Sp x Se 2 0.007 0.004 0.12 0.887 Residual Sp x Se 2 0.007 0.004 0.13 0.881 Residual

Residual 12 0.361 0.030 Residual 12 0.343 0.029

SNK tests SNK tests

Sp E . malaccana > E . radiata > E . vidua Sp E . malaccana > E . radiata > E . vidua

2.84 (0.19) 2.57 (0.21) 2.09 (0.17) 2.68 (0.14) 2.35 (0.19) 1.83 (0.10)

Se Winter > Summer Se Winter > Summer

2.70 (0.21) 2.30 (0.23) 2.42 (0.23) 2.16 (0.14)

StanleySouth Bay

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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 MS F p Fvs

Sp 2 266395.1 133197.6 38.60 <0.001** Residual

Se 1 3500.1 3500.1 1.01 0.334 Residual

Sp x Se 2 27200.4 13600.2 3.94 0.048* Residual

Residual 12 41403.3 3450.3

SNK tests

Se (Sp) E . malaccana

Winter < Summer

289.67 (91.45) 427.33 (69.25)

E . radiata

Winter = Summer

194.67 (85.34) 162.33 (9.28)

E . vidua Winter = Summer

73.67 (23.76) 52.00 (17.44)

Sp (Se) Summer

E . malaccana > E . radiata > E . vidua

427.33 (69.25) 162.33 (9.28) 52.00 (17.44)

Winter

E . malaccana = E . radiata > E . vidua

289.67 (91.45) 194.67 (85.34) 73.67 (23.76)

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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.


Sp 2 577354.8 288677.4 34.20 <0.001** Residual

Se 1 100650.9 100650.9 11.92 0.005** Residual

Sp x Se 2 32431.4 16215.7 1.92 0.189 Residual

Residual 12 101300.0 8441.7

SNK tests

Sp E . malaccana > E . radiata > E . vidua

475.50 (104.93) 252.00 (109.48) 36.83 (21.01)

Se Winter > Summer

329.56 (133.47) 180.00 (108.69)

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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

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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

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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).

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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 9 th 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

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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

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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.

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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 ofEchinolittorina

species on Hong Kong rocky shor es

3.2 MATERIALS AND METHODS 58

Preliminary studies as quali ty control of experimental protocol

Seasonal var iati on in lethal temperature

Seasonal var iation in Ar rhenius breakpoin t temperatur e of heart

rate

Acti vit ies of malate dehydrogenase and l actate dehydrogenase

Data analysis

3.3 RESULTS 72


Seasonal var iation in Ar rhenius breakpoin t temperatur e of heart

rate

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Acti vit ies of malate dehydrogenase and l actate dehydrogenase

3.4 DISCUSSION 84

Lethal temperature in r elation to verti cal distri bution

Arrhenius breakpoint temperature of heart rate in relation to

ver tical distri bution

Acti vit ies of malate dehydrogenase and lactate dehydrogenase in

relation to vertical distri bution

Seasonal variati on of LT 50 and ABT of HR

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Chapter 3: Thermal tolerance of Echinolittorina species in Hong Kong

3.1. INTRODUCTION

Thermal tolerance and species distributi on 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

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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 (Muñoz et al., 2005) or even

migrating downshore (Williams and Morritt, 1995; Harper and Williams, 2001) to

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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 2-6o

C 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).

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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

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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 Pörtner, 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

Pörtner, 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

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(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 Pörtner, 2001, 2003; Dahlhoff et al., 2002; Lee and

Lim, 2009; see Pörtner, 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

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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 Pörtner, 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 Pörtner, 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).

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Thermal tolerance and vertical distribution ofEchinolittorina species on Hong

Kong rocky shor es

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 58°C (Williams et al.,

unpubl data) and exceed their optimal range, or even be close to their survival

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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.

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57

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

(Factor(s) in relation to thermal tolerance)

Species (no. of species tested) Heating

Condition

Thermal

Tolerance

Reference

Water Air HCT LT50

Vertical distribution Molluscs 11 Evans, 1948

Environmental temperatures and geographical distribution Molluscs and barnacles 15 Southward, 1958

Illustration of temperature adaptation Cod, littorinid and hermit crab 3 Fraenkel, 1960

Geographical distributions Dog whelks and littorinids 5 Sandison, 1967

Season, vertical and geographical distribution Gastropods 22 Stirling, 1982

Vertical distributions Gastropods 9 Cleland and McMahon, 1990

El Nino effect Bivalves 10 Urban, 1994

Shore height and geographical distribution Littorinids 7 Clarke et al, 2000b

Habitats types, vertical and geographical distribution Gastropods 60 McMahon, 2001

Shore height, wave exposure, geographical distance Gastropods, bivalves & barnacles 10 Davenport and Davenport, 2005

Vertical distributions Littorinids 3 Lee and Lim, 2009

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3.2. MATERIALS AND METHODS

Preliminary studies as qual ity control of experimental protocol

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 Student’s 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,

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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

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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 Student’s t-test and

indicated by asterisk(s): * (< 0.05).

Factor Mean S.D. p

Size 6-7mm 55.92 0.07 0.001*

7-8mm 57.22 0.31

Sample Size 10 individuals 56.68 0.23 0.072

5 individuals 56.99 0.02

Acclimation 0 day 56.35 0.50 0.072

7 days 56.91 0.16

Recovery Time 13 hrs 56.68 0.23 0.482

24 hrs 56.68 0.09

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61


0

52

53

54

55

56

57

58

Species

L T 5 0 (

C )

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.

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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).


Sp 2 37.1931 18,5965 70.06 0.003* Si (Sp)

Si (Sp) 3 0.7963 0.2654 1.02 0.399 Residual

Residual 20 7.8324 0.2611

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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 d’Aguilar, Hong Kong) within 70 minutes of collection.

Littorinids were rehydrated in Petri dishes for two hours in natural seawater

collected from the respective site.

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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

Shell length

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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

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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 variati on 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®

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(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).

Acti vit ies of malate dehydrogenase and lactate dehydrogenase

Echinolittorina species were collected from Tai Tau Chau on 15th July, 2011

during low tide (tidal height ≦ 1.2m above C.D.). Approximately 120

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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 µl 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.

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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. (μmol substrate converted to product min−1) 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 µmol 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), 150µmol 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/µl. Enzyme

assays and protein quantifications were done in duplicate for all samples, and the

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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. Cochran’s 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

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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.

Cochran’s test was used to check for homogeneity of variances (Cochran, 1951)

and data were treated as above where variances were heterogeneous. If any

significant difference(s) for fixed factors was detected, post-hoc

Student-Newman-Keuls (SNK) tests were performed to identify differences by

multiple comparisons of treatment means.

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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, (1.15°C, South Bay and 1.53°C Stanley), followed by E . malaccana

(0.74°C, South Bay and 1.14°C Stanley), with E . vidua having the smallest

difference (0.57°C South Bay and 0.5°C Stanley).

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0

52

53

54

55

56

57

58South Bay

L T 5 0 ( C )

0

52

53

54

55

56

57

58Stanley

E . malaccana E . vidua E . radiata

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) ( ) and summer (Jun - Jul, 2011) ( )

at South Bay and Stanley.

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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 byasterisk(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 F P Fvs

Sp 2 6.00 6.00 167.24 <0.001** Residual

Se 1 49.14 24.57 684.55 <0.001** Residual

Sp x Se 2 0.54 0.27 7.49 0.002* Residual

Residual 30 1.08 0.04

SNK tests


Summer > Winter

56.93 (0.07) 56.19 (0.12)

E . radiata

Summer > Winter

56.06 (0.07) 54.91 (0.03)

E . vidua

Summer > Winter

54.01 (0.02) 53.44 (0.28)

Sp (Se) Summer


56.93 (0.07) 56.06 (0.07) 54.01 (0.02)

Winter


56.19 (0.12) 54.91 (0.03) 53.44 (0.28)

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Seasonal variati on 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.

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042

43

44

45

46

47

48

49

50

Species

A B T ( C )

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) ( ) and summer (June - July, 2011) ( ) at Tai Tau Chau,

Hong Kong.

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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. Significantdifferences 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 F P Fvs

Sp 2 54.43 27.22 12.28 <0.001** Residual

Se 1 108.42 108.42 48.92 <0.001** Residual

Sp x Se 2 41.51 20.76 9.37 <0.001** Residual

Residual 78 172.85 2.22

SNK tests


Summer = Winter

48.76 (1.05) 47.71 (1.53)

E . radiata

Summer > Winter

48.58 (1.09) 44.34 (2.13)

E . vidua

Summer > Winter

47.36 (1.83) 45.84 (0.86)

Sp (Se) Summer


48.76 (1.05) 48.58 (1.09) 47.36 (1.83)

Winter

E . malaccana > E . vidua > E . radiata 47.71 (1.53) 45.84 (2.13) 44.34 (0.86)

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Acti vit ies of malate dehydrogenase and lactate dehydrogenase

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).

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80

Figure 3.5 Activities of malate dehydrogenase (MDH) for Echinolittorina species along a thermal gradient. Mean MDH activity (x 10 3 I.U. per

g protein ± S.D., n = 3, with each replicate pool with 5 individuals) at 30, 35, 40, 45, 50 and 55°C for Echinolittorina malaccana, E . radiata and

E . vidua collected on 15th July, 2011 at Tai Tau Chau, Hong Kong. Bars filled in white ( ) = E . malaccana, in grey ( ) = E . radiata, and

in dark grey ( ) = E . vidua.

30 35 40 45 50 55

0

20

40

60

80

Temperature ( C)

M D H A c t i v i t y

( x 1 0 3

I . U . p e r

g p r o t e i n )

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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

(Cochran's test: C = 0.4445; p < 0.01); therefore, p-value for significant differencewas 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.


Sp 2 105600731.28 7800365.64 18.37 <0.001** Residual

Te 5 903956115.45 180791223.09 6.06 <0.001** Residual

Sp x Te 10 231327791.26 23132779.13 0.78 0.651 Residual

Residual 36 1073764820.57 29826800.57

SNK tests

Sp E . vidua > E . malaccana > E . radiata

60.2 x 103

(2989.66)

54.1 x 103

(2947.55)

9.2 x 103

(2007.81)

Te 30°C = 35°C = 0°C =

55.1 x 103

(3406.42)

53.4 x 103

(2532.34)

52.9 x 103

(4378.68)

45°C = 50°C < 55°C

50.8 x 103

(3718.04)

50.8 x 103

(2694.53)

63.2 x 103

(6461.85)

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82

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 55°C for Echinolittorina malaccana, E . radiata and

E . vidua collected on 15th July, 2011 at Tai Tau Chau, Hong Kong. Bars filled in white ( ) = E . malaccana, in grey ( ) = E . radiata, and

in dark grey ( ) = E . vidua.

30 35 40 45 50 55

0

25

50

75

100

125

Temperature (

C)

L D H A

c t

i v i t y

( I . U . p e r g p

r o t e i n )

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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). Significantdifferences 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.


Sp 2 0.069 0.035 3.00 0.062 Residual

Te 5 0.226 0.451 3.92 0.006** Residual

Sp x Te 10 0.065 0.007 0.57 0. 828 Residual

Residual 36 0.414 0.01 2

SNK tests

Te 30°C = 35°C = 0°C =

4.38 (0.05) 4.36 (0.05) .35 (0.05)

45°C = 50°C < 55°C

4.36 (0.03) 4.34 (0.07) .53 (0.09)

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3.4 DISCUSSION

Lethal temperature in relation to verti cal distri bution

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 ~ 2.5°C, and that this

group generally went into coma only when temperatures reached > 40°C. 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 53°C. This lethal temperature is slightly below the maximum rock

surface temperatures recorded at +2 m above CD (~ 57.6°C) in summer at Cape

d’Aguilar, 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

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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

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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 1-3˚C 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

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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. 6˚C/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

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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).

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89

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 LT 50 of E . malaccana and E .

radiata determined in present study.

Shore

height

Species Heat coma temperature (oC) LT50 (

oC)

Stirling, 1982 McMahon, 2001 Present study

Echinolittorina malaccana 48.5 46.3 56.95

Echinolittorina radiata 46 44.8 56.17

Planaxis sulcatus 50.5 40.8 -

Monodonta labio 43 37.4 -

Morula musiva 44.5 39.6 -

Lunella coronate 43.5 38.8 -

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90

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

(mm)

Heating

Condition

Thermal tolerance

HCT/LT50

(˚C± S.D.)

Reference

Echinolittorina

malaccana

Japan Constant ˚C/hr - Air LT50

48.5 Fraenkel, 1966

Hong Kong 1˚C/10 mins - Water HCT 48.5 Stirling, 1982LT

50 56.5

Hong Kong 1˚C/5 mins - Water HCT 46.5 ± 0.75 Cleland & McMahon, 1986

Hong Kong 1˚C/5 mins - Water HCT 46.31 ± 0.71 McMahon, 2001

Singapore Constant ˚C/hr 7-11 Water LT50

50.4 Lee & Lim, 2009

Hong Kong 5˚C/10 mins;

1˚C/10 mins

7-8 Air LT50

56.95 ± 0.11 Present study

Echinolittorina

radiata

Hong Kong 1˚C/10 mins - Water HCT 46.0 Stirling, 1982

LT50

56.5

Hong Kong 1˚C/5 mins - Water HCT 44.8 ± 1.46 Cleland & McMahon, 1986

Hong Kong 1˚C/5 mins - Water HCT 44.8 ± 1.46 McMahon, 2001


1˚C/10 mins

7-8 Air LT50


Echinolittorina

vidua

Singapore Constant ˚C/hr 7-11 Water LT50

48.1 Lee & Lim, 2009


1˚C/10 mins

7-8 Air LT50


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Table 3.11 Lethal temperature (LT50) of (a) E . malaccana and (b) E . radiata

under different heating conditions and heating rates. Student’s 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 Mean S.D. n p

Condition Air 56.99 0.02 3<0.001*

Water 55.99 0.06 3

Rate 5°C /10 min 56.89 0.13 3

0.2991°C /5 min 56.95 0.02 35°C /10 min (25-50°C);

1°C / 10 min (50-58°C)56.99 0.02 3

(b) E . radiata

Factor Mean S.D. n p

Condition Air 56.59 0.49 3 0.077

Water 55.95 0.06 3

Rate 5°C /10 min 56.98 0.04 3 0.02*

1°C /5 min 56.05 0.02 3

5°C /10 min (25-50°C);

1°C / 10 min (50-58°C)56.59 0.49 3

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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 > 6o

C 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

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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 littorinid’s heart is linked to their survival limits under thermal

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stresses.

Acti vit ies of malate dehydrogenase and lactate dehydrogenase in relation to

verti cal distri bution

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 55°C. 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

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thermally liable and denature at extreme temperatures (see Mathews et al., 2000;

Hochachka and Somero, 2002; Angilletta, 2009). Sokolova and Pörtner (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 20-25°C to 45°C at a constant rate of 1°C 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 Pörtner, 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

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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 40°C has been shown to be lower than at 30°C 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

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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 (Simpfendörfer et al., 1995; Sokolova and Pörtner, 2001;

Rinke and Lee, 2009). Sokolova and Pörtner (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 Pörtner 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.

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Seasonal variati on of LT 50 and ABT of HR

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 ~ 0.7-1.6 oC 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

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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). Both hsp 38 and 70, for

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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 individual’s 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 . malaccana’s

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,

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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.

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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).

Species Thermal

Tolerance

Mean (o

C) Reference

Winter Summer

Limpets Patella vulgata HCT 40.3 42.0 Evans, 1948

P. depressa 41.0 42.6

P. athletica 39.3 41.0

Acmaea pelta LT 34-35 35-36 Wolcott, 1973

A. scutumn 34-35 35-36

A. persona 37-38 38-39

A. digitalis 38-39 39-40

A. scabra 40-41 41-42

Littorinids Littorina littorea LT 39.0 40-41 Fraenkel, 1960; 1968

HCT 32.6 37.6 Hamby, 1975

28.0 27.0 Clarke et al., 2000b

L. neglecta LT 36.0 38.0 Cannon and Hughes, 1992

L. saxatilis 37.0 39.0

HCT 27.0 30.0 Clarke et al., 2000b

L. arcana HCT 27.5 31.0 Clarke et al., 2000b

L. obusata 28.5 32.5

L. fabalis 29.0 31.0

Echinolittorina. malaccana LT 56.0 56.9 Present study

E. radiata 54.3 56.2

E. vidua 53.5 54.0

Top shells Nerita plicata LT 50.0 50.5 Stirling, 1982

N. textilis 51.5 52.5

Crabs Cancer pagurus LT 20.4 24.9 Hopkin et al., 2006

Carcinus maenas 31.9 34.1

Hyas araneus 24.5 26.1

Liocarcinus depurator 27.7 28.0

Necora puber 26.4 29.5

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42

44

46

4850

52

E . vidua

E . radiata

E . malaccana

y = 0.4948 x + 20.67

r 2 = 0.9804

p = 0.0893

42

44

46

48

50

52

E . vidua

E . radiata

E . malaccana

y = 0.6939 x + 7.959

r 2 = 0.2693

p = 0.6526

42

44

46

48

50

52

E . vidua E . radiata

E . malaccana

y = 0.5171 x + 18.54

r 2 = 0.5268

p = 0.4829

54 56 5852

A B T o f H R ( o C

)

Summer

Winter

Overall

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).

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CHAPTER 4:

GENERAL DISCUSSION

4.1 DISCUSSION 105

Vertical and seasonal distribution of Echinolittorina species

in Hong Kong

Thermal tolerance of Echinolittorina species and their

seasonal variation in Hong Kong

Present study: impli cations for fu ture investigations

Conclusions

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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 ~

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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). A

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

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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

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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 0.8o

C 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,

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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 Pörtner (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

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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 Pörtner, 2001; Rinke and Lee, 2009; Ombres et

al., 2011), in order to acquire a more general picture of how their activities

contribute to an individual’s 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

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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 spot’s by Helmuth and colleagues (2002), as

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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 spot’s. 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 spot’s 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 spot’s 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

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secondary purpose was to provide basic information on species distribution limits

and acclimation potential of these species. Such information will allow

predictions of the littorinid’s 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 ‘winner s’ or ‘loser s’ ( 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.

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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.

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115

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138

APPENDIX 1

NAME USED FOR ECHI NOLITTORINA MALACCANA, E . RADIATA AND E . VIDUA

IN PREVIOUS LITERATURE

Scientific name Reference

Current Past

Echinolittorina malaccana Nodilittorina pyramidalis Fraenkel, 1966

Stirling, 1982

Ohgaki, 1985

Yipp and Dudgeon, 1990

Yipp et al., 1990

Cleland and McMahon, 1990

McMahon, 2001

Nodilittorina trochoides Williams, 1994a

Mak, 1996

Walters, 2002

Echinolittorina radiata Nodilittorina exigua Ohgaki, 1985, 1988a,b, 1989

Yipp and Dudgeon, 1990

Yipp et al., 1990

Ohgaki, 1988, 1989

McMahon, 2001

Nodilittorina millegrana Stirling, 1982

Nodilittorina radiata Williams, 1994a

Mak, 1996

Walters, 2002

Echinolittorina vidua Nodilittorina millegrana Ohgaki, 1985

Nodilittorina vidua Williams, 1994a

Mak, 1996

Walters, 2002

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APPENDIX 2.1

HEART RATE OF ECHINOLITTORINA SPECIES (n=14 per species, illustrated by

different symbols) VS TEMPERATURE (WINTER)

0

50

100

150

200

250

300

350

0

50

100

150

200

250

300350

0

50

100

150

200

250

300

350

H e

a r t r a t e ( b p m )

20 30 40 50 60

Temperature (oC)

E . malaccana

E . r adiata

E . vidua

WINTER

For all species

n=14

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APPENDIX 2.2

HEART RATE OF ECHINOLITTORINA SPECIES (n=14 per species, illustrated by

different symbols) VS TEMPERATURE (SUMMER)

0

50

100

150

200

250

300

350

150

200

250

300

350

a t e ( b p m )

E . malaccana

E . radiata

SUMMER

For all speciesn=14

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