TEMPERATURE TOLERANCE AND POTENTIAL IMPACTS OF...

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

TEMPERATURE TOLERANCE AND POTENTIAL IMPACTS OF

CLIMATE CHANGE ON MARINE AND ESTUARINE ORGANISMS

Diana Sofia Gusmão Coito Madeira

MESTRADO EM ECOLOGIA MARINHA 2011

UNIVERSIDADE DE LISBOA

FACULDADE DE CIÊNCIAS

DEPARTAMENTO DE BIOLOGIA ANIMAL

TEMPERATURE TOLERANCE AND POTENTIAL IMPACTS OF

CLIMATE CHANGE ON MARINE AND ESTUARINE ORGANISMS

Dissertação orientada pelos:

Professor Doutor Henrique Cabral

Doutora Catarina Vinagre

Diana Sofia Gusmão Coito Madeira

MESTRADO EM ECOLOGIA MARINHA 2011

i

Acknowledgements

I wish to express my gratitude to all people that guided, helped and contributed to this

work, in particular to:

Professor Doutor Henrique Cabral for kindly accepting me as his student and for believing

in me. Thank you for all your supervision, trust and support, kindness, friendship and

understanding. All of your knowledge and expertise was invaluable: it helped me grow as a

scientist and it made me take my professional skills one step further.

Doutora Catarina Vinagre for her unconditional guidance, supervision and enthusiasm.

Thank you for showing me the beauty of this work, for supporting me through the joyful and

harsh times, for giving me strength, for believing in my skills and for sharing your friendship

and companionship with me. This would not have been possible without everything you taught

me.

Doutor Mário Diniz for taking me into his guard at Faculdade de Ciências e Tecnologia da

Universidade Nova de Lisboa and for teaching me all about molecular techniques and

proteomics. His guidance, attention, friendship and support gave me the determination to

overcome hurdles along the way and keep the perseverance necessary to reach the finish line.

Thank you for all the opportunities you kindly offered me.

Professor Doutor Luís Narciso for his precious and joyful help during field and laboratory

work. Your experience and teaching helped me through this trial and your cheerful moods and

companionship surely made this journey much more fun.

All MSc, PhD and post-doc fellows at Faculdade de Ciências e Tecnologia, especially Joana

Lourenço, Marco Galésio, Ricardo Carreira, Gonçalo Vale, Pedro Costa and Íris Batalha, for

their warm welcome, sympathy and kindness. All your help unquestionably improved my work

and sharing ideas with you was enriching, both personally and professionally.

All team at Laboratório Marítimo da Guia, in particular Ana Pêgo and Doutor Rui Rosa for

all their understanding, encouragement and help in field work and aquaria maintenance.

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The crew of Albacora II and the team of Aquário Vasco da Gama, in particular Fátima Gil,

for their priceless help in the capture of the organisms and for providing us several species

when our field work was less successful. Without you this thesis would not have proceeded

according to plan.

Miguel Leal, who supported my work enthusiastically and gave me valuable advice

throughout this work, helping me out with tough decisions and doubts along the way. It was

really useful to have another friend and biologist’s eye on these matters.

To all my dearest friends, old ones from childhood and new ones from FCUL, who

encouraged me and showed me unreserved support. Your joyful company made me happy and

made me believe in myself when things did not go so well.

My family for everything they’ve done for me and everything they have taught me. To my

mother and father because they taught me to be hard working, to fight for what I want, to be

strong and patient. Your support, care and advice were really important. To my sister who

shares with me this passion for biology and gave me tones of advice, shared hard and

frustrating times with me and joked around to cheer me up when necessary.

At last, to Alexandre, who gave me his unconditional support, concern, care and love.

Thank you for your understanding, for giving me motivation, encouraging my work, cheering

me up and sharing your scientific expertise with me.

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Resumo

Este projecto tem como principais objectivos determinar as tolerâncias térmicas

superiores e os padrões de expressão das proteínas de choque térmico de várias espécies de

estuário e da costa Portuguesa com interesse comercial, com a finalidade de entender os

impactos da temperatura e das alterações climáticas sobre a fauna marinha. Este é um aspecto

particularmente importante pois sabe-se que a temperatura influencia processos bioquímicos,

fisiológicos, comportamentais e ecológicos, determinando assim parâmetros populacionais,

distribuição e abundância de espécies, estabilidade da teia trófica e potencialmente a futura

capacidade de exploração dos stocks de pesca. A variação espacio-temporal da temperatura

medeia os efeitos de todos os factores bióticos e abióticos e determina a diversidade de

adaptações dos organismos.

As principais questões relativas a alterações climáticas, e que têm sido alvo de grande

controvérsia, passam por entender como é que cada espécie reage às elevadas temperaturas,

como é que os aspectos ambientais e genéticos influenciam as respostas dos organismos e

quais os cenários faunísticos expectáveis tendo em conta a vulnerabilidade/resistência de cada

espécie. Como tal, o estudo da tolerância térmica é o primeiro passo para compreender esta

vulnerabilidade/resistência das espécies às alterações climáticas. O método escolhido para

estudar esta questão foi o Critical Thermal Maximum (CTM), em que os organismos são

expostos a um gradiente de temperatura com aumento de 1°C/h até atingirem o seu limite

térmico máximo. Este método permitiu ordenar as espécies em termos de vulnerabilidade a

temperaturas elevadas. Os resultados mostraram que espécies de diferentes taxa que vivem

em habitats semelhantes têm CTMs similares, uma vez que evoluíram em condições abióticas

semelhantes e potencialmente desenvolveram adaptações celulares e fisiológicas

semelhantes. O CTM é mais elevado para espécies típicas de ambientes quentes, instáveis e

muito variáveis e.g. intertidal/supratidal e para espécies migradoras, que têm de conseguir

atravessar inúmeras condições de temperatura ao longo dos seus movimentos para garantir o

sucesso reprodutivo. Relativamente às espécies de águas mais frias e com distribuição mais a

norte, o CTM foi mais baixo. Interespecificamente, o CTM foi mais variável em peixes do que

em caranguejos e camarões, possivelmente devido à grande capacidade locomotora dos

primeiros, que lhes permite colonizar inúmeros tipos de habitats. Os resultados permitiram

ainda concluir que a variabilidade intraespecífica é baixa e que, para espécies com uma larga

distribuição, não houve aclimatação ou adaptação local do limite térmico, o que pode indicar

pouca plasticidade nas respostas e pouca capacidade de adaptação a novas condições

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térmicas. De todas as espécies avaliadas, identificaram-se duas potencialmente vulneráveis às

alterações climáticas (Diplodus bellottii e D. vulgaris).

Um outro objectivo foi avaliar que espécies de peixes, temperadas/subtropicais ou

tropicais, é que vivem mais próximas do limite térmico, de forma a compreender quais serão

as mais vulneráveis ao aquecimento global. Concluiu-se que não existiam diferenças entre

espécies demersais mas que as espécies intertidais temperadas/subtropicais vivem mais

próximas do limite térmico uma vez que as temperaturas máximas do habitat podem

ultrapassar o seu CTM, enquanto que o CTM das espécies intertidais tropicais é 2-5°C mais

elevado do que a temperatura máxima do habitat.

Em resumo, nesta primeira parte do trabalho determinaram-se os CTMs de 16 espécies

com distribuição temperada/subtropical duma variedade de taxa (peixes, caranguejos e

camarões) e avaliaram-se diferenças inter e intraespecíficas. Foi a primeira vez que se fez uma

abordagem deste género para espécies marinhas com esta distribuição, visto que a maior

parte dos estudos tem sido focado em espécies tropicais. Assim, o presente trabalho fornece

resultados facilmente comparáveis com outros estudos, possibilitando uma avaliação da

vulnerabilidade das espécies de diferentes latitudes.

Na segunda parte do trabalho, a investigação foi direccionada para os mecanismos

celulares de defesa contra o stress térmico, com especial foco nas proteínas de choque

térmico (HSPs). Tendo em conta que a temperatura afecta os processos bioquímicos e provoca

stress proteotóxico através da desnaturação proteica e formação de agregados citotóxicos,

estas proteínas (chaperonas) são a componente de defesa que assegura a estabilização de

polipéptidos desnaturados e proteínas nascentes. Como tal, o objectivo foi determinar os

padrões de expressão da HSP de peso molecular 70 kDa, em várias espécies marinhas de

diferentes taxa, ao longo de um gradiente de temperatura e no limite térmico máximo (CTM).

Os métodos de análise proteica utilizados foram o ELISA (Enzyme Linked Immunosorbent

Assay), Western Blot e 1D SDS-PAGE (one-dimension sodium dodecyl sulfate polyacrylamide

gel electrophoresis). Foram identificadas quatro tendências nos perfis de resposta dos

organismos: aumento na produção de HSP70 à medida que a temperatura aumenta, seguido

de um decréscimo próximo dos limites térmicos (Liza ramada, Diplodus sargus, Pachygrapsus

marmoratus, Liocarcinus marmoreus); manutenção dos níveis de HSP70 ao longo de todo o

gradiente de temperatura (Diplodus vulgaris, Dicentrarchus labrax, Palaemon longirostris,

Palaemon elegans, Carcinus maenas); aumentos e decréscimos na produção de HSP70 ao

longo do gradiente de temperatura ( Gobius niger); aumento na produção de HSP70 ao longo

de todo o gradiente de temperatura (Crangon crangon). No geral, os padrões identificados são

independentes do taxon, CTM e tipo de habitat. No entanto, os resultados apontam para uma

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relação entre a magnitude da expressão, as condições térmicas do habitat, o CTM e os limiares

de indução, uma vez que na maioria dos casos os organismos que habitam locais muito

quentes apresentaram maior quantidade de HSP70, limiares de indução mais elevados e maior

CTM. Relativamente a espécies de água mais fria, verificou-se que ou a expressão de HSP70

tem uma estreita amplitude no gradiente de temperatura ou que não existe sequer uma

produção induzida destas proteínas, indicando que são espécies potencialmente vulneráveis

ao aquecimento dos oceanos. Ainda assim, a magnitude da expressão e o tipo de padrão

apresentado estão muito relacionados com características específicas. Espécies congenéricas

foram comparadas de forma a testar as influências genéticas/filogenéticas e ambientais na

produção de HSPs. Os resultados mostraram que no género Diplodus parece existir uma

influência ambiental enquanto que no género Palaemon tudo aponta para uma influência

genética. Isto indica que poderá haver espécies com respostas mais plásticas e outras com

respostas geneticamente determinadas pelo que nestas questões é muito importante

considerar não só as condições ambientais mas também os múltiplos factores inerentes à

espécie, de forma a compreender as estratégias usadas para lidar com o stress. Verificou-se

que existe não só uma variabilidade interespecífica no tipo de resposta mas também uma

elevada variabilidade intraespecífica na quantidade de HSP70 produzida.

Concluindo, este projecto mostra que as espécies mais vulneráveis às temperaturas

elevadas e ao aquecimento glocal são espécies de águas frias e ambientes estáveis, espécies

sobre exploradas e espécies intertidais, que vivem próximo dos seus limites térmicos. Há que

ter também em conta outros factores nesta vulnerabilidade, tais como a idade da primeira

maturação, estratégia de reprodução (semelparidade ou iteroparidade) e capacidade de

adaptação dos organismos, que podem determinar se a população tem a capacidade de se

manter ou não. O estudo dos mecanismos de resistência à temperatura integra conhecimentos

de diversas áreas, pelo foi necessária uma abordagem multidisciplinar para desvendar

processos bioquímicos e celulares e avaliar os padrões dentro de um gradiente

ecologicamente relevante. Assim, este estudo contribui com informação importante para o

conhecimento de processos ecofisiológicos e pode ser relevante para a gestão dos recursos

marinhos, o que é um ponto essencial, especialmente para países com uma economia ligada

ao mar, como é o caso de Portugal.

Palavras-chave: alterações climáticas, stress térmico, Critical Thermal Maximum, defesa

celular, proteínas de choque térmico, organismos marinhos, variabilidade inter/intra-

específica.

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vii

Abstract

This project aimed to determine the thermal tolerances and uncover the Heat Shock

Protein 70 patterns of expression in several marine and estuarine species of commercial

interest. Once temperature affects biochemical, physiological, behavioral and ecological

processes, the purpose of this study was to understand the impacts of temperature and

climate changes on marine communities.

Firstly, through the method of Critical Thermal Maximum (CTM), the species were ranked

in terms of their vulnerability. Results showed that species from different taxa inhabiting in

similar thermal conditions have CTM values alike. CTMs are higher for warm/unstable

environment and migratory species. Local adaptation was not verified for wide distributed

species. Two potentially vulnerable species were identified (Diplodus bellottii and Diplodus

vulgaris). Also, results showed that intertidal temperate/subtropical fish are more vulnerable

than tropical intertidal fish because they live closer to their CTM. Also, maximum habitat

temperatures can surpass their thermal limits. On the other hand this was not observed for

tropical intertidal fish. For demersal species no differences were found.

Secondly, cellular mechanisms of defense against stress were analyzed, in particular

HSP70 production along a temperature gradient and at CTM. Protein analysis was performed

through ELISA, Western Blot and SDS-PAGE. Four trends, indepently of taxa, CTM and habitat

type, were identified in the response profiles. Results also point towards a correlation between

HSP70 amounts, thermal conditions, CTM and thresholds of induction. Cold water species

either lack inducible HSP70 or have a narrow range for its induction, potentially making them

vulnerable to sea warming. Some congeneric species showed an HSP production influenced by

environment while others showed a response influenced by genetic features.

Concluding, this work shows that cold/stable water species, over-exploited and intertidal

species might be more vulnerable to climate warming. Some species present more plastic

responses while others are more genetically determined so environmental and phylogenetic

influences may account for the type of response. To address questions on this research area

one must focus on a multidisciplinary approach in order to link biochemical mechanisms to

ecological patterns within relevant gradients. This investigation contributes to the knowledge

of marine ecophysiological processes which is important to countries with a sea-based

economy, like Portugal.

Keywords: Climate change, thermal stress, Critical Thermal Maximum, cellular defense,

Heat Shock Proteins, marine organisms, inter and intraspecific variability

Table of contents

Acknowledgements ................................................................................................................ i

Resumo ................................................................................................................................. iii

Abstract .................................................................................................................................vi

CHAPTER 1 ............................................................................................................... ix

General Introduction ..................................................................................................... 1

1. Coastal and estuarine ecosystems, environmental stress and climate change .................. 1

2. Temperature tolerance and heat stress studies ................................................................. 4

3. Molecular mechanisms behind thermal tolerance and adaptation .................................... 5

3.1. Heat shock proteins and resistance to stress ............................................................................. 5

3.2. Physiological and ecological relevance of HSPs ........................................................................ 12

4. Aims and scopes of the dissertation ................................................................................. 12

References ................................................................................................................... 13

CHAPTER 2 .............................................................................................................. 21

Thermal tolerance and potential impacts of climate change on coastal and estuarine

organisms (submitted to Marine Biology) ................................................................. 23

Abstract ....................................................................................................................... 23

Introduction ................................................................................................................. 23

Materials and methods ............................................................................................... 26

Study area and sampling method ......................................................................................... 26

Thermal tolerance method ................................................................................................... 26

Data analysis ......................................................................................................................... 27

Results ......................................................................................................................... 28

Discussion .................................................................................................................... 32

References ................................................................................................................... 39

CHAPTER 3 .............................................................................................................. 45

Heat Shock Protein 70 patterns of coastal and estuarine organisms facing increasing

temperatures ........................................................................................................... 47

Abstract ....................................................................................................................... 47

Introduction ................................................................................................................. 47

Materials and methods ............................................................................................... 51

Species collection and acclimation conditions...................................................................... 51

Thermal tolerance method ................................................................................................... 52

HSP70 extraction and quantification .................................................................................... 53

SDS-PAGE .............................................................................................................................. 54

Western Blot ......................................................................................................................... 54

Statistical analysis ................................................................................................................. 55

Results ......................................................................................................................... 55

Discussion .................................................................................................................... 62

References ................................................................................................................... 69

CHAPTER 4 .............................................................................................................. 75

Final considerations ................................................................................................ 77

CHAPTER 1

CHAPTER 1 – General introduction

1

General Introduction

1. Coastal and estuarine ecosystems, environmental stress and climate change

Coastal and estuarine communities are extremely important both socially and

economically. They are amongst the most productive ecosystems, serving as nursery areas,

depuration systems and providing crucial food stocks. Therefore, research projects on the

molecular mechanisms, physiology and ecology of inhabiting organisms are extremely relevant

as they provide information that can be used to protect and manage these ecosystems.

Coastal and estuarine waters are far more variable than ocean waters, which imply that

organisms must go through some degree of environmental stress. Environmental variables

such as temperature, salinity, tides, oxygen levels, etc can vary both seasonally in the

temperate latitudes as well as daily, especially in estuaries and intertidal areas. It is known that

temperature is one of the main factors affecting marine communities since it is one of the

variables defining the structure of the water column, the dynamics of the aquatic systems and

the planktonic production (Badillo et al. 2002). Considering marine ectotherms, the effects of

temperature can be very pronounced. For example, at the molecular level, temperature

affects the biochemical reactions leading to physiological and behavioral changes (Mora and

Ospina 2001) which have consequences on individual fitness and performance. At a higher

level of organization, temperature affects recruitment success, distribution, abundance,

migrations, and biological interactions. As such, the knowledge on how species react to their

changing environment allows us not only to understand adaptations (Lutterschmidt and

Hutchinson 1997) but also functional and genetic constraints (Huey and Kingsolver 1989). It

allows the prediction, to some extent, of population changes that might occur when

communities face disturbance. These changes might involve for instance stock redistribution,

invasion of exotic species (Bennett et al. 1997, Kimball et al. 2004) and resilience of native or

threatened species (Walsh et al. 1998). With this in mind, the molecular and physiological basis

for the observed ecological patterns might be associated with the temperature tolerance and

its location on the temperature scale (Pörtner 2002).

In the face of climate change, these issues are particularly important. In order to protect

ecosystems we need to understand the causal-effect relationship between climatic changes

and ecosystem changes. This can be a very hard task since there are multiple factors

introducing noise and masking patterns, such as overfishing, diversity and complexity of

habitats, trophic relations and biological interactions (IPCC 1997, Roessig et al. 2004). Besides,


2

we must keep in mind that in order to address the impacts of climate forcing on marine

organisms and make realistic predictions for the future, it is essential to know the species

current vulnerability status. This vulnerability is dependent not only on the thermal limits but

also on factors like fishing pressure (once it alters the genetic structure of the population and

leads to a fragmentation in the food web - Perry et al. 2010), duplication time, capacity to

adapt, regional rate of temperature increase and predicted changes in food availability due to

climate forcing. Nevertheless, the study of thermal limits and heat tolerance is essential to

uncover mechanisms of resistance or sensibility to heat (Pörtner and Knust 2007). Moreover,

Mora and Maya (2006) refer that knowing the levels of thermal tolerance allows us to

understand how deep the climatic events will affect mortality rates, recruitment, distribution

and abundance.

Several studies already show and predict the effects of ongoing environmental changes.

At the physiological level, the oxygen availability can limit the aerobic metabolism and thus the

thermal tolerance (Frederich and Pörtner 2000, Pörtner et al. 2004, Pörtner and Knust 2007,

Melzner et al. 2007, Rosa and Seibel 2008). Sea warming may therefore decrease oxygen

availability and increase the metabolic rates, leading to a decline in aerobic performance with

consequences on the species abundance and distribution. If these parameters are altered by

the thermal regime, then the survival, reproduction, recruitment and structure (Mora and

Ospina 2001) of the populations will be affected because temperature has direct effects not

only on time and frequency of spawning and survival of eggs, larvae and juveniles (IPCC 2001)

but also on temperature-dependent sex determination (Ospina-Alvaréz and Piferrer 2008). At

the behavioral level, there could be changes in reproductive strategies (Angilletta et al. 2006)

and life history patterns. This is justified by the fact that temperature affects the differential

allocation of energy to several activities (growth, reproduction, food intake, metabolism, and

excretion), the size and number of eggs and the size at which fish reach sexual maturation.

There is special concern on benthic and native organisms because they cannot escape

thermal conditions by moving to other areas (Mora and Ospina 2001). Native species are

highly adapted to the habitat and environmental conditions in which they occur, living close to

their tolerance limits and having limited potential for migration. However, some small bodied

species with short generation time might be able to adapt to new conditions (Munday et al.

2009). Species with long generation times and late maturation are in disadvantage and have

low adaptation potential. Additionally, changes at the phenological and planktonic events and

on the North Atlantic Oscillation (Rosenzweig et al. 2007) are also of special interest since


3

several impacts are expected: a trophic mismatch, changes in advection rates and changes on

larval transport and retention. These impacts clearly affect trophic relations, patterns of

migration and larval production of commercial species (IPCC 1997, 2002, 2007, Pörtner et al.

2008). There is one other factor of concern related to commercial species which is the

predicted loss of 20% of the wetlands until 2080 (IPCC 2002) with direct impacts on fish

production (Costa et al. 1994, Cabral et al. 2001, Costa et al. 2007) and thus economy and

society.

More specifically, and besides the impacts already mentioned, predictions for Portugal

refer a +2°C increase, at the most, in water temperature until the year of 2100 (Miranda et al.

2002; scenario A2 from the Special Report on Emission Scenarios, Nakicenovic et al. 2000,

coupled with the regional climatic model HadMR3, Reis et al. 2006). Considering this scenario,

Vinagre et al. (in press), predict an increase in species richness with a gain in tropical and

subtropical species, and a decrease in temperate ones, essentially demersal. An exception

seems to be the south coast of Portugal which, according to the authors, will have more losses

than gains if a 2°C increase occurs. Nevertheless it is expected a shift from a subtropical-

temperate fish assemblage towards a more subtropical-tropical fish assemblage (Vinagre et al.,

in press). The gain in more subtropical species is already taking place, according to the work of

Cabral et al. (2001). This shows how species biogeographical ranges might shift northward and

ultimately influence competition interactions because if the arriving species are more tolerant,

they will outdo the native ones.

Gathering all this information, and considering that Portugal has an economy highly

dependent on marine resources, there is a need to develop research projects concerning the

marine environment and its response to global changes. Taking into account that there is a

lack of knowledge on the adaptation capacity of marine organisms to environmental change,

the results obtained from these research projects should be an imperative point on stock

management, as suggested by Munday et al. (2009). This way, fishing might be more

sustainable and impacts of climate change can be potentially mitigated. Moreover, the

response mechanisms that accompany the change on environmental variables are still poorly

understood at several levels of organization: individual, population, community and interaction

between abiotic-biotic factors (Munday et al. 2009). On this perspective, knowing the species

thermal limits and the molecular mechanisms responsible for the thermal tolerance are critical

steps on the stress and climate change research.


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2. Temperature tolerance and heat stress studies

There is a considerable amount of literature on temperature tolerance, however most of

it is focused on fish species from the orders Cyprinifomes and Perciformes and crustacean

species from the class Malacostraca (Lutterschmidt and Hutchinson 1997). According to the

same authors, the less studied fish groups include Pleuronectiformes, Osmeriformes,

Atheriniformes, Beloniformes, Anguilliformes and Scorpaeniformes. Furthermore, most of the

tolerance studies have been performed in species from the tropical regions (reef, estuarine

and intertidal fish species) and fresh water species (Becker and Genoway 1979, Lutterschmidt

and Hutchinson 1997, Rajaguru and Ramachandran 2001, Mora and Ospina 2001, 2002,

Rajaguru 2002, Badillo et al. 2002, Eme and Bennett 2009). Compiling these studies, a pattern

is revealed: most of the tropical species have thermal limits close or higher than 40°C and can

survive in temperate waters. As such, due to the gap of knowledge related to temperate and

subtropical species, it is relevant to explore how these groups react and face induced change.

By doing so, it will be possible to define patterns for temperate species and compare them

with the tropical ones.

The most used method to study temperature tolerance of fish has been the Critical

Thermal Maximum (Bennett and Judd 1992, Lutterschmidt and Hutchinson 1997, Bennett et al.

1997, Mora and Ospina 2001, 2002) whereas on crustaceans it has been the Lethal

Temperature Method. Differences in the measured parameters and experimental conditions

have made it difficult to compare results between different species and higher taxonomic

groups. In this view, the method was standardized for all the examined species in this project,

allowing a comparable approach between several marine taxa since.

When conducting tolerance trials it is necessary to consider that the tolerance to one

single factor is higher than when several factors are simultaneously applied. Therefore, the

response of the organisms might be slightly different when comparing laboratory conditions to

natural conditions. Nonetheless, it is important to know what the response to each factor

isolated is, so that future studies can combine several factors and acknowledge the matching

impacts occurring in the natural environment. It is known that the thermal regime occurring in

the habitat and the thermal conditions of microhabitats can lead to the selection of certain

physiological set-points, so correlations between habitat use and physiological parameters

(e.g. metabolism and thermal tolerance) can be established (Dent and Lutterschmidt 2003).

The thermal properties of water masses can lead to an adaptive window of thermal tolerances


5

and considering that ectotherms can, to a certain extent, behaviorally select thermal

conditions that help them get the greatest metabolic advantage, it is expected that habitat

conditions are somehow related to the thermal physiology of each species (Dent and

Lutterschmidt 2003).

Notwithstanding, there is some controversy about the capacity of the organisms to adapt

genetically and physiologically (Angilletta 2009). It is hard to predict whether the evolution of

thermal tolerance and adaptation can keep the fitness before the selective and esthocastic

processes lead some populations to reduced numbers or extinction. Even so, tolerance studies

provide insight into how organisms relate to their environment and cope with extreme

conditions, and if they are potentially vulnerable species or not, considering a climatic scenario

of water-temperature increase.

3. Molecular mechanisms behind thermal tolerance and adaptation

3.1. Heat shock proteins and resistance to stress

Throughout their lifetime, organisms are exposed to several stress factors. Environmental

conditions tend to be stressful when they reach values outside the tolerance limits of the

organisms, causing a decrease in fitness. Fitness is dependent on the capacity for adaptation,

which in turn can be related to the maintenance and integrity of the protein pool through the

expression of several proteins, including Heat Shock Proteins (HSPs). Shortly, organisms might

adjust their genetic expression to acquire physiological plasticity in response to physical and

chemical factors (Hofmann and Todgham 2010). Nonetheless, stressful conditions induce

consequences at the cellular, physiological and individual levels. They can lead to great

changes in the metabolic processes, disturbing vital functions and thus the survival, growth,

reproduction, biological interactions and ultimately the community and ecosystem’s structure.

In regard to this, organisms must have several mechanisms that allow them to cope and

survive stressful events; otherwise population numbers may suffer a strong downfall.

In general, the stress response occurs at 3 levels:

1. Primary response – perception of an altered state and activation of the

neuroendocrine/endocrine response, characterized by a rapid production of stress hormones

(Iwama 1999).

2. Secondary response – includes the several physiological and biochemical adjustments and

it is regulated by stress hormones (adrenaline and cortisol) which activate metabolic pathways


6

that lead to biochemical and hematological alterations (Barton and Iwama 1991), changes in

the hydromineral balance and cardiovascular, respiratory and immune functions (Barton

2002). During stressful conditions, organisms mobilize their energy stores in order to provide

energy for tissues, to deal with an increased need of energy. The increase in glucose

production covers this need, through gluconeogenesis and glycogenolysis, which are triggered

by adrenaline and cortisol (Iwama 1999). In summary, this response results in metabolite and

ion changes and in the induction of HSPs (Barton 2002). Nevertheless, it is important to

consider that the nutritional state of the organism (Vijayan and Moon 1992, 1994) and its age

(Hall et al. 2000, Snoeckx et al. 2001, Kregel 2002) can highly affect responses to stress. This

verifies because the quantity of available energy can compromise cellular defense and because

HSP expression tends to decline in aged individuals.

3. Tertiary response – changes occurring at the organismal and population levels, directly

linked to the alterations that occurred due to the primary and secondary responses. If the

organism can not acclimate, adapt or maintain homeostasis, several changes may occur: at the

behavioral level, resistance to disease, growth and reproduction capacity (Iwama 1999, Barton

2002). Regarding this, a severe or prolonged exposure to stress can eventually alter population

demographics and dynamics. Impacts can be critical when it comes to larvae and juveniles

because growth is of crucial importance to their fitness at these stages. If the growth is fast,

there are two advantages: there is a lower chance of being predated because a bigger size

reduces the range of predators, and the first maturation will occur faster leading to a higher

investment in reproduction (in iteroparous species). As such, if growth and reproduction

become energetically compromised due to stressful conditions, it is reasonable to expect

lower recruitment and production, altering the abundance and diversity of species in a

community (Barton 1997).

This project, focused on one of the secondary responses: induction of HSPs as a

mechanism of cellular response to stress, especially HSP70. The Heat Shock Proteins,

discovered in 1962 by Ritossa after heat shock treatment in Drosophila, are considered an

“ancient, primary system for intra-cellular defense” (Csermely and Yahara 2003). They have

been conserved through evolution in almost every living organism, from bacteria to human

(Kregel 2002). These proteins act as chaperones, stabilizing both denatured and nascent

proteins, stopping them from forming cytotoxic aggregations (Welch 1992, Hartl and Martin

1992, Becker and Craig 1994, Moseley 1997, Fink 1999). Their most relevant functions are to

mediate the folding process although they are not part of the new formed structures (Ellis and

Van der Vies 1991), and perform the translocation of other proteins between cellular


7

compartments also helping in the refolding process (Beckmann et al. 1990, Ellis 1990, Hartl

1996, Fink 1999). There is considerable knowledge about their cellular location, regulation and

function (e.g. Lindquist and Craig 1988, Hightower 1991, Welch 1992, Morimoto et al. 1994,

Benjamin and McMillan 1998) however the environmental factors and thus the physiological

and biochemical signals that induce their synthesis might be a field to keep exploring. It is of

great interest to know the mechanisms by which organisms maintain their homeostasis (Kregel

2002). Briefly, it is known that heat stress leads not only to ROS (Reactive Oxygen Species)

production and cellular damage in several components (mitochondria, Golgi complex,

cytoskeleton, DNA and proteins – Dubois et al. 1991, Vidair et al. 1996, Snoeckx et al. 2001)

but also to a slow-down or shut-down of most original cellular functions (Csermely and Yahara

2003) so the chaperoning function of HSPs is a mechanism of defense in order to maintain

cellular homeostasis. Nevertheless, it is important to refer that HSP production also takes

place in non stressful conditions, being a permanent process of “housekeeping” in the cell.

During stress, their production suggests that intrinsic mechanisms of defense have developed

in tissues in order to recover or destroy damaged proteins. Thus, HSPs may confer not only

heat tolerance but also tolerance to other stresses and cross-tolerance (Basu et al. 2002), since

they are induced by various physical, chemical and biological factors.

The next table summarizes the collected information about HSPs in terms of families,

functions and factors that trigger their production.


8

Table 1. HSPs’ characterization.

Family Protein Localization Function Expression Sensibility Other aspects References

HSP10 Hsp10 Mitochondria Promotes substrate release Constitutive x x Snoeckx et al., 2001

small HSP (sHSP)

Hsp27/Hsp28 Cytosol, nucleus, stress granules

Stabilization of microfilaments; anti-apoptosis; maintenance of the barrier functions (endothelial and

epithelial); cytoskeleton stabilization; control the

association of macro globular complexes (F-actin polymerization,

depending on the fosforilation state and the monomeric or multimeric state of Hsp27)

Constitutive Induced

Temperature

It can form aggregates; it binds to structural proteins in sarcomers, cytoskeleton and nucleus after thermal stress

Kim et al., 1984; Arrigo et al., 1988; Lavoie et al., 1993; De Jong, 1993; Takemoto et al., 1993; Caspers et al., 1995; Van de Klundert et al.,

1998; Kedersha et al., 1999; Snoeckx et al., 2001; Basu et al.,

2002 ; Kregel, 2002

αA-crystallin Cytosol, nucleus

Chaperone (protein folding, prevents protein aggregations)

Constitutive x x

αB-crystallin Cytosol, nucleus Constitutive x

It binds to structural proteins in sarcomers, cytoskeleton and nucleus after thermal

stress

Hsp32 Cytosol Heme-oxygenase Induced

Hypoxia, hyperthermia,

physical stress, heavy metals, Reactive Oxygen Species

(ROS), reperfusion

x

Ewing & Maines, 1991; Maulik et al., 1996; Essig et al., 1997;

McCoubry et al., 1997; Snoeckx et al., 2001


9

HSP40

Hsp40 Cytosol, nucleus Protein folding Constitutive x x

Nakai et al., 1992; Hosokawa et al., 1993; Snoeckx et al., 2001

Hsp47 Endoplasmic

reticulum

It binds to and transports collagen from the endoplasmic reticulum to

Golgi’s complex Constitutive x x

HSP60

Hsp58 Mitochondria Chaperonines; they receive

proteins that enter mitochondria and proceed with their refolding;

they prevent aggregation of denatured proteins; pro-apoptosis

functions


Ischemia, hypoxia; water contaminants

ATPase activity

Deshaies et al., 1988; Manning-Krieg et al., 1991; Clayton et al.

2000; Snoeckx et al., 2001; Kregel, 2002

Hsp65 Mitochondria

HSP70

Hsp72 Cytosol, nucleus Protein folding and refolding in intracellular compartments; maintenance of structural

proteins; protein translocation; maitenance of the adequate

protein conformation for transport; prevention of protein

Highly induced Hiperthermia; energy depletion; hypoxia;

acidosis; ischemia/reperfusion;

ROS; Reactive Nitrogen Species (RNS); viral and

ATPase activity; 60-80% of similarity between DNA

nucleotides in eukarya; binds to cytoskeleton proteins,

cellular surface glycoproteins, calmoduline and long chain

saturated fatty acids; involved

Guttman et al., 1980; Hahn & Li, 1982; Hughes & August, 1982;

Scieandra & Subjeck, 1983; Bardwell & Craig, 1984; Craig,

1985; Weitzel et al., 1985; Lindquist, 1986; Koyasu et al.,

1986; Clark & Brown, 1986; Barbe

Hsp73 (Hsc70) Cytosol, nucleus

peroxisome, lysosome

Constitutive

Hsp75 Mitochondria Constitutive


10

Hsp78(Grp78) Endoplasmic

reticulum

aggregations; targeting of unstable proteins for degradation in

peroxisomes and lisosomes; targeting proteins for ubiquitin

pathway; repair the centrosome after damage; anti-apoptose

functions; blockage of cytokine production and their deleterious

effects; functions in antigen presentation

Highly constitutive in

gland cells; induced

bacterial infection; UV radiation;

endotoxins; cytokines (TNF-Tumor Necrosis

Factor); physical exercise; anaerobic metabolism; heavy

metals; water contaminants; ;

ethanol; circulating hormones;

malnutrition; high population densities;

exposure to predators

in thermotolerance et al., 1988; Moalic et al., 1989; Chiang et al., 1989; Stevenson &

Calderwood, 1990; Löw-Friedrich & Schoeppe, 1991; Terlecky et al., 1992; Amici et al., 1992, 1993;

Hendrick & Hartl, 1993; Walton et al., 1994; Tsang, 1993; Sanchez et

al., 1994; Mivechi et al., 1994; Schoeniger et al., 1994; Herrmann et al., 1994; Myrmel et al., 1994;

Xu et al., 1995; Marber et al., 1995; Flanagan et al., 1995; Skidmore et al., 1995; Kregel & Moseley, 1996; Chi & Mestril, 1996; Terada et al.,

1996; Agarraberes et al., 1997; Oberdörster et al., 1998; Iwama et

al., 1999; Clayton et al., 2000; Kagawa and Mugiya, 2000;

Snoeckx et al., 2001; Ackerman & Iwama, 2001; Kregel 2002; Gornati

et al., 2004; Cara et al., 2005; Padmini et al., 2009

TRtC (T-ring complex)

Cytosol Chaperonine (protein folding) x x x

HSP90

Hsp90α Cytosol, nucleus,

endoplasmic reticulum

Regulation of hormone receptors (steroids); protein folding;

transport of kinases to the cellular membrane; links to intermediate filaments, microtubules and actin

microfilaments; protein translocation; important

component of the glucocorticoid receptor

Constitutive Induced Hyperthermia; energy

depletion; prostaglandins;

malnutrition; high population densities

ATPase activity; involved in thermotolerance; greater selectivity in terms of the proteins which it interacts

with; it interacts mostly with proteins in the regulatory pathways; involved in the

development of the ovaries; Hsp94 is commonly found in

the renal medulla

Welch & Feramisco, 1982; Collier & Schlesinger, 1986; Koyasu et al.,

1986; Anderson et al., 1991; Bansal et al., 1991; Amici et al., 1992,

1993; Pratt, 1993; Schoeniger et al., 1994; Jacob et al., 1995;

Moseley, 1997; Nayeem et al., 1997; Snoeckx et al., 2001; Kregel, 2002; Cara et al. 1995; Gornati et al., 2004; Ali et al., 2006; Zhao et

al., 2010

Hsp90β Cytosol, nucleus,

endoplasmic reticulum


Hsp94 x Protein folding Induced Hyperthermia; osmotic stress


11

HSP110 Hsp104 Cytosol

Protein refolding; binds to actin

Constitutive x x Koyasu et al., 1986; Snoeckx et al.,

2001 Hsp110 Cytosol,

nucleolous Constitutive

Induced x x

Ubiquitins Ubiquitins Cytosol, nucleus Targets abnormal proteins for

degradation by proteases Constitutive x x

Bond & Schlesinger, 1985; Snoeckx et al., 2001

x– No specific information was found

Table 2. Special group of HSPs: Glucose Regulated Proteins.

Group Protein Localization Function Expression Sensibility Other aspects References

GRPs (Glucose

Regulated Proteins)

Grp58

Endoplasmic reticulum

x Constitutiva x

They belong in several HSPs families (HSP60,

HSP70; HSP90; HSP110) Snoeckx et al., 2001

Grp78 Inhibits glycosilation; protein

folding Constitutive

Induced

Low concentrations of extracellular

glucose; depletion of intracellular

calcium stores

Grp94 Binds to calcium Induced in

skeletal muscle cells

Grp170 x Constitutive x

x – No specific information was found


12

3.2. Physiological and ecological relevance of HSPs

Heat Shock Proteins might be considered indirect biochemical indicators of the degree of

damage and protein unfolding that is occurring in the cell (Hofmann 2005). Studies concerning

these proteins give us clues about the temperature at which species become thermally

stressed. HSP production is related to the past thermal history (Hofmann 2005) and the

thermal regime and its variability occurring in the habitat (Tomanek 2010), and may partially

explain the thermal limits of the species and their resistance/vulnerability to heat. They

highlight the eco-physiological complex patterns occurring in nature and contribute to our

understanding of the stress defense mechanisms and the species’ potential responses to

further climate forcing. This is of special interest considering that we need to understand

molecular and physiological traits and responses to be able to decode and explain not only

microhabitat scale patterns but also the occurring large-scale, biogeographic and ecological

patterns (Hofmann 2005).

Therefore, HSPs can have pleiotropic effects on organisms, interacting with multiple

systems in diverse ways, so the cellular response has impacts at several levels and it is

influenced by processes at levels of biological organization (Basu et al. 2002), modulating

ecology and evolution of the biological systems (Sørensen and Loeschcke 2007).

4. Aims and scopes of the dissertation

The general objectives of the research developed in the present dissertation were to

evaluate the tolerance to high temperatures and uncover the Heat Shock Protein 70 patterns

of expression in several marine taxa of commercial interest. The knowledge of thermal limits

and the cellular responses to stress provided a basis to relate several aspects of thermal

physiology to ecological patterns in order to understand the species’ vulnerability/resistance

to heat and the potential impacts of temperature and climate change on marine communities.

The objective was to assess these issues in a broad and multidisciplinary approach, relating

physiology, behavior and ecology.

More specifically, the objectives of this research are as follows:

1. To test, in the laboratory, the tolerance to high temperatures of several species of fish, crab

and shrimp from the Portuguese coast. The aim was to obtain a ranking in terms of

vulnerability through the method of Critical Thermal Maximum and discuss potential impacts

of sea warming in the distribution and abundance of the studied species;


13

2. To investigate which species live closer to their upper thermal limits and compare the

results with available data for tropical species;

2. To compare inter and intraspecific variation in the thermal tolerance and HSP70 production;

3. To uncover patterns of HSP70 expression along a temperature gradient and at the upper

thermal limits;

4. To test the genetic influence on HSP70 production by analyzing whether congeneric species

have similar HSP70 expression patterns and thresholds of induction, maximal production and

shut-off;

5. To evaluate whether habitat features influence the species’ mechanisms of resistance;

6. To evaluate which species might be more vulnerable to climate change, particularly sea

warming.

This dissertation is presented in the form of two scientific articles (already submitted

to indexed scientific journals), the first one concerning thermal tolerances and the second one

concerning HSP70 expression patterns.

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CHAPTER 2

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Thermal tolerance and potential impacts of climate change on coastal

and estuarine organisms (submitted to Marine Biology)

Abstract

The study of thermal tolerance is the first step in understanding species vulnerability

towards climate warming. This work aimed to determine the upper thermal limits of various

fish and crustaceans in a temperate estuarine ecosystem and adjacent coastal area. Species

were ranked in terms of vulnerability to increasing temperatures and intraspecific variability

was evaluated. The method used was the Critical Thermal Maximum (CTM). CTM was found to

be higher for species typical of thermally unstable environments, e.g. intertidal/supratidal, or

which make reproduction migrations. Acclimation or local adaptation of species with a wide

distribution was not observed. It was confirmed that organisms that occur in thermally

unstable environments live closer to their thermal limits. Two fish species which are potentially

threatened by climate warming were identified. This was the first time such an approach was

carried out for marine species of the temperate/subtropical regions, since most studies focus

on the tropics.

Key words: global change, Critical Thermal Maximum, tropical species, temperate species,

intertidal, subtidal, local adaptation, intraspecific variability

Introduction

Temperature is one of the most important factors affecting organisms because it impacts

the kinetic energy of molecules, influencing biochemical reactions and thus the animal’s

physiology and behavior (e. g. Somero 1969; Fry 1971; Mora and Ospina 2001). Therefore,

fitness and performance of the species might be altered by the thermal regime and other

physical and chemical variables operating in the habitat, and dynamic fluctuations of these

variables can interfere and dominate the life history, demographics and competition between

species (Christian et al. 1983; Porter 1989; Huey 1991; Huey and Berrigan 2001; Munday et al.

2008) explaining a diversity of adaptations among organisms (Lutterschmidt and Hutchison

1997a). Individual parameters like growth rate, longevity, excretion rate, food intake and basic

metabolism as well as population parameters like mortality, reproductive rate, recruitment

and population size/distribution all depend on temperature (e.g. Shaw and Bercaw 1962; Brey

1995; Southward et al. 1995; Kröncke et al. 1998; Perry et al. 2005; Pörtner et al. 2008), which

Temperature tolerance and potential impacts of climate change on coastal and estuarine organisms

24

is heterogeneous in time and space (Re et al. 2005) structuring marine community

assemblages and ecosystems at the ultimate level (Glynn 1988).

Aquatic ectotherms such as fish and crustaceans should be studied carefully and get full

attention in temperature tolerance studies not only because they are particularly at risk from

thermal stress (Schaefer and Ryan 2006) but because there is a considerable lack of knowledge

on their thermal limits, especially for temperate species, pelagic and schooling fish and even

for some crustacean species that are widely distributed and easy to handle (Freitas et al.

2010). The tolerance window for each species can be described as a favorable range of

temperature or performance breadth and it is noteworthy that above or below that range,

performance is negatively affected and the species cannot survive unless it is for a limited

period of time. This means that ectotherms can only carry out behavioral thermoregulation

(Rozin and Meyer 1961; Neill et al. 1972; Neill and Magnuson 1974) which can imply habitat

selection based on the habitat’s thermal characteristics. Therefore, and in the light of climate

change scenarios, it is reasonable to expect inter and intraspecific competition to occur if the

thermal microhabitat is scarce.

Regarding the current concerns about future climate change scenarios, the knowledge of

thermal tolerance is the first step to understand how vulnerable species are. However it is

essential to consider that not only there is a great diversity of responses but also that global

warming tends to vary regionally (Rivadeneira and Fernández 2005) so there is a need to do

regional and population studies (McFarlane et al. 2000). That being said, most literature focus

on tropical regions maybe because predictions for temperate regions are the hardest to make

due to the diversity of life history patterns, complexity of trophic relations, habitat variability

and over-fishing (IPCC 1997; Roessig et al. 2004).

It has been proposed that the impacts of climate warming should be greatest on thermal

specialists that have limited acclimation potential because they live in aseasonal environments

(Hoegh-Guldberg et al. 2007; Tewksbury et al. 2008). According to Cuculescu et al. (1998), the

thermal limits of an organism are set genetically but in evolutionary terms the rate at which

temperature is increasing might not allow the organisms to adapt genetically. Thereby, the

ecosystems that have evolved in stable conditions for a long time such as cold environments or

tropical ones are especially at risk. Species living in the tropical regions are reported to have

the disadvantage of living close to their upper thermal limits (Jokiel and Coles 1977; Sharp et

al. 1997) although other authors present contradictory evidence (Mora and Ospina 2001). It

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25

has also been suggested that warm-adapted species of the intertidal/supratidal zone may be

particularly at risk since they live closer to their upper thermal limit and have limited

acclimation capacity (Hopkin et al. 2006; Somero 2010). Despite the fact that they are more

thermally tolerant there is a high probability that maximum habitat temperatures surpass their

upper thermal limit (Somero 2010) because they live in a hot and unstable environment with

wide daily and seasonal thermal amplitudes.

When studying the potential climate change impacts on marine fauna, marine coastal

waters and estuaries should be the focus of attention because they are amongst the most

productive ecosystems, are nursery areas and depuration systems. Furthermore, they are

shallow habitats, have little thermal inertia and will thus be the first to reflect atmospheric

temperature rise, acting as sentinels of climate change.

Besides the direct effects of warming, these areas will also be strongly affected by other

aspects of climate change. Intertidal communities, which play an important role as feeding

areas for fish and birds, may be compromised if sedimentation patterns change and/or if there

is an increase in erosion because these communities depend on the type and characteristics of

the substrate (Sagarin et al. 1999). Coastal ecosystems will be furthermore subjected to

increased eutrofication by anthropogenic activities and the situation should only get worse at

increasing temperatures (Officer et al. 1984; Kennedy 1990; Roessig et al. 2004; IPCC 2007),

leading to more frequent episodes of hypoxia and HABs (Harmful Algal Blooms) (Rosenzweig

et al. 2007). The hypoxia combined with warmer water results in lower oxygen availability,

which can aggravate the condition of these ecosystems once it affects the organisms’ aerobic

performance restricting the tolerance to extreme temperatures (Sommer et al. 1997; Frederich

and Pörtner 2000; Pörtner et al. 2004; Pörtner and Knust 2007).

With this in mind, the aim of this work was to determine the upper thermal limits of

various temperate and subtropical species, fish and crustaceans that are commercially

important and/or crucial in the temperate estuarine/coastal ecosystem studied. The species

were then ranked in terms of vulnerability to increasing temperatures and hypothesis on the

potential impacts of climate warming on the species studied were put forward. Another goal of

this work was to evaluate and compare intraspecific variability of the upper thermal limit in

order to discuss the species’ potential to adapt to ongoing global climate changes. Also, it was


26

investigated which species live closer to their upper thermal limits and comparisons with

tropical species were performed.

Materials and methods

Study area and sampling method

This study was carried out in the Tagus estuary and adjacent coast (from Vila Franca de

Xira to Cascais), at an approximate latitude of 38°N (Northeast Atlantic). This estuary is located

in the midwest coast of Portugal, and has an area of 320 km2, a length of 34 km and a

maximum width of 15 km. During the summer (June to September), the Tagus estuary and the

intertidal pools of the adjacent coast have a mean surface temperature of 24°C (Centro de

Oceanografia database).

This research was focused not only on marine and estuarine species of commercial

importance but also species that play an important role in the food web. The fish species

studied were Diplodus bellottii (N=17), Diplodus vulgaris (N=13), Diplodus sargus (N=28), Solea

lascaris (N=8), Dicentrarchus labrax (N=7), Gobius cobitis (N=4), Blennius trigloides (N=9),

Gobius niger (N=9) and Liza ramada (N=6). The crustacean species studied can be divided in

two groups: the crabs Liocarcinus marmoreus (N=17), Xantho incisus (N=16), Carcinus maenas

(N=25), Pachygrapsus marmoratus (N=26) and the shrimps Palaemon longirostris (N=14),

Palaemon elegans (N=25) and Crangon crangon (N=16). Sample sizes were similar to Mora and

Ospina (2001) for comparable analysis. Most of these species have a range that goes from

Northern Europe to North Africa, occurring in both cold and moderately warm waters, except

for Crangon crangon, Palaemon longirostris and Liocarcinus marmoreus which occur mainly in

cold waters (Froese and Pauly 2011, Palomares and Pauly 2011).

The species were caught during the summer months (from July to September of 2010).

Sampling was carried out using hand nets, dip nets, beam trawl and beach seine.

Thermal tolerance method

Thermal tolerance of these species was determined using the dynamic method described

in Mora and Ospina (2001). The goal was to determine the Critical Thermal Maximum (CTM),

which is defined as the “arithmetic mean of the collective thermal points at which the end-

point is reached” (Mora and Ospina 2001). This end-point can be either loss of equilibrium or

onset of muscular spasms. In fish both of these end-points were easily observed, while in

CHAPTER 2

27

crustaceans they were stimulated with a lab tweezer to force them to swim, allowing the

identification of equilibrium loss.

After capture, organisms were transported to the laboratory and placed in a re-circulating

system with aquaria of 70L with aerated sea water, a constant temperature of 24°C and

salinity 35. The water dissolved O2 level varied between 95% and 100%. They were left there to

acclimate for two weeks, being fed ad libitum twice a day. They were starved for 24h before

the experiments. To determine CTM, the organisms were subjected to a thermostatized bath.

During the trial, animals were exposed to a constant rate of water-temperature increase of

1°C.h-1, until they reached the end-point. In each trial 10 to 15 individuals were tested

simultaneously and observed continuously. The temperature at which each animal reached its

end-point was measured with a digital thermometer, registered and then CTM and its standard

deviation were calculated for each species. All experiments were carried out in shaded day

light (15L;09D). To prevent any additional handling stress, the total length of all individuals was

measured at the end of each trial using a slide caliper rule.

Data analysis

The upper thermal limits for each species were calculated through the equation:

1 int( )n

end po n

species

TCTM

n

Where Tend-point is the temperature at which the end-point was reached for individual 1,

individual 2, individual n, divided by the n individuals that were in the sample.

To determine intraspecific variability of CTM, coefficient of variation (in percentage) was

calculated for each species.

In order to evaluate which species live closer to their upper thermal limits, the difference

between CTM and mean surface water temperature (24°C) was calculated for each species

(fish and crustacean). Using the work of Mora and Ospina (2001), the same difference was

calculated for tropical species considering a mean surface temperature of 27°C. Then, the

results for temperate/subtropical and tropical fish were compared through a Student’s t-test,

since the data followed normality (Shapiro Wilk’s test) and homocedasticity (Levene’s test). A

significant level of 0.05 was considered in all test procedures. Additionally, the difference

between CTM and Maximum Habitat Temperature (MHT) was calculated for intertidal fish


28

species from temperate/subtropical and tropical regions (Mora and Ospina 2001). The

crustaceans were left out of this analysis since comparable data was only available for fish.

Results

All environmental variables known that might influence the results (e.g. oxygen levels,

salinity, food, pH, photoperiod, acclimation temperature) were monitored during the

acclimation and trials, so it is believed that observed results are due to temperature. Loss of

equilibrium was the most observed end-point, although S. lascaris and species from the genera

Diplodus also showed muscular spasms. But in all cases the first sign of stress was loss of

equilibrium.

Total length for each species is shown in table 1. The CTMs obtained for all species ranged

from 27.38°C for D. bellotti to 38.00°C for L. ramada (Fig.1). Considering the results by groups

of marine organisms, within crabs, the lowest CTM was observed for L. marmoreus (32.24°C),

followed by X. incisus and C. maenas. Finally, the highest CTM observed in crabs was for P.

marmoratus (35.73°C). Within shrimp, C. crangon had the lowest CTM (33.75°C) and Palaemon

longirostris had the highest (34.43°C). Considering fish, the lowest CTMs were observed for

two Diplodus species (D. bellottii and D. vulgaris) and the highest CTMs, besides L. ramada,

were observed for G. niger (34.10°C) and B. trigloides (34.00°C). The CTM was more variable

between fish species (10.62°C), followed by crabs (3.49°C) and less variable between shrimp

species (0.68°C).

Table 1. Mean total length and standard deviation for each species.

Species Total length (mm)

Mean±SD

Dincentrarchus labrax 86.00± 6.19

Diplodus bellottii 103.71± 8.91

Diplodus sargus 33.89±9.07

Diplodus vulgaris 74.62±7.76

Blennius trigloides 67.33±28.26

Gobius cobitis 46.00±29.41

Gobius niger 98.70±6.36

Liza ramada 44.00±3.90

Solea lascaris 184.38±39.70

Carcinus maenas 28.65±5.80

CHAPTER 2

29

Liocarcinus marmoreus 22.35±2.74

Pachygrapsus marmoratus 17.32±6.24

Xantho incisus 34.38±6.09

Crangon crangon 36.50±4.84

Palaemon elegans 32.52±7.34

Palaemon longirostris 43.79±8.94

Fig. 1 Critical Thermal Maxima of nine fish species, four crab species and three shrimp species from the Tagus

estuary and adjacent marine coastal waters. Straight line represents the mean surface temperature during the

summer (24°C) for the study area and the dashed line represents the temperature during heat waves (28°C). The

dotted line represents the maximum temperature for tide pools. CTMs of intertidal/supratidal species are tagged

with an arrow. Average total length ± standard deviation for each species is shown in parenthesis.

0

5

10

15

20

25

30

35

40

Tem

pe

ratu

re °

C


30

Intraspecific variability, given by %CV, was generally low, being the lowest for S. lascaris

and L. ramada and the highest for D. labrax (Table 2).

Table 2. CTM (Critical Thermal Maximum) intraspecific variability given by the coefficient of variation (in

percentage).

%CV Species

0 Solea lascaris

Liza ramada

1≤ %CV ≤2 Liocarcinus marmoreus

Crangon crangon

Gobius cobitis

Diplodus sargus

Blennius trigloides

2< %CV ≤3 Carcinus maenas

Pachygrapsus marmoratus

3< %CV ≤ 4 Xantho incisus

Palaemon elegans

Gobius niger

Palaemon longirostris

4< %CV ≤ 5 Diplodus bellottii

Diplodus vulgaris

> 5 Dicentrarchus labrax

The species living closer to its thermal limits is D. bellottii with only 3.38°C of difference

between the mean surface water temperature and its CTM. In second comes D. vulgaris with a

difference of 7.08°C. The species living farthest from its thermal limits is L. ramada with a

difference of 14°C. In between these values are the demersal and intertidal/supratidal species,

ranging from approximately 8-10°C and 9-11°C of difference between mean surface water

temperature and CTM (Fig 1).

When analyzing differences between CTM and mean surface temperature for

temperate/subtropical and tropical fish species (Table 3), no significant differences were found

(p-value˃0.53). However, when differences were analyzed dividing the fish in two groups

(demersal and intertidal) significant differences were found for intertidal species (p-

CHAPTER 2

31

value˂0.03), but not for demersal ones (p-value˃0.71). Thus, temperate/subtropical intertidal

fish live closer to their upper thermal limit than tropical intertidal fish.

Table 3. Differences between CTM (Critical Thermal Maximum) and mean surface water temperature for

temperate/subtropical and tropical fish species. First of all, the differences were calculated for all the species

included in this work and Mora and Ospina (2001). Following, differences were tested dividing the fish in two

groups: demersal and intertidal. Significant differences (p-value<0.03) are presented in bold.

Mean difference

(°C)

All species

Mean difference

(°C)

Demersal species

Mean difference

(°C)

Intertidal species

Temperate/subtro

pical

9.16 9.84 9.95

Tropical 9.73 9.17 12.00

Maximum Habitat Temperature can reach up to 35°C in the study area (Portugal’s

Meteorological Institute database and personal data) so organisms in the intertidal area can

be subjected to very high temperatures. For tropical areas, MHT was 36°C for tide pools (Mora

and Ospina 2001). The results show that CTM of tropical intertidal species is 2-5°C higher than

MHT while CTM of temperate/subtropical species is 1-2°C lower than MHT (Table 4).

Table 4. Differences between CTM (Critical Thermal Maximum) and MHT (Maximum Habitat Temperature) for

temperate/subtropical and tropical intertidal fish. The difference was calculated for each species and then a mean

difference and SD was calculated for each latitudinal group. CTMs of tropical fish used in these calculations were

obtained from Mora and Ospina (2001).

Temperate/Subtropical Tropical

Gobius

cobitis

Gobius

niger

Blennius

trigloides

Malacotenus

zonifer

Bathygobius

ramosus

Mugil

curema

CTM-MHT

(°C) -1.25 -0.90 -1.00 2.1 3.5 4.8

Mean±SD

(°C) -1.05±0.18 3.46±1.35


32

Discussion

In this study we have determined the Critical Thermal Maxima (CTM) of 16

temperate/subtropical species of a variety of taxa and in addition we have calculated the

intraspecific variability, which gives us an idea of the capacity of organisms to adapt to

environmental changes. This is the first time such an approach has been carried out for marine

species of the temperate and subtropical regions, since most of the work has been focused on

tropical and freshwater fishes (e.g. Becker and Genoway 1979; Lutterschmidt and Hutchison

1997a,b; Mora and Ospina 2001, 2002; Badillo et al. 2002; Eme and Bennett 2009). We also

determined which species live closer to their upper thermal limits and compared results for

temperate/subtropical versus tropical fish species, evaluating which ones are more vulnerable

to climate warming, which is an issue of current debate.

Our study also covered species from the less studied groups, such as Pleuronectiformes

(Lutterschmidt and Hutchison 1997a) as well as schooling species (Schaefer and Ryan 2006),

for instance sea breams. In terms of crustacean species, most of the previous studies used the

Lethal Temperature Method, so our work provides a means of comparison between fish and

crustaceans with the additional advantage that when CTM is reached, the behavioral response

is the same throughout a diversity of taxa (Lutterschmidt and Hutchison 1997b), making it easy

to identify the moment when the organisms start to lose function. This loss of function is

primarily detected by loss of equilibrium (LE), which indicates that nerve impulse transmission

is disturbed (Aslanidi et al. 2008) and that there is a pre-synaptic failure (Re et al. 2005). This is

a common response both in fish and crustacean whereas onset of muscular spasms is difficult

to see in crustaceans (Lutterschmidt and Hutchison 1997a). Nonetheless, sole and sea breams

showed muscular spasms rapidly after we first detected LE, maybe because they were among

the less resistant species to high temperatures so they may suffer rapid physiological and

cellular heat damage. It is known that exposure to extreme temperature affects a number of

cellular structures like Golgi’s complex, mytochondria, cytoskeleton and proteins (Mizzen and

Welch 1988; Dubois et al. 1991; Vidair et al. 1996; Kedersha et al. 1999; Snoeckx et al. 2001)

and causes low blood flow (Kregel 2002) to peripheral tissues, metabolic depression and ion

imbalance (Stanley and Colby 1971) leading to the onset of muscle spasms. However, the fast

occurrence of muscle spasms might also depend on species physiology because Diplodus

sargus was not one of the less resistant species and also showed onset of muscular spasms not

long after LE.

CHAPTER 2

33

These metabolic changes and the temperature at which they occur are most probably

related to the thermal regime operating in the habitat because organisms have been adapting

to a certain range of values of environmental variables, having different physiological set-

points (Dent and Lutterschmidt 2003), which actually points out that CTM and optimal

temperatures are co-adapted (Huey and Kingsolver 1993). For instance, among crab species,

the ones with lower CTM (L. marmoreus and X. incisus) are inhabitants of the subtidal zone

(Alvarez 1968, Hayward et al. 1996), which is constantly covered with water so the organisms

are not directly exposed to sun heat and live in a thermally stable environment.

Liocarcinus marmoreus is a swimming crab with demersal habits so is even less exposed to

heat because it can move and escape less favorable thermal conditions reaching down to

200m while X. incisus usually stays in relatively shallow waters (Hayward et al. 1996), which

can still slightly warm up during the day. Anyhow, L. marmoreus and X. incisus incisus (the

northern subspecies) are characteristically from relatively colder waters since their distribution

is mainly Northern/Eastern Atlantic (Alvarez 1968; Hayward et al. 1996; Ingle 1997), so it was

expected that their CTM would be lower when compared to other crab species with wider and

more southern distributional ranges.

As for C. maenas, it is known that it is an eurythermal species widely distributed (from

North Atlantic to Mauritania – see Ingle 1997) that can live in the subtidal but mainly occurs in

the intertidal area (Hayward and Ryland 1995; Ingle 1997; Flores and Paula 2001) so it is

exposed to high temperatures, having a higher CTM. The CTM we found was around 35°C,

which is in conformity with the study done by Ashanullah and Newell (1977) and Cuculescu et

al. (1998). The authors of the latter found a very similar CTM for this species even though crabs

they caught were from the North Sea. Some authors report that organisms suffer local

adaptation or acclimation to the temperature range on a regional scale, so crabs from the

North Sea would be expected to have lower CTMs than their conspecifics living more to the

south (e.g. Lutterschmidt and Hutchison 1997a; Beitinger et al. 2000; Mora and Ospina 2001;

Re et al. 2005; Schaefer and Ryan 2006; Eme and Bennet 2009; Freitas et al. 2010). Yet, the

present work shows that this is not always the case.

The high CTM found for C. maenas is probably due to the association of this species with

the intertidal zone, which is extremely variable, at several levels such as temperature, salinity,

and dissolved oxygen. Species inhabiting such an environment, specially the resident ones,

https://vpn.ul.pt/http/0/www.sciencedirect.com/science/article/pii/S0306456598000084#bbib1


34

should possess physiological and cellular adaptations that enable them to cope with such

extreme conditions. These adaptations lead to a higher CTM so organisms living in variable

environments have higher tolerances (e.g. Mora and Ospina 2001; Badillo et al. 2002; Schaefer

and Ryan 2006) than those coming from more stable environments, for example the subtidal

zone.

Pachygrapsus marmoratus was the crab species with the greatest upper thermal limit

certainly because it inhabits the most extreme environment: the supratidal zone, though it can

also occur in the intertidal zone (Ingle 1997; Flores and Paula 2001). The abiotic factors (mainly

temperature and insolation) occurring in the supratidal are crucial for littoral zonation of the

organisms living in that habitat, depending on how adapted they are to water loss and heat.

Our results, along with other studies (e.g. Davenport and MacAlister 1996; Davenport and

Davenport 2005) follow the perspective that organisms living higher in the shore are more

tolerant than those closer to low water, although other authors did not find such a connection

(e.g. Clarke et al. 2000).

Regarding the results for shrimp and fish species, a similar pattern occurs. Crangon

crangon has the lowest CTM among shrimps probably because it is a cold water species

(mainly from the Northeast Atlantic – see Hayward et al. 1996) and it is a demersal species

living in depths of 0-50m (Hayward and Ryland 1995), which means that it is exposed to lower

temperatures and lives in a more stable environment, although it can also occur in estuaries

(Boddeke 1989; data from Centro de Oceanografia) so it must be adapted to a certain degree

of environmental variability.

Palaemon longirostris and P. elegans inhabit the temperate and subtropical regions

(Alvarez 1968) and presented a higher CTM enabling them to live in warmer environments.

Palaemon longirostris is a catadromous migrating species (Cartaxana 1994; Barnes 1994; Paula

1998), which means it has to pass through several habitat types and thermal regimes to be

able to reproduce and keep the population numbers, so a high resistance for this species was

expected.

The linkage between thermal limits and aerobic scope (Pörtner and Knust 2007) is of

crucial importance in migrating species because fecundity and recruitment are connected to

aerobic scope (Farrell 2009). Organisms have a higher energy and oxygen demand due to an

additional swimming rhythm during the reproduction season (Cartaxana 1994) which means

CHAPTER 2

35

they need their full aerobic scope to carry out the migration and reproduce. According to

Farrell (2009), if warmer waters decrease aerobic scope, reproduction success might be

jeopardized, putting the population at risk. This is especially relevant considering that P.

longirostris has a one-year or two-years population cycle (Cartaxana 1994). Finally, with regard

to P. elegans, it lives in intertidal pools (Hayward et al. 1996) that have diel cycles of

temperature, oxygen availability, CO2 accumulation and pH fluctuation so it was also expected

to be one of the most heat resistant among shrimps.

At last, all fish species studied have a wide distributional range in the

temperate/subtropical region so besides species physiological performance, differences in

CTM should mainly come from habitat type, circulation and current patterns plus distributional

depth. In the case of sea breams, although they are found in estuaries as juveniles (Vinagre et

al. 2010), juveniles and adults also live in the continental shelf area so, in the Portuguese coast,

they are exposed to annual sea-surface temperatures that range from 15°C to 18°C (Levitus

and Boyer 1994) or even colder since adults can live in water depths down to approximately 50

m (D. sargus) to a 100 m (D. bellottii and D. vulgaris) (Bauchot and Hureau 1986). Besides, they

also suffer the influence of cold waters from coastal upwelling during the summer due to

northerly trade winds (Lemos and Sansó 2006). Knowing that D. bellottii is originally an African

species from the Mauritania upwelling system (Vinagre et al. in press), it was expected that its

upper thermal limit would not be one of the highest among the fish studied, even though the

African upwelling has waters that round a mean temperature of 20-25°C (Levitus and Boyer

1994), which are warmer than Portuguese waters. A similar situation occurs for D. vulgaris,

which has a discontinuous distribution, being absent from the warmer stretches of the African

coast (Bauchot and Hureau 1986). As for D. sargus, its wide distributional range is continuous

throughout the west coast of Africa indicating that it can live in tropical warm waters, so its

higher CTM than the other two Diplodus species was expected and is in accordance with these

species climatic envelopes. Vinagre et al. (in press) came to a similar conclusion. They found a

general trend of higher vulnerability to high temperatures in the species D. bellottii, unlike D.

sargus. According to the authors, the reason for this difference is related to a steep increase in

D. bellottii’s metabolism in high temperatures, a decrease in the aerobic scope and increased

mortality rate. On the contrary D. sargus maintained a low level of sensibility towards

increased temperatures, which is in accordance with our results.


36

Similar intermediate values of CTM were found for S. lascaris and D. labrax when

comparing to other fish, possibly as a result of their demersal life and the depth at which they

can be found, which can reach 350m for soles and 100m for sea bass (Quéro et al. 1986;

Tortonese 1986; FAO 2011). Notwithstanding, they are frequently found in relatively shallow

coastal waters and estuaries so they are physiological capable of inhabiting warmer waters,

not only as adults but also as juveniles because sole and seabass use estuaries as nursery

grounds (Barnes 1994; Hayward and Ryland 1995; Vinagre et al. 2008, 2009).

Amongst the most resistant fish species were G. cobitis, B. trigloides and L. ramada, which

followed the pattern already described for crabs and shrimps. Gobius and Blennius are genera

inhabiting either estuaries, surf zone waters or the intertidal pools so they undergo wide

variations in abiotic variables and are exposed to very high temperatures, which they can cope

with due to their high upper thermal limit. The case of L. ramada is less obvious but similar to

P. longirostris since it is a migratory species that can live in coastal waters and inshore waters,

entering estuaries, lagoons and lower parts of the rivers (Sauriau et al. 1994; Hayward and

Ryland 1995) so it must be adapted to a diversity of thermal regimes where temperature can

vary considerably and reach elevated values.

Overall, when analyzing interspecific differences, we might initially expect a greater

disparity as the mean genetic distance increases but this would only be if ecological niches

were very dissimilar. Our results shed some light on this matter since it appears that species

from different taxa living in identical habitat types have similar upper thermal limits (Fig. 1)

because they evolved in similar abiotic conditions, probably developing similar physiological

and cellular adaptations. Another important result is that upper thermal limits were more

variable between fish than between crabs and shrimps. This is probably because fish have a

great locomotory capacity and colonize all kinds of habitats.

Intraspecific variability was generally low, which is in concordance with Mora and Ospina

(2001). According to Cuculescu et al. (1998), thermal tolerance is subject to phenotypic

alteration within a genetically fixed range. This phenotypic plasticity is dependent upon several

factors but thermal history of individuals and parental effects seem to be the most important

ones (Cossins and Bowler 1987; Schaefer and Ryan 2006), inducing irreversible changes to the

thermal tolerance (Schaefer and Ryan 2006). Then, variability found for each species relates

not only to specific genetic features but also the previous influence of environmental variables.

Nevertheless, if genetic variability is in general low, this might be a concern because species

CHAPTER 2

37

might not be able to adapt to climate changes (Mora and Ospina 2001). Our data shows that D.

labrax, D. bellotti and D. vulgaris appear to have the highest variability of response towards

temperature and thus possibly the highest genetic variability concerning the genes involved in

such a response.

There are various untested factors that can potentially influence species upper thermal

limits. These factors can be sex, reproductive state, nutritional condition, diseases and

parasites, inter-population variability, age and size (Cox 1974; Copeland et al. 1974; Hutchison

1976; Becker and Genoway 1979; Lutterschmidt and Hutchison 1997b) as well as thermal

history of individual animals (Schaefer and Ryan 2006) and also seasonal variation (Cuculescu

et al. 1998; Hopkin et al. 2006). Yet, there seems to exist a certain degree of controversy on

the influence of these factors since some authors found significant differences for instance

between different sized individuals (e.g. Cox 1974; Copeland et al. 1974; Peck et al. 2004,

2007, 2009) while others did not (e.g. Ospina and Mora 2004), nor for males versus females

(Badillo et al. 2002). Even though we limited the influence of some of these factors by, for

example, restricting sampling to the summer and testing individuals of approximately the

same size, this might have a disadvantage which is missing important intraspecific variability

patterns. Further studies should address this issue and CTM should be tested for different life

stages, different seasons and different habitats used by conspecifics, amongst other factors.

In this study it was confirmed that CTM of marine ectotherms is higher for species which

inhabit variable environments, like the intertidal or the supratidal and/or make reproduction

migrations that require the passage through different habitats with potentially very elevated

temperatures. Acclimation or local adaptation of species with a wide distribution was not

confirmed since C. maenas displayed the same CTM as found in the North Sea.

In order to know which species might be more vulnerable to temperature, we calculated

the difference between thermal limit and mean surface water temperature for each species.

Mora and Ospina (2001) conducted a similar study on tropical reef fish and concluded that the

CTM of the least tolerant species was 8ºC above the current mean sea temperature, while in

the present study the CTM of the least tolerant species, D. bellottii, is only 3°C above the

average summer estuarine temperature (it is in summer that this species juveniles occur in

estuaries). In fact, this species is already under stress during current heat waves, which makes

it clear that its presence in estuarine nursery areas (where juveniles are very abundant


38

nowadays) is under threat by climate warming, as observed by Vinagre et al. (in press) through

experimental studies. In a climate warming perspective, if we add 2°C to the current

temperature attained by the waters during heat waves (28°C+2°C), it is clear that another

seabream, D. vulgaris, may also be under threat.

When we compared mean differences between CTM and mean water temperature for

temperate/subtropical and tropical fish, no significant differences were found considering all

species. When we evaluated demersal and intertidal species separately, demersal ones didn’t

show significant differences as well but intertidal species did. The intertidal

temperate/subtropical fish have an upper thermal limit on average 9.95°C above mean water

temperature (24°C) while tropical intertidal fish have an upper thermal limit on average 12°C

above mean water temperature (27°C). This result may indicate that temperate/subtropical

intertidal species might be more vulnerable to further increases in temperature.

In fact, one of the main findings of this study was the confirmation that organisms that

occur in thermally unstable environments, e.g. intertidal/supratidal habitats, live closer to their

thermal limits because maximum habitat temperatures may reach or exceed their CTM.

Comparing the results obtained for temperate/subtropical and tropical species, we can see

that the former species have CTMs on average 1.05°C under the maximum habitat

temperature while tropical species have CTMs on average 3.46°C above maximum habitat

temperatures. Therefore, maximum habitat temperatures in temperate/subtropical regions

may exceed the upper thermal limits of intertidal fish species, making them especially

vulnerable to high temperatures and climate warming. Additionally, since it is the extreme

values of environmental conditions that exert the most selective pressures (Lutterschmidt and

Hutchison 1997a), this result might indicate that temperate/subtropical intertidal fauna may

be exposed to strongest selection when it comes to temperature conditions.

This is indicative of a probable higher vulnerability of temperate/subtropical fish species,

and it is in concordance with the predictions that tropical fish might be affected through

indirect effects of climate change rather than direct ones (Mora and Ospina 2001). The

statement that tropical species live closer to their thermal limits is supported through the

studies of Jokiel and Coles (1977) and Sharp et al. (1997). However, these apply to corals, so in

order to understand climate impacts on marine communities, more species of various

taxonomic groups should be tested.

CHAPTER 2

39

Nonetheless, predicting the impacts of climate change on particular species is not a

simple task and requires in depth knowledge on several subjects from molecular biology,

physiology, ecology and evolution. Thus, realistic predictions will necessarily be the result of

multidisciplinary and integrative approaches. Nevertheless, since sea warming effects are

already clear throughout ecosystems and base studies are urgently needed, we consider this

work a necessary first step in the investigation of climate change impacts upon marine biota

and ecosystems.

Acknowledgements

Authors would like to thank everyone involved in the field work, maintenance of the experimental

tanks and in the feeding of the organisms. Authors would like to express their gratitude to “Aquário

Vasco da Gama” for the collaboration in the sampling and maintenance of organisms, in particular to Dr

Fátima Gil. This study had the support of the Portuguese Fundação para a Ciência e a Tecnologia (FCT)

through the funding of projects and the grant SFRH/BPD/34934/2007 awarded to C. Vinagre.

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Heat Shock Protein 70 patterns of coastal and estuarine organisms facing

increasing temperatures (submitted to Molecular Ecology)

Abstract

Heat Shock Proteins are a crucial component of the cellular defense against proteotoxic

stress. This worked aimed to uncover HSP70 expression patterns in several marine species

along a temperature gradient and at the upper thermal limit. Congeneric species were

compared to test genetic versus environmental influences on HSP production. Exposure trials

were performed through the Critical Thermal Maximum (CTM) method and protein analysis

was performed using ELISA, Western Blot and SDS-PAGE. Several trends in HSP70 expression

profiles were identified in several species independently of taxa, CTM and habitat type.

Magnitude of expression seems to correlate with thermal conditions, with some exceptions. In

Diplodus genus HSP70 production seems to be influenced by thermal conditions while in

Palaemon genus it seems to be more genetically fixed. Cold and stable environment species

that lack an inducible Heat Shock Response or have a narrow range for HSP70 production may

be vulnerable to sea warming.

Key words: climate change, thermal stress, cellular defense, heat shock proteins, marine organisms

Introduction

Ectothermic organisms are subjected to variable environmental temperatures which

impact their function at several levels during their lifetime, including molecular, physiological,

organismal and behavioral levels (Mora and Ospina 2001, Hochachka and Somero 2002, Dent

and Lutterschmidt 2003). Therefore, temperature has great impacts on performance and

fitness so it is reasonable to expect larger patterns to be partially determined by this variable,

such as species biogeographic distribution, biological interactions and community structure

(Glynn 1988, Huey 1991, Sanford 1999, Gaston 2003, Pörtner and Knust 2007). Since the

mechanisms connecting molecular and physiological responses to ecological patterns are still

poorly understood (Hofmann 2005), it is necessary to uncover and deepen the knowledge on

this matter. This implies that basic molecular information must be obtained (Basu et al. 2002),

analyzed and correlated with physiological and ecological information.

The research of the effect of thermal stress on the molecular functioning of marine

organisms has focused on the expression of Heat Shock Proteins (HSPs) (Tomanek 2011). HSPs

Heat Shock Protein 70 patterns of coastal and estuarine organisms facing increasing temperatures

48

act as chaperones, stabilizing not only denatured polypeptides but also nascent proteins,

mediating their folding and preventing their interaction and the occurrence of cytotoxic

aggregations (Hightower 1991, Welch 1992, Becker and Craig 1994, Moseley 1997, Fink 1999).

They are also important in the targeting of non-native or aggregated proteins for degradation

(Feder and Hofmann 1999), in DNA repairing processes (Zou et al. 1998), as well as regulatory

pathways, cellular energetic processes such as protein translocation between cellular

organelles (Beckmann et al. 1990, Ellis 1990, Hartl 1996, Fink 1999) and immune responses

(Jacquier-Sarlin et al. 1994, Bachelet et al. 1998). HSPs are a ubiquitous conservative group of

proteins with constitutive and induced expression, playing an important role in cell functioning

(during both regular and stressful conditions) and cell protection (Baler et al. 1992, Currie et al.

1999, Kregel 2002). In addition, these proteins are important in an ecological perspective since

their production occurs amongst almost every taxa and it has been conserved through

evolution (Parsell and Lidquist 1993, Feder and Hofmann 1999).

Within HSPs, the most studied group is the 70kDa, which contains proteins that can be

located in the cytosol, nucleus, peroxysome, lysossome, mitochondria and endoplasmic

reticulum (Snoeckx et al. 2001). This group has a constitutive expression as well as a highly

inducible expression (Morimoto et al. 1990) triggered not only by temperature (Koban et al.

1991, Iwama et al. 1998, Currie and Tufts 1997, Kregel 2002) but other factors such as hypoxia

(Mestril et al. 1994, Ramaglia and Buck 2004), acidosis (Kregel 2002), oxidative stress (Polla et

al. 1998, Snoeckx et al. 2001, Kregel 2002), Reactive Nitrogen Species (Kregel 2002), cellular

energy depletion (Feder and Hofmann 1999), viral and bacterial infection (Forsyth et al. 1997,

Kregel 2002), endotoxins and cytokines (Moseley 1997, Kregel 2002), anaerobic metabolism

(Myrmel et al. 1994), heavy metals and other aquatic contaminants (Renfro et al. 1993, Ryan

and Hightower 1994, Williams et al. 1996, Clayton et al. 2000), prostaglandins (Amici et al.

1992), UV and γ-radiation (Kedersha et al. 1999, Yamashita et al. 2010), high population

densities (Iwama et al. 1999, Gornati et al. 2004), exposure to predators (Kagawa and Mugiya

2000) and malnutrition (Cara et al. 2005). In addition, there are some studies suggesting that

HSPs may play specific roles during phases of ontogeny, for instance myogenesis (see Feder

and Hofmann 1999, Basu et al. 2002, Deane and Woo 2010 reviews).

The production of HSPs is part of the Heat Shock Response (HSR) (Yamashita et al. 2010)

which, according to Parsell and Lindquist (1993), is an important mechanism in the cellular

defense against proteotoxic stress. This mechanism consists in a coordinated up-regulation of

several genes, functionally related, after the protein pool is exposed to stress factors that lead

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49

to protein denaturation. This response is elicited a few minutes after stress exposure because

HSP’s mRNAs lack introns, which makes the translation a fast process (Basu et al. 2002). HSR is

dependent upon the stimulation of membrane receptors, changes in the physical properties of

the cellular membrane or intracellular changes (e.g. temperature, pO2) (Snoeckx et al. 2001).

The HSR’s set points and intensity are determined not only by the environmental temperature

but also by its rate of change (Snoeckx et al. 2001), so HSP production might differ according to

species thermal history and habitat conditions and variability.

It is known that hyperthermia is a strong activator of the HSR since it strongly affects the

organization of several cellular components: Golgi’s complex suffers fragmentation,

mithocondria’s cristae change, intermediate filaments from the cytoskeleton aggregate,

cytosolic proteins aggregate and become poorly soluble (Dubois et al. 1991, Vidair 1996) and

there is an increased Reactive Oxygen Species production leading to great damage in DNA,

RNA, fatty acids, proteins and enzyme co-factors. In addition, Mizzen and Welch (1988) refer

that hyperthermia might cause a translational arrest which is proportional to the intensity and

time of exposure to the stress. However, Kedersha et al. (1999) refer that mRNAs from HSPs

are not included in this arrest and can keep being translated in a cell under stressful

conditions. This period of translational arrest might be shortened if the cells are already

thermotolerant because if they already contain a certain level of HSPs, they don’t need to

produce high quantities in a second exposure to the stress, suggesting that HSP production

depends on its current cellular concentration (Kregel 2002).

Focusing on the marine domain, studies about HSPs have been performed at several

levels, from cell lines and primary cultures of various cells to tissues of whole organisms

(Iwama 1998). It is acknowledged that when it comes to HSP expression, there are 3 important

temperature values: the inducing temperature (Ton), the temperature of maximal response

(Tmax) and the shut-off temperature (Toff) (Barua et al. 2004). These set the patterns of HSP

expression and depend on the species, tissue (Dietz and Somero 1993, Chang 2005, Yamashita

et al. 2010), acclimation temperature (Dietz and Somero 1992, Hofmann and Somero 1995,

Tomanek and Somero 2002) and evolutionary history of the organism (Tomanek and Somero

1999, Hofmann 2005). Moreover, most studies reveal a great plasticity on the HSR (Dietz 1994,

Buckley and Hofmann 2002), which is related to the thermal sensitivity of transcriptional

factors of the HSP genes (Buckley and Hofmann 2002, 2004). This sensibility results in response

plasticity dependent on the thermal history of the organism once the activity of transcriptional


50

factors is correlated with acclimation temperature (Buckley and Hofmann 2002). This enables

HSPs to be expressed accordingly to the occurring temperature and the cell’s needs,

depending on the amount of denatured or unstable proteins. This may be an adaptive

mechanism in order to prevent an excessive energy cost and excessive over-expression

because very high amounts of these proteins can be deleterious to cells (Feder et al. 1992,

Lindquist 1993, Feder and Hofmann 1999). Nevertheless, some other studies refer that there is

a lack of plasticity in HSR, and that it is more genetically fixed (see Barua et al. 2004). Hence,

further research is needed since ecological and evolutionary significance of the plasticity in

HSP gene expression remains partially unknown (Buckley and Hofmann 2002, Tomanek and

Somero 2002) and might be especially important when it comes to predicting the impacts of

climate change on natural populations (Barua et al. 2004).

Despite this, some authors have come forward about the physiological and ecological

significance of HSP. Hofmann and Somero (1995) refer that heat damage of proteins can

significantly impact the organism’s energy budget. Accordingly, the authors mention that

habitat temperature might cause an energetic cost due to the amount of energy necessary to

maintain the integrity of the protein pool (trough ubiquitin pathway, HSP rescue or de novo

synthesis). This cost may have metabolic and physiological impacts on the organisms, affecting

their growth, performance and fitness and thus the species abundance and distribution

patterns. Since organisms may be exposed to values of environmental variables outside their

tolerance window, HSP expression might be a mechanism with a selective value contributing

to the organism’s and species’ success across an environmental gradient (see Hofmann 2005).

HSPs are often seen as adaptations that are maintained via natural selection but this requires

both genetic intra-population variation and effects on individual fitness (Feder and Hofmann

1999). In general, it is stated that stress responses enable marine organisms to adapt or

acclimate to new conditions, being critical for the organism’s survival (Yamashita et al. 2010).

Furthermore, there is a growing body of literature in respect to the benefits of HSPs on

the organisms, especially focused on HSPs and acquired tolerance to extreme values of

environmental variables. Basu et al. (2002) refer that tolerance and cross-tolerance (“ability of

one stressor to transiently increase the resistance of an organism to a subsequent

heterologous stressor” – Basu et al. 2002) given by HSPs may be a critical trait of the stress

response considering the natural environment. It is so because when organisms are exposed to

one stress factor, their capacity to tolerate a subsequent stressor increases, improving their

capacity to cope with environmental change (Basu et al. 2002).

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51

In summary, our understanding of the stress response has been greatly improved through

HSP studies, although there are some points to further explore, in relation to their ecological

significance and historical variation in natural populations (Feder and Hofmann 1999). Once we

fully understand the molecular and physiological traits and responses, large scale patterns can

be explored and explained. Also, these proteins are considered biomarkers of cellular injury

(Iwama et al. 1998, Feder and Hofmann 1999, Deane and Woo 2010) and therefore can be

useful on immediate practical aspects such as biomonitoring and environmental toxicology.

The aim of this work was to test the intensity of one feature of the Heat Shock Response

through the quantification of HSP70 expression along a temperature gradient and at the upper

thermal limit in several taxa, ranging from various fish to shrimps and crabs. Inter-specific

comparisons of the amount of HSP70 and expression patterns were made in order to address:

1) Whether congeneric species have similar HSP70 expression patterns and temperatures of

induction, maximal production and shut-off;

2) Whether habitat features might influence the species’ mechanisms of resistance;

3) Which species might be more vulnerable to climate change, in particular climate warming.

Materials and methods

Species collection and acclimation conditions

Marine and estuarine organisms were captured during the summer months (from July to

September of 2010) in the Tagus estuary and adjacent coast (Portugal), at approximate

latitude of 38ºN (Northeast Atlantic). The capture was carried out using hand nets, dip nets,

beam trawling and beach seine.

The species collected are of commercial importance or play an important role in the food

web and can be divided in the following groups: the fish Diplodus vulgaris (N=19), Diplodus

sargus (N=25), Dicentrarchus labrax (N=19), Gobius niger (N=18) and Liza ramada (N=23); the

crabs Liocarcinus marmoreus (N=20), Carcinus maenas (N=30), Pachygrapsus marmoratus

(N=26); and the shrimps Palaemon longirostris (N=32), Palaemon elegans (N=34) and Crangon

crangon (N=18). Most of these species occur in cold and moderately warm waters (their


52

distributional range goes from temperate to subtropical latitudes). The distributional range of

most of these species is in both temperate and subtropical latitudes with the exception of

Crangon crangon, Palaemon longirostris and Liocarcinus marmoreus which occur mainly in

northern temperate regions. Some of the species are intertidal (Gobius niger, Carcinus

maenas, Pachygrapsus marmoratus and Palaemon elegans) while others can occur in marine

and brackish estuarine waters (Diplodus vulgaris, Diplodus sargus, Dicentrarchus labrax, Liza

ramada, and Liocarcinus marmoreus) or river and estuarine waters (Palaemon longirostris)

(Bauchot and Hureau 1986, Cartaxana 1994, Hayward and Ryland 1995, Hayward et al. 1996,

Ingle 1997, Flores and Paula 2001, Vinagre et al. 2010).

After capture, organisms were transported to the laboratory and placed in aquaria of 70L

with aerated sea water, a constant temperature of 24ºC (similar to their natural environment)

and salinity 35. The water dissolved O2 level varied between 95% and 100%. They were left

there to acclimate for two weeks, being fed twice a day.

Thermal tolerance method

Thermal tolerance of these species was determined using the dynamic method described

in Mora and Ospina (2001). The goal was to increase temperature until they reached the

Critical Thermal Maximum (CTM), which is defined as the “arithmetic mean of the collective

thermal points at which the end-point is reached” (Mora and Ospina 2001). This end-point can

be either loss of equilibrium or onset of muscular spasms.

During the trial, a thermostatized bath was used to increase water temperature at a

constant rate of 1ºC/h, stopping when the organisms reached their end-point. In each trial

individuals were tested simultaneously and observed continuously. All experiments were

carried out in shaded day light (15L:09D). Muscle samples from fish and haemolymph samples

from crab and shrimp were taken every other 2ºC or every other 4ºC depending on how many

individuals were captured for each species. Three individuals were sacrificed at each

temperature point for every species. Samples were also taken when animals reached their

end-points. In this case the number of individuals sacrificed varied between species depending

on sample size. Fish were killed by cervical transection and crustaceans, after haemolymph

collection, were placed in an ice-slurry immersion for 20 minutes, following the European Food

Safety Authority (2005) and National Aquaculture Council of Australia recommendations. The

shrimps were killed by splitting and crabs were killed by spiking, which is thought to cause

minimal pain (Baker, 1975).

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All samples were immediately frozen in liquid nitrogen and then stored in a regular

freezer at -20ºC.

HSP70 extraction and quantification

Samples from muscle were homogenized in 1mL of sodium phosphate buffer solution (pH

7.4) to extract the cytosolic HSP70 proteins, using a glass/teflon Potter Elvejhem tissue grinder.

Then they were centrifuged for 5 min at 13000 rpm. The supernatant was collected to new

eppendorfs and frozen immediately until further analysis. Haemolymph samples were

centrifuged as described previously without need for prior processing. Fish samples were

diluted 1:200 and crab and shrimp samples were diluted 1:100 in 0.05M carbonate-

bicarbonate buffer (Sigma-Aldrich, USA). Heat Shock Protein 70 was quantified through an

Enzyme Linked Immunosorbent Assay (ELISA) (Njemini et al. 2005) using 96 well microplates

(Nunc-Roskilde, Denmark). Three replicates of 50 µL were taken from each diluted sample,

transferred to the microplate wells and incubated overnight at 4ºC. The microplate was

washed (3X) in PBS 0.05% Tween-20 and then blocked by adding 200 µL of 1% BSA (Bovine

Serum Albumin, Sigma-Aldrich, USA). The microplate was incubated at 37ºC for 1h30min.

Aftermicroplate washing, the primary antibody (AM03140PU-N anti-HSPA8/HSC70, Acris USA)

was added to microplate wells (50µL each) after diluted to an appropriate concentration

(0.5µg/mL in 1% BSA). Then microplate was incubated for 1h30min. After a new washing step,

the secondary antibody (anti-mouse IgC, fab specific, alkaline phosphatase conjugate, Sigma-

Aldrich, USA) was diluted (1µg/mL in 1% BSA) and added (50 µL) to each well followed by

incubating the microplate for 1h30min. After the wash up step, a 100µL of substrate (SIGMA

FASTTM p-Nitrophenyl Phosphate Tablets, Sigma-Aldrich, USA) was added to each well and

incubated for 30 min at room temperature. Fifty µL of stop solution (3N NaOH) were added

and absorbance was read in a 96 well microplate reader at 405nm (BIO-RAD, Benchmark,

USA). For quantification porpuses, a calibration curve was constructed using serial dilutions of

purified HSP70 active protein (Acris, USA) to give a range from 2000 ng to 0 ng/mL.

The Bradford Assay was used to quantify the total amount of protein in each sample. The

analysis was carried out in 96well microplates (Nunc-Roskilde, Denmark) by adding 200 µL of

Bradford reagent in each well and 10 µL of each sample or standards. After 10 minutes of

reaction, the absorbance was read at 595nm in a microplate reader (BIO-RAD, Benchmark,

USA). A calibration curve was calculated using BSA standards.


54

SDS-PAGE

Only a few samples were chosen for this procedure in order to give complementary

information. Considering the total amount of protein calculated via the Bradford method,

different volumes correspondent to 20-30 µg of protein were taken from the samples and put

in eppendorfs. The same volume of sample buffer was added to the samples and the

eppendorfs were put in boiling water for 3-5 minutes in order to denaturate the protein. Then

the mixture was loaded into a 10% Laemmli (Tris-HCl) polyacrylamide gel and electrophoresis

was run for 70-85 minutes at 120V and 400mA. The procedure was performed following Mini-

PROTEAN® 3 Cell Assembly Guide, from BIO-RAD. When electrophoresis finished, gels were

stained with Coomassie Blue R-250 for 24h and then de-stained with a solution containing

glacial acetic acid, methanol and water.

Western Blot

Only a few samples were chosen for this procedure in order to give complementary

information regarding protein identity. Gels that were intended for western blot (one 7.5%

polyacrylamide and the other one 10% polyacrylamide) followed the procedure described

above but were not stained. Instead they were put in transfer buffer for 30 minutes. Before

assembling the electroblotting sandwich, sponges, filter paper and nitrocellulose membrane

were first put in transfer buffer as well but for 10 minutes only. Then, electroblotting sandwich

was assembled following MitoSciences Protocol and protein transference was performed for

2h at 150mAmp and 120V. Then, the membranes were blocked in a 5% solution of milk/PBS

for 3h and then washed in PBS 0.05% Tween-20 for 10 minutes. Membranes were then

incubated with diluted (1/1000 in 1% non-fat milk/PBS solution) primary antibody (anti-

HSPA8/HSC70, Acris, USA). Five mL were used per membrane and membranes were incubated

for 2h with constant agitation. Membranes were then washed 3 times (5 minutes each time) in

a PBS 0.05% Tween-20 solution. Following they were incubated with the secondary antibody

(anti-mouse IgC fab specific) diluted to 1/1000 in a 1% non-fat milk/PBS solution. Five mL were

used for each membrane and they were left incubating for 2h with constant agitation.

Membranes were washed again 3 times in a PBS 0.05% Tween-20 solution and then another 3

times in PBS solution to remove Tween-20. Finally the membranes were incubated in a

BCIP/NBT premixed solution (Sigma-Aldrich, USA) until satisfactory signal was achieved. The

reaction was terminated with 1mM EDTA (Riedel-de Haën, Sigma-Aldrich, Germany).

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Statistical analysis

Data was analyzed for normality and homocedasticity through Shapiro-Wilk and Levene’s

test, respectively. Depending on the result for the assumptions, a one-way ANOVA or Kruskal-

Wallis was performed for each species in order to detect significant differences in HSP

expression among temperature groups. When results were significant, the Tukey post-hoc test

was performed. The statistics were carried out using the Software Statistica (Version 8.0

StatSoft Inc., USA).

Results

During all the acclimation period and trials, the environmental variables which possibly

affect the results (e.g. oxygen levels, salinity, food, pH, photoperiod, and acclimation

temperature) were carefully monitored. Additionally, handling stress does not affect HSP levels

(Vijayan 1997) so it is believed that observed results are due to the influence of temperature.

Depending on the species, different ranges were obtained considering the average

amount of HSP70 produced, although control HSP70 levels were moderately elevated in the

species studied with the exception of Liocarcinus marmoreus (Fig. 1). Every species, except

Liocarcinus marmoreus, showed a somewhat high standard deviation, indicating a moderately

elevated intraspecific variability. Regarding fish, Liza ramada produced an average amount of

HSP70 (µg HSP70/µg total protein) that ranged from approximately 0.102 to 0.56; in Diplodus

sargus it ranged from 0.031 to 0.12; in Diplodus vulgaris it ranged from 0.027 to 0.037; in

Gobius niger it ranged from 0.064 to 0.107; and in Dicentrarchus labrax it ranged from 0.015 to

0.026. Considering crabs, in Carcinus maenas it ranged from 0.005 to 0.027; in Pachygrapsus

marmoratus it ranged from 0.009 to 0.09; and finally in Liocarcinus marmoreus it ranged from

0.002 to 0.917. At last, in shrimp species, the amounts of HSP in Palaemon longirostris ranged

from 0.005-0.017, in Palaemon elegans ranged from 0.141-0.328 and in Crangon crangon it

ranged from 0.011-0.041 µg HSP70/µg total protein (Fig. 1).

Considering the analysis of variance (for all cases α=0.05 and p<0.05 for significance) Liza

ramada (p<0.021), Diplodus sargus (p<0.014), Gobius niger (p<0.002), Crangon crangon

(p<0.05), Liocarcinus marmoreus (p<0.002) and Pachygrapsus marmoratus (p=0.003) showed

significant differences among temperature groups. Diplodus vulgaris (p>0.9), Dicentrarchus

labrax (p>0.08), Palaemon elegans (p<0.3), Palaemon longirostris (p<0.2) and Carcinus maenas

(p>0.07) showed no significant differences among the temperature groups.


56

Focusing on the patterns of HSP70 expression (Fig. 1), four trends were identified. A

general trend was identified among various species in which there is an increase in the amount

of HSP70 as temperature increases, followed by a slight or steep decrease close to the thermal

limits. Species with this kind of response are Liza ramada, Diplodus sargus, Pachygrapsus

marmoratus and Liocarcinus marmoreus. Another pattern identified, in Gobius niger, is the

occurrence of several increases and decreases in the amount of HSP70 produced along the

temperature gradient, with a final decrease close to the thermal limits. The third identified

trend occurs in Crangon crangon and it consists in an increase in the amount of HSP along the

temperature gradient without a decrease close to the thermal limits. Finally, Diplodus vulgaris,

Dicentrarchus labrax, Palaemon longirostris, Palaemon elegans and Carcinus maenas seem to

maintain HSP70 levels constant along the temperature gradient.

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Fig. 1 Levels of HSP70 along a temperature gradient ranging from the mean water temperature in summer to the critical upper thermal limit of each species. The value for each temperature

group is shown as mean±SD. Not all temperature groups are equally represented in all of the species due to variable sample size. a) Liza ramada; b) Diplodus sargus; c) Diplodus vulgaris; d)

Dicentrarchus labrax; e) Gobius niger; f) Carcinus maenas; g) Pachygrapsus marmoratus; h) Liocarcinus marmoreus; i) Crangon crangon; j) Palaemon longirostris; k) Palaemon elegans.


60

In regard to the gels and western blots, it is observable that there is some intraspecific

variability in the patterns and amounts of total (Fig. 2) as well as HSP70 (Fig. 4) protein

expression. Additionally, differences in the protein expression patterns in control versus

stressful conditions (at the upper thermal limit) are also clear (Fig. 3). In crustaceans, it is

visible a very high haemolymph protein concentration approximately between 97.0 and 66.0

kDa (Fig. 3). More specifically, in Carcinus maenas there also seems to be an under expression

of some proteins, in particular <45.0 kDa proteins but the blur between 97.0- 66.0 kDa seems

to be slightly increased, the same happening in Palaemon longirostris.

Fig. 2 SDS-PAGE for Liza ramada at 30ºC and upper thermal limit (38ºC) evidencing intraspecific variability within

each temperature group. a) Individual 1 expressing proteins at 30ºC; b) Individual 2 expressing proteins at 30ºC; c)

Individual 3 expressing proteins at 38ºC; d) Individual 4 expressing proteins at 38ºC.

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Fig. 3 SDS-PAGE evidencing differences in protein expression between temperature groups: controls versus upper

thermal limit in C. maenas and P. longirostris. a1) C. maenas control, a2) C. maenas upper thermal limit, b1) P.

longirostris control, b2) P. longirostris upper thermal limit.

Fig. 4 Western Blot for L. ramada evidencing intraspecific differences in HSP70 production at the upper thermal

limits.

In the second western blot (Fig. 5) two intermediate temperature groups were chosen

and we can see that at 32ºC, HSP70 seems to be more concentrated than at 28ºC. Also, one of

the individuals at 32ºC shows two bands of HSP70 while the others show only one band.


62

Fig. 5 Western Blot for D. labrax evidencing intraspecific differences in HSP70 as well as differences between

temperature groups. a) HSP70 for 2 individuals at 28ºC, b) HSP70 for 2 individuals at 32ºC.

Discussion

In this study we have uncovered the HSP70 expression patterns at increasing

temperatures for several temperate/subtropical species of different taxa. The analysis

comprised species from several habitats and also some congeneric species in order understand

the potential influence of phylogenetic/genetic versus habitat features in the heat resistance

mechanisms. Several trends were identified. In general, such trends support a correlation

between HSP70 expression and a gradient of temperature stress within an ecological relevant

range (in accordance with Basu et al. 2002).

We verified that almost every species showed relatively high levels of HSP70 in the

controls, which is in accordance with other studies referred in Iwama et al. (1998) review (e.g.

Misra et al. 1989, Koban et al. 1991, Yu et al., 1994). This may be explained by the fact that

HSPs perform chaperone functions both in unstressed and stressed cells. However, since

sampling was performed during summer, HSP production may suffer acclimation, so it is

possible that endogenous levels were higher in that season (Dietz and Somero 1992, Sanders

et al. 1992, Fader et al. 1994, Hofmann and Somero 1995, Basu et al. 2002). This may be an

adaptive mechanism to maintain native protein structures intact during the hottest season

conferring a mechanism of seasonal thermotolerance (Hofmann and Somero 1995).

There was also elevated intraspecific variability. This is in agreement with other studies

that mention that within a population or species the HSP expression or genes that determine it

may vary (see the review by Feder and Hofmann 1999) and have significant polymorphism

(Iwama 1998). This variation might be due to genetic variation among individuals, variable

number of copies of HSP genes and their organization in chromosomal loci (Ish-Horowicz and

Pinchin 1980, Leigh-Brown and Ish-Horowicz 1981, Feder and Hofmann 1999) as well as

environmental conditions, leading to differences in the amount of HSP70 expressed by

individuals when exposed to stressful conditions. The western blot for D. labrax also showed

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63

another trait in this variability, which is the 2 bands that represent different HSP70 isoforms, as

it has already been found in various animal cell types (Yamashita et al. 2010).

The gel results also showed that there was an over expression and sub-expression of

several proteins during stress. According to Teranishi and Stillman (2007) there are genes

upregulated during heat stress, generally involved with protein folding (such as HSPs), protein

degradation (for instance ubiquitin pathway), protein synthesis and gluconeogenesis,

suggesting that heat stress accelerates protein turnover. There are also genes downregulated

that are involved in detoxification, oxygen transport, oxidative phosphorylation and lipid

metabolism, suggesting the avoidance of ROS generation. Nevertheless, the under expression

at the upper thermal limits might also be related to the exhaustion of the biological system

and potential translational arrest, leading to a general downfall in protein synthesis and

integrity.

One of the main objectives of this work was to evaluate whether congeneric species have

similar HSP70 patterns and estimate the influence of genetic versus environmental features.

Considering the results obtained for congeneric species, the fish Diplodus sargus and Diplodus

vulgaris showed different amounts of HSP70: the first one had mean amounts of HSP70 higher

than the second. Statistical analysis revealed significant differences among treatment groups

in D. sargus but not in D. vulgaris. D. sargus has an HSP70 expression pattern characterized by

high amounts of HSP70 at 24ºC and 26ºC followed by a steep decrease at 28ºC and

maintenance of that level until upper thermal limit (33.82ºC – Madeira et al. unpublished) is

reached. D. vulgaris HSP70 levels were quite constant, although standard deviation and thus

intraspecific variability was much higher when temperatures were elevated, which may be due

to differences in individual responses. Also, due to a smaller sample size in D. vulgaris, samples

were not taken at all temperature groups and this may be masking the pattern. Despite this,

results make sense at the light of each species habits and distribution. In general, sea breams

maybe found in estuaries as juveniles (Vinagre et al. 2010) but then move to the continental

shelf area where they experience temperatures that round 15ºC to 18ºC in this region (Levitus

and Boyer 1994). D. sargus lives in relatively shallow waters while D. vulgaris can reach deeper

water masses (Bauchot and Hureau 1986), dealing with colder temperatures, so it may not

need as high amount of HSPs as D. sargus. Since deeper waters are more stable than shallower

ones, D. vulgaris may have a slower mechanism of reaction and could not respond as rapidly to

such sudden changes. In fact, this species’ response is in agreement with other case studies


64

(see Tomanek 2010) that indicate that most organisms inhabiting stable environments, in

general, do not respond with an inducible HSR. The lack of an inducible HSP70 expression upon

heat stress might be of future concern if we consider that sea temperatures continue to rise

due to climate warming, putting species at risk. Also, it is important to refer that upper

thermal limits are lower in D. vulgaris when comparing with D. sargus (Madeira et al.

unpublished) and this might actually be related to the amount of HSP70 expressed. This is in

accordance with distributional limits for each species because, although they are both

considered subtropical species, D. sargus has a continuous distribution along the west coast of

Africa, while D. vulgaris is absent from the warmer stretches thus indicating that this species is

not very resistant to heat, probably because it is adapted to colder and less variable

environments. In this case, HSP70 expression appears to be influenced by environment and

not genetically fixed, following the general acknowledge model (Feder and Hofmann 1999,

Basu et al. 2002, Hofmann 2005).

Taking into consideration Palaemon elegans and Palaemon longirostris we can see that

mean amounts of HSP70 were higher for P. elegans, which may be a species specific feature

but it also can relate to the fact that this species inhabits highly variable intertidal pools

(Hayward et al. 1996) which have changes of >20ºC (Tomanek 2010) while P. longirostris may

occur in rivers and estuaries which are moderately variable environments thus the lesser

magnitude of expression. Nevertheless, both species rely on the same pattern of expression to

deal with thermal stress. They maintain a high baseline level of HSP70 throughout the

temperature gradient. This may enable them to process a high amount of aberrant proteins

denaturated due to the very high temperatures their body experiences thus keeping the

integrity of the protein pool. This pattern is in concordance with other studies for resistant

species (Botton et al. 2006). HSP70 induction in P. longirostris may also be critical to its

reproduction because this species is catadromous (Cartaxana 1994, Barnes 1994, Paula 1998),

undergoing several temperature changes during migration. As such, high constitutive levels

may provide a means for this species resistance. Considering that the pattern of response is

similar in both species, we may infer that HSP70 production may be more genetically hard-

wired in this genus and hence may be correlated to phylogeny (see Dietz and Somero 1993).

This may also indicate a limited acclimatory plasticity, which in fact has been shown for species

from highly variable environments (Tomanek 2010), possibly making them vulnerable to

climate changes.

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65

Another two species following the pattern of constant levels along the temperature

gradient were C. maenas and D. labrax. Dicentrarchus labrax has a demersal life at relatively

high depths (Tortonese 1986; FAO 2011) but they also frequently occur in other warmer

habitats such as estuaries (Hayward and Ryland 1995). If the encountered stressor is found

with frequency it can take place desensitization towards that stressor leading to lesser

inducible responses (Reid et al. 1998), as it already has been shown for other stress factors

such as hypoxia (Ferguson et al., 1989; Currie and Tufts, 1997). Also, organisms can rely on

other defense mechanisms that confer thermotolerance (e.g. homeoviscous adaptation,

compensatory expression of isozymes and allozymes, induction of superoxide dismutase,

glutathione system and cytochrome P450 - Feder and Hofmann 1999).

Carcinus maenas is an eurythermal inhabitant of the intertidal zone (Hayward and Ryland

1995; Ingle 1997; Flores and Paula 2001). According to some authors intertidal organisms

frequently induce the HSR within the range of body temperatures they normally experience

therefore this response may be an effective biochemical strategy to live in this thermal niche

and deal with intertidal cycles (Botton et al. 2006, Tomanek 2010). A high constitutive level of

HSP70 may be the most energy saving strategy to deal with the extremes occurring in this

zone, preventing not only a high cost of protein damage but also the existence of a lag time

between stress exposure and response.

Results for Pachygrapsus marmoratus, which also inhabits the intertidal and mostly the

supratidal zone (Ingle 1997; Flores and Paula 2001), an extreme and highly variable

environment, showed a different pattern. While intertidal P. elegans and C. maenas lack

inducible HSP70, P. marmoratus showed not only higher mean amounts of HSP70 but also a 10

fold increase in HSP70 at 34ºC comparing to control levels, which might in fact explain the high

thermal resistance and the upper thermal limit determined for this species (35.73ºC) (see

Madeira et al. unpublished). The organisms occupying these niches undergo temperature

changes in the order of >20ºC (Tomanek 2010) so it is possible that their thermal history and

environmental conditions influence their thermal tolerance (Basu et al. 2002) and HSP70

production. Regarding the two species of crabs, Pachygrapsus marmoratus and Carcinus

maenas, the results are in accordance with the general statement of the Partnership for

Interdisciplinary Studies of Coastal Oceans (McClintock 2009) which report that HSP levels

increase with height on shore but responses differ between sites. As such, it is clear that

intertidal organisms may have common trends in HSP70 responses but they don’t necessarily


66

apply to all species; responses may vary according to littoral zonation, genetic features,

thermal history, etc. Although they have high baseline levels of HSP or have inducible HSP it is

relevant to acknowledge that they live closer to their thermal limits (Tomanek 2010) so an

additional sudden increase in temperature might make them vulnerable.

Other species following this same pattern of increase and decrease close to thermal limits

were Liocarcinus marmoreus and Liza ramada. Liocarcinus marmoreus had a very strong

response to temperature with a huge increase in HSP70 levels at 26ºC followed by a steep

decrease at 28ºC. Induction of HSP expression occurred at a relatively low temperature which

is in accordance with the fact that this species is a northern swimming crab with demersal

habits that can reach down to the depth of 200m (Hayward et al. 1996). Consequently, if

habitat temperatures are lower, the threshold for HSP induction occurs at lower temperatures

as well. Also, HSP70 production decreases very soon indicating that the capacity for HSP70

induction does not have a wide range in the temperature gradient, which might be concerning

considering a climate change scenario of increasing sea temperature. This species may then

rely in a single, strong induction in order to help maintain the protein pool before exhaustion

takes place and protein synthesis is stopped.

Liza ramada presented relatively high average amounts of HSP70 when compared to

other fish. Post-hoc tests revealed that treatment group of 34ºC had a significantly different

amount of HSP70 when comparing to treatment groups at the beginning of the thermal

gradient (28ºC) and at the end of the thermal gradient (36ºC). Both of these results were

expected because L. ramada is a very resistant species, with an upper thermal limit of 38ºC

(Madeira et al. unpublished). Additionally, this is a species that inhabits water masses of the

coastal shelf, as well as inshore waters like estuaries, lagoons and lower parts of the rivers

(Sauriau et al. 1994; Hayward and Ryland 1995), adapted to a diverse set of thermal

conditions, because these habitats are highly variable and reach elevated temperatures

(Madeira et al. unpublished) so HSP70 expression is probably a mechanism that enables L.

ramada to perform its migratory movements. HSP70 expression seems to have its onset at

approximately 30ºC and a temperature of maximal HSP70 expression at 34ºC which

corresponded to the expectations, because high habitat temperatures may set HSP induction

thresholds higher in the stress gradient. The temperature of shut-off is approximately 36ºC,

possibly due to high oxidative stress via ROS production (Tomanek and Zuzow 2010) and

exhaustion of the biological system. At the temperature of critical thermal maximum the

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67

amount of HSP70 seems to slightly increase but standard deviation is high so this might be due

to different levels of individual resistance/exhaustion. Since this is a highly resistant species

used to hot environments, the onset of response occurs further ahead in the thermal gradient,

which is in accordance with Buckley and Hofmann (2002) and Barua et al. (2004). This shows

that the correlation between threshold for heat shock expression and its maximal amount are

correlated with the levels of stress that the organism experiences in its natural environment,

which is in accordance with Hightower et al. (1999) and Feder and Hofmann’s (1999) review.

This also suggests that translation might have a specific upper thermal limit, according to the

thermal regime that the species experiences (Feder and Hofmann 1999).

G. niger and C. crangon expressed HSP70 in patterns different from all the other species

analyzed. Gobius niger occurs in estuaries, lagoons and inshore waters, which are hot and

moderately variable environments. However, HSP70 production didn’t follow the patterns

obtained for other species inhabiting these environments. The HSP70 levels were amongst the

highest considering the group of fish species studied. High amounts of HSPs might be

correlated with their high thermal tolerance of 34.10ºC (Madeira et al. unpublished), enabling

them to live in these hot environments. Statistic results show that there is a significant

decrease in the amount of HSP70 from 24ºC to 28ºC followed by a significant increase at 32ºC

and a subsequent significant decrease at the upper thermal limit. Therefore the expression

pattern for this species follows a series of increases and decreases in the amount of HSP70,

which may reflect the HSPs’ turnover rate.

Finally, results for Crangon crangon showed significant differences among temperature

groups, which did not happen for the other shrimp species (P. elegans and P. longirostris). A

gradual increase in HSP production takes place along the temperature gradient, following the

intensity of the stress and thus protein damage. HSP levels did not decrease at temperatures

close to the thermal limit therefore indicating that the organism may maintain some protein

synthesis functions at elevated stress levels perhaps due to other mechanisms of stress

defense (e.g. homeoviscous adaptation and high levels of ROS scavangers) protecting cell

integrity. Crangon crangon is a cold water demersal species but it also occurs in estuaries

(Boddeke 1989, Hayward and Ryland 1995, Hayward et al. 1996) so having an inducible HSR

through a 3.7 fold increase in HSP70 levels may be a way to cope with warmer and more

variable estuarine waters.


68

Conclusions

Our work has shown that there are several response strategies and patterns to increasing

temperatures, and that these strategies are mostly independent of taxa (fish, shrimp or crab),

upper thermal limits (for instance species with lower CTM like D. vulgaris showed the same

pattern as C. maenas which has a high CTM), and habitat type (subtidal species like D. vulgaris,

D. labrax and P. longirostris have similar patterns to intertidal ones like P. elegans and C.

maenas).

There seemed to be a correlation between habitat temperatures, upper thermal limits,

amounts of HSP70 and thresholds for induction and maximum production. For instance L.

ramada and G. niger are resistant species with high upper thermal limits living in variable

environments and they showed the highest mean amounts of HSP70 while less resistant

species such as D. vulgaris showed lower amounts. However, D. labrax is an exception because

it is somewhat resistant but showed HSP70 amounts lower than D. vulgaris. This may thus

indicate the genetic influence and phylogenetic factors affecting the HSP70 production. HSP70

amounts for species from the genus Palaemon also seem to correlate with habitat

temperature and variability, because the intertidal P. elegans has the highest amounts.

Considering crabs, we could not find any correlation. In relation to thresholds, species

inhabiting warmer habitats have set-points for induction at higher temperatures and vice-

versa. With all of this in mind, we must always consider not only environmental influence but

also multiple factors inherent to the species in order to understand the strategy of coping with

stress.

Finally, besides molecular defense mechanisms, organisms resort to behavioral (habitat

selection, mobility) and/or physiological strategies to deal with stress. These will certainly be at

play in the survival and adaptation of these species to future climate change.

Acknowledgements

Authors would like to thank everyone involved in the field work, maintenance of the experimental

tanks and in the feeding of the organisms. Authors would like to express their gratitude to “Aquário

Vasco da Gama” for the collaboration in the sampling and maintenance of organisms, in particular to Dr

Fátima Gil. In addition authors would like to thank Dept of Chemistry – Requimte from FCT-UNL, for

allowing the use of the equipments and materials and for the financial support given to this work. This

study also had the support of the Portuguese Fundação para a Ciência e a Tecnologia (FCT) through the

funding of projects and the grant SFRH/BPD/34934/2007 awarded to C. Vinagre.

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69

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CHAPTER 4

CHAPTER 4 – Final considerations

77

Final considerations

Marine organisms inhabiting coastal and estuarine waters are of great importance

because they are important food resources. Hence, their maintenance and integrity have

impacts on both economic and social levels. This study covered species from different taxa and

different trophic levels in order to give us a general picture of stress responses across a diverse

set of the ecosystem’s biological components. Moreover, marine ectotherms are very good

models to study the thermal stress response because they inhabit a medium with very high

temperature conductivity, which is reflected on their biochemistry, physiology and

biogeographic distribution. Thus, the study of thermal tolerance and HSP70 production

enables us to understand if species are resistant or vulnerable to potential temperature

changes and what mechanisms they use to cope with those changes.

This work determined the CTM of 16 species from a variety of taxa (including fish, crabs

and shrimps). This research showed that CTMs are similar for species inhabiting similar

habitats, probably due to the fact that they have evolved in alike abiotic conditions. CTM is

higher for species living in warm/unstable environments (e.g. supratidal, intertidal) or

migratory species and lower in species from cold/stable water habitats. Intraspecific variability

was generally low which also might explain why there was no local adaptation in CTM for wide

distributed species. This may point to a low plasticity in CTM, which is a matter of concern

considering the ongoing and future predictions for sea warming. Moreover, sea breams were

identified as potentially vulnerable to global climate change, in particular D. bellotti and D.

vulgaris due to their relatively low CTM and because they live at temperatures close to their

thermal limits, which also verifies for intertidal species. Also, since there is controversy on the

vulnerability of temperate versus tropical species, we analyzed intertidal species for which

there was comparable information. We concluded that intertidal fish species from

temperate/subtropical regions are more vulnerable than tropical ones because the former

may be subjected to maximum habitat temperatures that surpass their CTM.

To understand the molecular mechanisms behind thermotolerance and vulnerability,

HSP70 expression profiles were uncovered. The results were not as linear as for CTM. Although

one of the patterns (high baseline of HSP70 along a temperature gradient) was common in

some but not all intertidal species, the expression patterns identified were generally

independent from taxa, CTM and habitat type. Nevertheless the referred pattern also occurred

in non intertidal species.

CHAPTER 4 – Final considerations

78

There is a correlation between magnitude of expression, thermal conditions, CTM and

induction thresholds because organisms from warmer habitats present higher CTMs, higher

amounts of HSP and thresholds for induction occur at higher temperatures. In the littoral zone

it was acknowledged that HSP70 amounts are also higher for species inhabiting warmer

streches but response profiles might differ according to site and specific features. HSP70

results also point to an increased vulnerability of cold/stable water species (e.g. D. vulgaris,

Liocarcinus marmoreus) because they either lack and inducible HSR or they have a narrow

range in the temperature gradient for inducing it.

Molecular responses may undergo the influence of genetic versus environmental

conditions and this may in fact depend on the taxon. For instance, while Diplodus genus has a

response that seems to be influenced by environmental conditions, Palaemon genus results

point towards a more gentic/phylogenetic influence. Therefore, the pasticity of the response

may depend on environmental and specific features.

The present work has shown which species may be more vulnerable to climate changes

and how the molecular mechanisms may account for thermotolerance. It is believed that

climate change has impacts at several biologic organizational levels which, at the ultimate level

will lead to changes in the marine communities and ecosystems’ structures. The first steps in

understanding the causal-effect relationship between sea warming and ecosystem change

requires the study of thermal limits and molecular mechanisms behind the organisms’

vulnerability or resistance. However, this vulnerability/resistance may also depend on other

aspects such as exploitation, generation time, age at first maturation, reproduction strategy

(semelparous or iteroparous), adaptation capacity, endemism and rate of regional warming. As

such, the study of thermal tolerance should rely on a multidisciplinary approach, considering

and correlating biochemistry, physiology and ecology. This research followed this rationale and

contributed with relevant new information to the understanding of ecophysiological

processes. Additionally it can be useful in marine resources management, which is crucial in

countries highly connected to the sea, like Portugal.

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