Document 2010140

Integrating biochemical and growth responses in ecotoxicological assays with
copepods
Ulrika Dahl
Department of Applied Environmental Science (ITM)
Stockholm University
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Doctoral Thesis, 2008.
Ulrika Dahl
Department of Applied Environmental Science (ITM)
Stockholm University
S-10691 Stockholm
Sweden
© Ulrika Dahl
ISBN 978-91-7155-699-8
Printed by US-AB
Cover by Joakim Larsson,
including modified figures from Göte Göransson
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ABSTRACT
The understanding of effects of chemical exposure in nature is lagging behind.
Predictions of harmful effects of chemicals on aquatic organisms rely mainly on
ecotoxicity tests. To improve the understanding of the biological linkage between
the cellular and organismal responses to a chemical in an ecotoxicological test, the
major aim of this doctoral thesis was to investigate the usefulness of two
biochemical endpoints, contents of RNA and ecdysteroids, by incorporating them
with life-history traits of copepods (Crustacea). To do so, the two methods needed
to be established at our laboratory. Both biochemical methods are more commonly
used in basic biological research, but I here present its usefulness in
ecotoxicological testing. It was found that individual RNA content as a
biochemical endpoint was significantly altered in the brackish water harpacticoid
copepod Nitocra spinipes when exposed to the pesticide Lindane (paper IV) and low
concentrations (0.16µg . L-1) of the pharmaceutical Simvastatin (paper I) during
partial life cycle tests. However, the RNA content was insensitive as endpoint in
the fresh water harpacticoid Attheyella crassa during multigenerational exposure (2 –
3 generations) to naturally contaminated sediments (paper III). The second
biochemical endpoint, ecdysteroid content (a crustacean growth-hormone), was
shown to be a useful tool for ecotoxicological studies using N. spinipes (paper IV),
as well as for mechanistic understanding of lipid turnover and reproduction of the
marine calanoid copepod Calanus finmarchicus (paper V). In paper I and IV, I
present a balanced ecotoxicological test, useful for substances with suspected
developmental disruptive effects. In this type of test, a balance between test
adequacy, exposure time, and costs has been proven useful. Further, the reliability
of tests (paper II) with N. spinipes was increased by optimizing its food regime. In
paper II, 25 different combinations of micro-algae were tested during short- and
long time exposure and a suitable food source (Rhodomonas salina) was identified,
whilst poorer development and reproduction, malformations, and even mortality
was induced by other algae. In conclusion, my studies provide useful tools for
ecotoxicological testing, as well as for basic understanding of developmental
biology of different copepod species.
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ABSTRACT
INDEX
ABBREVIATIONS
LIST OF PAPERS
STATEMENT
AIM OF THE STUDY
The specific objectives of each paper
INTRODUCTION
BACKGROUND
1. Environmental stress
2. Fundamentals of ecotoxicological tests
TEST ORGANISMS
1. Crustaceans as test animals
1.1. Copepods
1.1.1. Harpacticoid copepods
1.1.1.a. Nitocra spinipes
1.1.1.b. Attheyella crassa
1.1.2. Calanoid copepods
1.1.2.a. Calanus finmarchicus
SOME BIOCHEMICAL GROWTH VARIABLES OF CRUSTACEANS
1. Contents of RNA and protein
2. Crustacean hormones – an overview
2.1. Ecdysteroids
ENDPOINTS AND BIOCHEMCIAL METHODS USED IN THE EXPERIMENTS
1. RNA content measurements
2. Ecdysteroid content measurements
2.1. Enzyme immunoassay of ecdysteroids
2.1.1. Antigens and antibodies
2.1.2. Immunoassays
3. Mean development time
4. Growth rate and somatic measurements
5. Population abundance
TEST SYSTEMS USED IN THE EXPERIMENTS
1. Acute toxicity tests
2. Life cycle tests
2.1. Partial life cycle tests
2.2. Full life cycles tests
2.3. Multigenerational tests
RESULTS AND DISCUSSIONS
1. RNA content and somatic growth
1.1. Partial life cycle exposure
1.2. Full life cycle exposure
1.3. Multigenerational exposure
2. Ecdysteroid content
3. A balanced ecotoxicological test
4. Mean development time
5. Sub-optimal conditions
CONCLUSIONS
FUTURE WORK AND PERSPECTIVES
ACKNOWLEDGEMENTS
REFERENCES
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ABBREVIATIONS
20E
CHH
CI
CIII
CIV
CV
CYP
DNA
dsRNA
E
EIA
ELISA
EPA
Fab
Fc
Fv
hnRNA
Hsp
IgG
ISO
JH
JHIII
MF
MIH
miRNA
MOIH
mRNA
NI
NVI
PoA
RIA
RNA
rRNA
SDS-PAGE
sFv
siRNA
SIS
snRNA
stRNA
tRNA
VIH
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20-hydroxyecdysone
Crustacean hyperglycaemic hormone
Copepodite stage one
Copepodite stage three
Copepodite stage four
Copepodite stage five
Cytochrome
Deoxyribonucleic acid
Double stranded ribonucleic acid
Ecdysone
Enzyme immunoassay
Enzyme-linked immunoassay
Environmental Protection Agency
Fragment antigen binding
Fragment crystallisable
Fragment variable
Heterogenous nuclear ribonucleic acid
Heat shock protein
Immunoglobulin G
International Organization for Standardization
Juvenile hormone
Juvenile hormone methylepoxyfarnesoate
Methyl farnesoate
Moulting inhibiting hormone
Micro ribonucleic acid
Mandibular organ inhibiting hormone
Messenger ribonucleic acid
Nauplii stage one
Nauplii stage six
Ponasterone A
Radioimmnoassay
Ribonucleic acid
Ribosomal ribonucleic acid
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Single chain fragment variable
Small interfering ribonucleic acid
Swedish Standards Institute
Small nuclear ribonucleic acid
Small temporal ribonucleic acid
Transfer ribonucleic acid
Vitellogenesis inhibiting hormone
LIST OF PAPERS
This doctoral thesis is based on the following papers, which are referred to in
the text by their roman numbers:
Paper I
Dahl U., Gorokhova E., Breitholtz M., 2006. Application of growth-related
sublethal endpoints in ecotoxicological assessments using a harpacticoid copepod.
Aquatic Toxicology, 77: 433-438.
Paper II
Dahl U., Rubio Lind C., Gorokhova E., Eklund B., Breitholtz M., (in press). Food
quality effects on copepod growth and development: implications for bioassays in
ecotoxicological testing. Ecotoxicology and Environmental Safety (2008).
doi:10.1016/j.ecoenv.2008.04.008.
Paper III
Gardeström J., Dahl U., Kotsalainen O., Maxson A., Elfwing T., Grahn M.,
Bengtsson B.-E., Breitholtz M., 2008. Evidence of population genetic effects of
long-term exposure to contaminated sediments – a multi-endpoint study with
copepods. Aquatic Toxicology, 86: 426-436.
Paper IV
Dahl U., Breitholtz M., 2008. Integrating individual ecdysteroid content and
growth-related stressor endpoints to assess toxicity in a benthic harpacticoid
copepod. Aquatic Toxicology, 88: 191-199.
Paper V
Hansen B.H., Altin D., Hessen K.M., Dahl U., Breitholtz M., Nordtug T., Olsen
A.J., 2008. Expression of ecdysteroids and cytochrome P450 enzymes during lipid
turnover and reproduction in Calanus finmarchicus (Crustacea: Copepoda). General
and Comparative Endocrinology, 158: 115-121.
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STATEMENT
I, Ulrika Dahl, was involved in the following parts of the present papers:
Paper I, I was responsible for all laboratory work as well as for writing of the
paper. I performed all analyses (somatic, biochemical, and mathematical).
Paper II, I was responsible for the first life cycle test and involved in the
second two tests, mainly by supervising Charlotta Rubio Lind, a master thesis
student and co-author, but also by maintaining the second life cycle test on all
weekends. I performed the biochemical analyses. Additionally, I was
responsible for writing the paper.
Paper III, I was involved in the experimental set up and at both sampling
occasions of the experiment, as well as maintenance of the animals during the
experiment. I was responsible for supervising Anders Maxson, a master thesis
student and co-author, in teaching him biochemical analyses. I was involved in
writing the paper.
Paper IV, I was responsible for establishing the enzyme immunoassay (EIA)
in our laboratory. I was further responsible for the experimental set up, when
investigating usefulness of the EIA, and I performed all the laboratory work,
both during the partial life cycle experiment as well as all analyses (somatic,
biochemical, and mathematical) of the animals. I was responsible for writing
the paper.
Paper V, I was responsible for the ecdysteroid analyses. I was involved in
writing the paper.
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AIM OF THE STUDY
The major aim of the thesis was to incorporate biochemical responses with
copepod life history traits to improve our understanding about the biological
linkage between the cellular and organismal responses to stressors. This was
performed by laboratory exposure of copepods to single chemicals and natural
sediments.
A secondary aim was to establish two methods to detect biochemical responses
in copepods (Crustacea), i.e. fluorescence detection measurements of total
RNA and ecdysteroid content, in individual copepods with diminutive biomass
(1 – 2µg dry weight).
The specific objectives of each paper were:
Paper I: To investigate four growth-related endpoints (development time,
growth rate, body length, and individual RNA content) for their usefulness in
ecotoxicological tests using the copepod Nitocra spinipes exposed to low
concentrations of the pharmaceutical Simvastatin.
Paper II: To increase the reliability of tests with N. spinipes, by finding suitable
algal food for laboratory testing. This was performed by the use of the growthrelated endpoints investigated in paper I. N. spinipes were exposed to 25
different combinations of micro-algae during a number of screening tests and
two full life cycle tests.
Paper III: To increase the ecological realism of the tests by using natural
sediments collected from polluted and clean sites, and for exposure times
covering several generations. Cephalothorax length and RNA content of
individual Attheyella crassa were measured and integrated together with
population dynamic and genetic endpoints.
Paper IV: To establish a protocol for a enzyme immunoassay (EIA) for
analysis of ecdysteroid levels in individual N. spinipes. The ecdysteroid content
was further investigated for its usefulness in relation to the other growthrelated endpoints, presented in previous papers.
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Paper V: To investigate ecdysteroid involvement together with cytochrome
P450, in reproduction and lipid storage consumption of Calanus finmarchicus
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INTRODUCTION
The number of chemicals is increasing rapidly, as is their use, but the
understanding of how they affect the nature is lagging behind. In
ecotoxicology, which is the knowledge about ecology in the presence of
toxicants (Chapman, 2002), a major aim is to study potential anthropogenic
impact on the environment. This may e.g. be performed by the use of shortterm acute toxicity tests (described below) on individual organisms (Calow and
Forbes, 2003), and an estimate of a safety level may be provided by dividing
the effect concentration by so called safety factors (Calow, 1992; European
Commission, 2003). Acute toxicity tests, however, do not give information
about effects on long-term life history traits, such as reproduction (Schindler,
1987; Bechmann, 1994), and there is consequently a need for test methods that
include more of ecological realism for ecotoxicological assessment of
chemicals (Calow et al., 1997). For a thorough understanding of stress
responses, it is additionally wise to study test organisms on more than one level
of biological organization (Heckmann et al., 2008), e.g. by adding cellular
responses to the life history responses (Korsloot et al., 2004), such as growthrelated responses of RNA contents (Dahlhoff, 2004) or hormone levels (Block
et al., 2003). These measured responses are more commonly used in basic
biological research of crustaceans, and I here present their usefulness in other
areas, such as ecotoxicological testing.
BACKGROUND
1. Environmental stress
Stress is often defined as an environmental change that could lead to
alterations in community structures and biological functions of an organism
(e.g. reproduction and growth) (Korsloot et al., 2004). The concept of stress is
however not absolute, it should be defined with reference to the normal range
of ecological function of a species (van Straalen, 2003), meaning that what
could be extremely stressful for one organism may be the normal for another.
There are two major causes of cellular stress - endogenous factors (e.g.
pathogens) and environmental factors (e.g. physical, such as heat, cold, osmotic
conditions, or chemical, such as heavy metals, organic chemicals). Chemical
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factors may disturb the organism (i.e. cause cellular stress) by transferring
incorrect signals within the cell, or by penetrating the cell (Korsloot et al.,
2004), leading to responses such as enzyme inhibition, interaction with
receptors, macromolecules, or organelles (Timbrell, 2001).
2. Fundamentals of ecotoxicological tests
The foundation of an ecotoxicological test is reliability, repeatability, sensitivity and
relevance, of which the three former are strongly related to the statistical power of
the experiment. That is, if the number of replicates is high, it is more likely to be a
reliable, repeatable, and sensitive test method. For example in life cycle tests, the
number of individuals will often diminish during its course. This may be a result of
uneven sex ratios, decreased survival, and unsuccessful fertilization, which in the
end could lead to a poor statistical power of the experiment if the starting number
of individuals is not high enough (Breitholtz et al., in press). At the same time, the
number of replicates may be restricted by too heavy workload among
experimenters, inadequate equipment and/or lack of time and money. However, if
the test method is insensitive for the chosen endpoint, there will be no or little
response no matter how many replicates is used.
With this in mind, a test system is reliable if the researcher can assure that a e.g.
chemical-induced response of a test animal is not a false positive (e.g. a
reflection of additional pressure on the organisms and thus an interactive
product of wanted and unwanted stressors), or a false negative (often a
function of too few replicates in the test system). Repeatability means that the
test should be able to be repeated with an acceptable variation (Environment
Canada, 1999). In addition to these prerequisites, an ecotoxicological test also
needs to be sensitive. The meaning of sensitivity is twofold; i) the test should have
such sufficient statistical power that even a rather small effect is revealed (e.g.
Forbes et al., 2001; Breitholtz et al., 2006), and ii) the test needs to focus on the
most fragile life stages, such as juvenile development and/or reproduction of
the tested organism (e.g. US EPA, 1992; Medina et al., 2002), or else ecologically
important endpoints may be missed. Finally, a test also needs to be relevant,
which means that i) the test is appropriate for measuring the area that is in
need of protection (e.g. an ecosystem, a population, a receptor within a cell),
and ii) the test is appropriate for the measurement of a potential hazard in the
environment (e.g. Solomon et al., 1996; Calow, 1998). Hence, researchers that
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develop new test methods need to consider a range of factors related to the
quality of the actual testing, and at the same time calculate if the tests are
performed at a reasonable cost (e.g. Hanson and Rudén, 2006 a; b), something
which has also been implicated in the present thesis.
TEST ORGANISMS
1. Crustaceans as test animals
Risk assessment of chemicals has to consider millions of species, with
extremely diverse morphology, physiology etc. Invertebrates comprise
approximately 95% of all known animal species in the environment (deFur et
al., 1999). They have unique physiological characteristics, and are often crucial
components of aquatic as well as terrestrial ecosystems. Crustaceans are the
second largest subphylum after the insects, comprising about 42,000 described
species, and they are common in both fresh and sea waters (Ruppert et al.,
2004). The enormous morphological and ecological heterogeneity includes
animals less than a millimetre in length (such as copepods), as well as giant
spider crabs with a leg span of 3m.
1.1. Copepods
The word copepod originates from the Greek words kope (an oar) and podos (a
foot), referring to the swimming legs of the animals. They are consumed by a
vast variety of invertebrates, as well as fish and whale species (Mauchline,
1998). The copepod body is generally small (0.5 – 2mm), however, in
occasional deep sea samples copepods have been recognized with a body
length of 18mm (Owre and Foyo, 1967). Copepod species have paired
appendages that function for swimming, detection of food, and mating. It is
possible to distinguish between males and females by sexually dimorphic
characteristics, usually emerging during the later stages of the development.
Additionally, the males are usually smaller than the females. With few
exceptions, the copepods reproduce sexually (Gilbert and Williamson, 1983),
and the copulation often involves the attachment of a sperm sac
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(spermatophore) by the male to the copulatory pore of the female (Huys et al.,
1996). The eggs of a copepod may be carried by the female or dispersed into
the free water column (Mauchline, 1998). The copepod usually develops
through six naupliar stages (NI to NVI) followed by five copepodite stages (CI
to CV). As other arthropods, they increase their body size by moulting, which
consists of post moult, inter-moult and pre-moult stages, the new exoskeleton
forming under the old one (Mauchline, 1998). Between the last naupliar stage
and the first copepodite stage, the copepods usually undergo considerable
metamorphosis (Gilbert and Williamson, 1983).
There is little doubt that copepods, with 12,000 described species (Ruppert et
al., 2004) are among the most abundant metazoan (i.e. multi cellular animal)
creatures on our planet (Fryer 1986, reviewed in Hopcroft and Roff, 1998;
Mauchline, 1998; Miller and Harley,. 1999). Since the copepods are of great
significance as prey for young and adult stages of ecologically and economically
important species of fish (e.g. Westin, 1970; Aneer, 1975; Sundby, 2000;
Skreslet et al., 2005), they are important to protect, in order to maintain stable
aquatic ecosystems. Copepods are also suitable in the laboratory as model
invertebrates in ecotoxicological tests, in order to find potential effects of
chemicals (e.g. Andersen et al., 2001; Breitholtz et al., 2003; Karlsson et al.,
2006), as well as complex matrixes such as oil, effluents or contaminated
sediments (Green et al., 1996; Bejarano et al., 2006). Due to their relatively short
generation times, it is feasible to study them for full life cycles, which includes
sensitive life events such as juvenile development and sexual reproduction.
1.1.1. Harpacticoid copepods
Harpacticoid copepods are usually benthic organisms (Rupert et al., 2004). Hatched
from an egg, NI is unsegmented with three pairs of appendages. The appendages
develop at each moult by the addition of setae (hair-like parts on limbs and mouth
parts) and/or segments (Huys et al., 1996). Rudimentary forms of other
appendages and additional body segments (somites) also develop during the
development of the nauplius stages. An adult bears six pairs of appendages and
consists of ten somites (Huys et al., 1996). The main food source is organic material
and presumably also the micro-biofilm associated with it (e.g. Dole-Olivier et al.,
2000).
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1.1.1.a. Nitocra spinipes
N. spinipes is commonly found in the benthic meiofauna. It is found in shallow
waters worldwide, including the Baltic Sea (Lang, 1948; Noodt, 1970; Wulff;
1972). It reaches sexual maturity within 10 – 12 days, and it has a total
generation time of 16 – 18 days at 20°C. The adults are less than 1 mm long
(~0.75mm for the female and ~0.45 – 0.56mm for the male; Abraham and
Gopalan, 1975). Since it has a good ability to adapt to salinity fluctuations (0 –
30‰) and temperature (0°C – 26°C) (Noodt, 1970; Wulff, 1972), it is easy to
keep in the laboratory and is useful in various kinds of experiments. It has
been used as a test species for 60 years (Barnes and Stanbury, 1948), and it has
been in use for toxicity testing in our lab since 1975; Bengtsson (1978)
developed a lethal toxicity test, which has been established as Swedish (SIS,
1991), Danish and International Standards (ISO, 1997). Further, N. spinipes has
successfully been used as a test organism in chronic tests (e.g. Breitholtz and
Bengtsson, 2001; Breitholtz et al., 2003; Ek et al., 2007).
1.1.1.b. Attheyella crassa
A. crassa is a fresh water species, belonging to Canthocamptidae, the most species-rich
family of harpacticoid copepods in fresh waters (Dole-Olivier et al., 2000). It is
found all over Europe, in North Africa, and in Asia in a wide variety of habitats
and seems to prefer muddy substrates and responds positively to eutrophication
(Dole-Olivier et al., 2000). It has a generation time of 6 – 8 weeks when cultured in
the laboratory at 20 – 20.6ºC (Sarvala, 1977). The body length of a newly hatched
nauplius is 0.076 – 0.079 mm (Sarvala, 1977). The adults are sexually dimorphic
and their average adult body length 0.65 mm (Enckell, 1980).
1.1.2. Calanoid copepods
The calanoid copepods can be found in pelagic and benthopelagic regions, as well
as in coastal, shelf and oceanic waters (Mauchline, 1998). They are omnivores, and
may feed on particles (e.g. microalgae) of a few microns in size (Poulet, 1983).
Many calanoids produce diapause eggs, i.e. resting eggs, in which growth and
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development are suspended and physiological activity is diminished to often nondetectable levels. Those eggs sink to the seabed (usually occurring at high
population densities or seasonal variations [Ban and Minonda, 1994]), and may be
viable for up to 40 years (Marcus et al., 1994). Many calanoids have lipid storage, of
which shape and position differ between different species (Sargent and Henderson,
1986).
1.1.2.a. Calanus finmarchicus
C. finmarchicus has been found in oceanic waters from e.g. Norway, Greenland, and
the Barents Sea towards Russia (Hirche and Kosobokova, 2007). It may, represent
up to 90% of the entire zooplankton biomass in the Barents Sea during summer
(Sakshaug et al., 1994), and they are an important component of the North
Atlantic food web (Planque and Batten, 2000). One generation takes 30 – 40 days
and the adults are 2 – 5 mm long (Ruppert et al., 2004). They are rich in lipids and
fatty acids and are therefore high quality food for many fish species (Sundby, 2000;
Skreslet et al., 2005). C. finmarchicus is able to hibernate without feeding for up to six
month during winter time (Mauchline, 1998).
SOME BIOCHEMICAL GROWTH VARIABLES OF CRUSTACEANS
1. Contents of RNA and protein
The use of RNA as an indicator of growth in different species has been of
interest for a long time. Sutcliffe (1965) proposed more than 40 years ago
that the RNA content could be an estimate of growth in small copepods.
Identification of biochemical changes, such as RNA content (Dahlhoff,
2004), can be used to determine if an organism has been exposed to a
stressor, including environmental pollutants (Feder and Hofmann, 1999;
Yang et al., 2002). The rationale is based on the fact that the RNA content of
tissues or whole organisms consists primarily (~80 – 90 %) of ribosomal
RNA (rRNA) (Alberts et al., 1983), which is the most stable class of RNA,
whereas tRNA represents about 10 – 20%, and mRNA accounts for less then
10% (e.g. Becker et al., 2000), together with small RNA´s (e.g. hnRNA,
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miRNA, siRNA, snRNA, dsRNA and stRNA; Dreyfuss, 1986; Downward,
2004; Matzke and Birchler, 2005; Grennan, 2005). Taken together, levels of
rRNA, at any given time, are directly related to the protein synthesis of a cell,
and thus to the growth of the individual (Elser et al., 2000). For example, in
small metazoans, which have high metabolic rates of biosynthesis, a high
portion of rRNA is required (Brown et al., 2004). Indeed, nucleic acid content
may be an advanced indicator to evaluate the condition and growth rates of
copepods (e.g. Saiz et al., 1998; Gorokhova, 2003).
Toxic chemicals may bind to molecules, such as nucleic acids, lipid
membranes, hormones and proteins, and either destroy or change their
structures, which in turn may lead to physiological dysfunctions. On the other
hand, this could also result in a protective response of the cell (e.g. stress
protein induction). Stress proteins differ between phyla, but are highly
conserved within taxa through evolution (Korsloot et al., 2004). They are a
ubiquitous family of proteins, present within cells at constitutive levels. During
unstressed conditions, they are participating in protein folding and assembling,
metabolic processes, as well as in cell growth and development (Lindquist,
1986; Ellis and van der Vies, 1991; Elefant and Palter, 1999). Upon exposure
to stress, the production of stress proteins is induced to perform different
functions against cellular damage. This may be performed by e.g. assisting in
reparation of damaged proteins, or degradation of abnormal folded proteins
(Feder and Hofmann, 1999; Korsloot et al., 2004). By doing so, they are
minimizing the risk of proteins with impaired functionality to further engage in
synthetic or regulatory processes (Feder and Hofmann, 1999; Becker et al.,
2000; Tomanek and Somero, 2002) and thus minimizing the cellular damage.
At the same time, normal functions are halted, and the energy is channelled
into surviving and homeostasis restoration (Korsloot et al., 2004).
2. Crustacean hormones – an overview
Hormones regulate physiological processes in invertebrates as well as
vertebrates. For an outline of the crustacean endocrine system, see Figure 1.
Even though the crustacean endocrine system regulates many processes also
seen in vertebrates (i.e. reproduction, growth, development), there are some
endocrine processes that are unique (i.e. moult, diapause).
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The reference information for the endocrine system of crustaceans is mostly
based on the larger crustacean species. In crabs, for instance, the sinus gland (a
neurohemal organ which contains the nerve endings of the X-organs) acts as
storage and release site for neuropeptides, which are synthesized in the eyestalk
(Liu et al., 1997). The X-organ/sinus gland complex is responsible of the
regulation for important physiological functions, such as reproduction,
moulting, metamorphosis, pigmentation, and metabolism (Homola and Chang,
1997). The neuropeptides includes hormones, such as moulting inhibiting
hormone (MIH), vitellogenesis inhibiting hormone (VIH), crustacean
hyperglycaemic hormone (CHH), and mandibular organ inhibiting hormone
(MOIH) (Coast and Webster, 1998). The Y-organs synthesize the ecdysteroids
(Subramoniam, 2000). Y- and X -organs control ecdysis (moult) as follows: in
absence of appropriate stimuli, the X-organs produce MIH, which is released
by the sinus gland. The target of MIH is the Y-organ. At high titers of MIH,
the Y-organ is inactive. Under internal or external stimuli, MIH release is
inhibited and the Y-organ releases ecdysone (E) followed by ecdysis (Miller
and Harley, 1999). It should however be mentioned that even though it is
generally established that MIH plays a crucial role in Y-organ ecdysteroid
genesis regulation, the endocrine control of the Y-organs is probably far more
complex (Lee et al., 2007).
It is well known that insect moult and reproduction are under control of
juvenile hormones (JHs) (Lomas and Rees, 1998; Lafont, 2000), and JH and its
analogues have also been observed to have some reproductive effects in
crustaceans (Homola and Chang, 1997). In crustaceans, it is believed that
methyl farnesoate (MF) fulfils similar functions as those of JHs in insects, but
it is also possible that MF has novel functions (Homola and Chang, 1997). MF
is structurally related to the JHIII of insects (Huberman, 2000), and it has been
implicated in crustacean reproduction and development (Borst et al., 1987).
Tamone and Chang (1993) provide direct evidence for a stimulatory effect of
MF on ecdysteroid secretion from crab Y-organs in vitro (Figure 1). They
conclude that it is not surprising that growth is not only regulated by inhibitory
factors such as MIH, but also by stimulatory factors such as MF. However, the
results are contradictory, since Mu and LeBlanc (2004) report that MF has an
inhibitory effect on ecdysteroid production. Again, this reflects that the
crustacean endocrine system is hardly as simple as presented in Figure 1, and
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that there are still knowledge gaps in the understanding of pathways between
crustacean hormones.
X--organ/sinus gland
storage for neurosecretory products
mandibular organ
inhibiting factor
-
+/--
mandibular organ
methyl farnesoate secretory organ
MIH
Y-organ/moulting gland
ecdysone seccretory organ
MF
acting as a JH
MF
?
+
20E
target
target tissues
tissues
ecdysis
ecdysis
Figure 1. Outline of the crustacean endocrine system. Revised version based on
deFur et al., 1999. Abbreviations: MIH – moulting inhibiting hormone; MF –
methyl farnesoate; 20E – 20-hydroxyecdysone; JH – juvenile hormone.
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2.1. Ecdysteroids
The ecdysteroids are a group of polyhydroxylated ketosteroids (392 isoforms
identified so far; Lafont et al., 2008) essential for moulting (Charlisle and
Dohrn, 1953; reviewed in Fingerman, 1987) and reproduction (Subramoniam,
2000). Being a precursor of ecdysteroids, cholesterol is the dominant sterol in
crustaceans (Goad, 1981). Yet, crustaceans are not able to synthesize
cholesterol de novo (Goad, 1981), which means that the animals are in need of
dietary intake of cholesterol.
As mentioned above, post-embryonic crustaceans have to moult, i.e. renew their
exoskeleton, in order to grow. The Y-organs release E into the heamolymph where
it is converted into the active hormone 20-hydroxyecdysone (20E) by
hydroxylation (LeBlanc et al., 1999). Additional suggestions for ecdysteroid
production centers (in arthropods) are the ovary and the epidermis (Delbecque et
al., 1990; reviewed by Subramoniam, 2000). It seems that the ecdysteroids circulate
freely and enter cells by diffusion (Huberman, 2000). At least two other
ecdysteroids are additionally released by the Y-organs: 3-dehydroxyecdysone and
25-deoxyecdysone, where the latter is a precursor to the active ponasterone A
(PoA; 25-deoxy-20-hydroxyecdysone) (Lachaise et al., 1989; Spaziani et al., 1989;
reviewed by Subramoniam, 2000). The circulating titers of ecdysteroids vary along
the moult cycle in larger crustaceans (reviewed in Chang, 1993). Immediately after
ecdysis, the titer is low and generally remains so during intermoult. A major
increase occurs at beginning of premoult, followed by a steep drop just before the
actual moult.
ENDPOINTS AND BIOCHEMICAL METHODS USED IN THE
EXPERIMENTS
1. RNA content measurements
Individuals were randomly selected for analysis and preserved in RNAlater for no
longer than 3 months. RNAlater is a tissue storage reagent, used to permeate tissue
for stabilization and protection of RNA from RNAse attacks (RNAse is a nuclease
that catalyses hydrolysis [i.e. breakdown] of RNA). Extraction with N20
laurylsarcosine (i.e. a detergent to solubilize membranes) in ice cold (to ensure
minimal RNA breakdown) ultrasonic bath was followed by loading of plates.
Fluorescence detection of total contents of RNA and DNA was performed by
RiboGreen labeling. RiboGreen is a dye used for detection and quantification of
nucleic acids. In its free form it exhibits a little fluorescence, but at binding to
nucleic acids its fluorescence is several orders of magnitude greater than that of the
unbound form. RNAse digestion of RNA ensured only DNA to remain in the
microplate wells; hence after a second detection, a subtraction between the two
detections resulted in total RNA content. The endpoint of RNA content, which
was used in paper I, II, III, and IV, was measured in individual copepods at stage
CIII. CIII was used since it has been shown to be the copepodite stage with the
lowest within-stage variability (paper III).
2. Ecdysteroid content measurements
Individuals were randomly selected for analysis and preserved in ice cold
methanol. Extractions with pellet pestle tissue grinder and ice cold incubation
were followed by removal of supernatants and additional extractions with ice
cold methanol - water suspension. Fc-specific goat anti-rabbit IgG-coated high
binding plates were loaded with extractions and standards, together with anti20E rabbit polyclonal antisera and 20E-horseradish peroxidase conjugate, and
a competitive reaction equilibrated before QuantaBlu Fluorogenic Peroxidase
Substrate labeling and fluorescence detection. Individual content of
ecdysteroids were used in paper IV and V. In paper IV, the copepods were
measured at stage CIII for comparison with the RNA content of animals
sampled in the same test. In paper V, copepods at different life stages (i.e. CV,
females with small developing gonads, females with fully developed gonads,
post-spawning females with no or few visible eggs, and males) were analysed as
blind samples.
21
2.1. Enzyme immunoassay of ecdysteroids
2.1.1. Antigens and antibodies
In the type of studies presented in the current thesis, an antigen is the molecule
under investigation, in this case the ecdysteroids. Immunization, i.e. utilizing a host
animal’s (often a goat, rabbit, or sheep) immune response, is performed by
injection of an immunogen, followed by bleeding for antibodies from the host.
An immunogen is a molecule capable of eliciting an immune response when
injected (i.e. an antigen is capable of binding to an antibody, not necessarily to elicit
an immune response [Wild, 2001; Eales, 2003]). The host starts to develop
antibodies against the immunogen (Wilson and Walker, 2000). Antibodies are a
group of globular proteins known as immunoglobulins, produced by plasma cells,
circulating throughout the blood and the lymph, where they bind to the antigens
(Becker et al., 2000).
When raising antibodies, there is a number of things to consider, e.g. the size of the
immunogen should preferably be larger than 2 kilodaltons (Wild, 2001), and the
length of exposure needs to be during a certain time length (in order to prolong
the exposure of the immunogen in vivo, it may be administrated with an adjuvant
[i.e. a depot of immunogens, which slowly are secreted into the host]) (Wilson and
Walker, 2000). Further, by nature, immunogens are normally proteins and
polysaccharides (Coico et al., 2003), but e.g. lipids and nucleic acids can also be
immunogenic (Wild, 2001). The immunogen also needs to be recognized by the
host as foreign; generally, the greater the phylogenetic difference, the better
(Wilson and Walker, 2000).
The part of an antibody that recognizes an antigen is called paratope, and the part
of the antigen that binds to the paratope is called epitope. An epitope has no
intrinsic property; it is only defined by its binding to the antibody (Harlow and
Lane, 1999). An antigen can also have several epitopes. The epitope and paratope
interactions are non-covalent and reversible; binding involves the establishment of
multiple hydrogen bonds, ionic bonds and van der Waals attractions (Harlow and
Lane, 1999). The epitopes on protein molecules may be either contiguous or noncontiguous amino acid sequences (Wild, 2001). Antibodies recognize relatively
small regions of an entire antigen, and occasionally they find related structures on
other molecules, i.e. cross-reactivity (Coico et al., 2003). Cross-reactivity is helpful
22
in finding related protein family members, but also distracting when they recognize
unrelated proteins with a shared structural feature.
Affinity is a measure of the strength of the binding of one epitope to one
paratope. The time to reach binding equilibrium depends on diffusion (Wilson and
Walker, 2000), but the time to reach equilibrium is also affected by temperature,
pH, ionic strength and solvents used (Harlow and Lane, 1999). Since many
antibodies usually are di- or multivalent, and antigens usually have more than one
epitope (Coico et al., 2003), this concept may be quite complex. Avidity is a
measure of the overall stability of the antibody – antigen complex, governed by e.g.
intrinsic affinity between paratope and epitope, and the geometric arrangement of
the interacting components (Wilson and Walker, 2000; Coico et al., 2003). One way
of increasing the avidity is to use two or several antibodies for the same antigen (i.e.
polyclonal antibodies). The polyclonal antibodies have generally less affinity, but
higher avidity.
Polyclonal antibodies are a heterogeneous mixture of antibodies of varying
binding affinities against the epitopes, and also with different specificities
recognizing different epitopes (Coico et al., 2003). The antibody profile of each
bleed of an individual host animal will change, which means that the health
condition of the host animal also has to be taken into consideration. The timing
and amount of immunogen injections into the host is important. Monoclonal and
recombinant antibodies are derived in vitro, and thus show homogenous
characteristics. There may be an advantage in using these antibodies if they are
carefully selected and characterized (Wilson and Walker, 2000; Wild, 2001), since
they have high specificity towards the chosen antigen. There are however
drawbacks, such as the fact that these antibodies may be specific for a particular
epitope, not necessarily the whole molecule (Wilde, 2001) and that the costs are
usually high.
There are at least six different types of antibody fragments used in immunoassays
(fragment antigen binding [Fab and Fab’], fragment crystallisable [Fc], fragment
variable [Fv], single chain fragment variable [sFv, sFv-effectors, of which both are
made in vitro]; Schots et al., 1992; Wilson and Walker, 2000; Eales, 2003), and five
different classes of antibodies (IgG, IgM, IgA, IgD, and IgE), which differ on the
basis of size, charge, amino acid composition and carbohydrate content (Coico et
al., 2003). Additionally, there are different subclasses (e.g. in mice: IgG1, IgG2a,
23
IgG2b, and IgG3), but the number of subclasses vary between species (Wilson and
Walker, 2000; Wild, 2001). The IgG used in the present thesis consists of a four
chain structure: two heavy chains and two light chains (Harlow and Lane, 1999).
The nature of the antibody is important for its sensitivity of the process (i.e. the
ability to detect low concentrations of the antigen), and for its specificity of the
methods (i.e. the ability to discriminate between the desired antigen and other
substances that may be present) (Wilson and Walker, 2000). The avidity of the
antibody (see above) is important for the former.
2.1.2. Immunoassays
There are a number of different immunoassays: e.g. label free (agglutination [i.e.
cells lumped together], precipitation and immunosensors), reagent excess
(competitive; one or two site), reagent limited (labeled antigen or antibody),
ambient analyte (microarray), all which include well-known techniques such as
radioimmunoassay (RIA), SDS-PAGE, rocket immunoelectrophoresis, and
enzyme linked immunoassays (ELISA). The latter method is frequently confused
with a number of other enzyme-based immunometric assays, and thereby often
distinguished by different names such as sandwich ELISA or two-site ELISA. The
immunometric assay used in the current thesis is an enzyme immunoassay (EIA,
described below). In many immunoassays, the binding of antibody – antigen
complexes can only be visualized by labeling the antibody or antigen with a marker
that can be quantitatively detected (Harlow and Lane, 1999). Antibodies are thus
labeled with e.g. radioactive isotopes, enzymes for colored products, or
fluorochromes; the latter two are referred to as conjugates (Wilson and Walker,
2000).
There are many different methods for the different immunoassays (Wild, 2001),
and I will not enumerate them all. In the method used in the current thesis (paper
IV and V), walls of microplates were coated with unlabelled, so called captured
immobilized primary IgG (goat anti rabbit; Fc-specific to ensure the captured
antibody to be immobilized in the correct orientation, for the most favourable
interaction with the antigen). The captured antibody was immobilised by a covalent
attachment. A secondary antibody, specific for the antigen, was added for
attachment to the primary IgG. The antigens (i.e. ecdysteroids), and a horseradishperoxidase labelled antibody were allowed to compete for attachment on the
24
secondary antibody. Detection for labelled antibodies was performed with
QuantaBlu Fluorogenic Peroxidase Substrate. The output measurements as
fluorescence was inversely proportional to the concentration of the antigen present
in the samples.
3. Mean development time
For investigation of mean development time, each individual was tracked daily
during development to a specific life stage. Since there is always natural
variability in coping with chemical exposure within a population, individuals
will react differently despite the fact that they are exposed to the same chemical
concentration. Hence, by following each individual’s development the duration
of a test may be relatively long, especially if the chemical of concern is affecting
the developmental rate negatively. On the other hand, the mean development
time is a way of revealing effects that may be of high concern in real
ecosystems. In this thesis, the mean development time was used as endpoint in
paper I, II and IV.
4. Growth rate and somatic measurement
The growth rates (i.e. the growth of an individual copepod per day until a
chosen life stage) were calculated according to Winberg (1971). The length
(µm) of NI, the length of an individual copepod at the chosen life stage, and
the individual mean time (days) the copepod used for reaching the same
chosen stage, were used for calculations. Gauss approximation of variance was
used, and differences between treatments were compared using approximate
test of significance (z-test). The growth rate was used as endpoint in paper I,
II, and IV in this thesis.
5. Population abundance
The long-term changes (over generations) in numbers and age composition of
individuals in a population were studied. The true total mean abundance was
counted on an individual basis. In these tests, the direct mortality is not
studied; it is rather an indirect function of reduced individual numbers in the
25
mean population abundance calculations, together with reduced fecundity. In
this thesis, the population abundance was used in paper III.
TEST SYSTEMS USED IN THE EXPERIMENTS
1. Acute toxicity tests
The acute toxicity test is a common tool within environmental risk assessment
(e.g. European Commission, 2003). The aim of an acute toxicity test is to
investigate the condition of a test organism after short-term exposure of a
toxic agent, usually at high doses or concentrations. The endpoints differ
between species: e.g. luminescent inhibition in bacteria (Coleman and Qureshi,
1985), growth inhibition in plants or algae (Blankenship and Larson, 1978;
Abou-Waly et al., 1991), lethality or immobility in invertebrates (Karlsson et al.,
2006; Penttinen et al., 2008), and lethality in vertebrates (Pielou, 1946; Winkaler
et al., 2007). Acute toxicity tests may further be carried out on both aquatic and
terrestrial species, and may be used on a variety of exposures regimes such as
single chemicals, mixture matrixes (e.g. paint, oil, effluents) or natural
sediments. In this thesis, acute toxicity tests have been used only for
concentration determinations of chemicals to be used in partial life cycle tests
(paper I and IV), described below.
2. Life cycle tests
2.1. Partial life cycle tests
In partial life cycle tests, the animals may be exposed to a chemical during at
least one sensitive life stage (EPA, 1992). The concentrations of the tested
chemical are usually low (Bechmann, 1994) (if possible down to concentrations
found in nature). For further details, see Figure 2. In this thesis, partial life
cycle tests were used in paper I and IV.
26
2.2. Full life cycle tests
In a full life cycle test, a whole life cycle is closely investigated instead of
choosing one life stage. This is preferable in ecotoxicology since different life
stages may respond differently depending on species and the chemical tested
(Ingersoll et al., 1999). Full life cycle tests were used in paper II.
2.3. Multigenerational tests
In a multigenerational test, the exposure time covers all life stages, over several
generations of a population, which means that transgenerational effects, such
as offspring affected by parental contaminant exposure, may be shown (Vogt
et al., 2007). In this thesis, a multigenerational test was used in paper III.
Survival success
Reproductive success
Day
-1
0
Ovigerours Newborn
females
nauplii
are allowed individually
allocated
to hatch
into wells
Hatchingtime Ecdysteroid content
Development times in different stages
Growth rate Body length
RNAcontent Malformations
6-9
--9
Stage CIII
in F0
generation
PaperI
PaperI, IV
12-14
14-16
-16
Adults
Newborn
paired in
nauplii
order to individually
mate
allocated
into wells
22-24
22--
42
Stage CI
in F1
generation
PaperII
II
Figure 2. Overview of time for events in life cycle tests of an unexposed N. spinipes
(below arrow). Endpoints used are shown above arrow. Broken arrow indicates
actual time for the life cycle tests in treatments with suboptimal food conditions
(paper II).
27
RESULTS AND DISCUSSION
The major aim of this thesis was to integrate responses on the biochemical level
with copepod life history traits, to improve the understanding of the connection
between two levels of organisation: the cellular and the individual. Biochemical
indicators, together with indicators at higher levels of biological organisation, may
provide a good measurement of an organism’s altered state due to toxicant
exposure (Gardeström et al., 2006). Papers I-IV show the usefulness of using a
RNA content assay as a measurement of growth-related variables of individual
copepods. In papers IV-V, an ecdysteroid content assay improved the
understanding of the copepod endocrine system. In paper I, II, and IV, contents
of ecdysteroids and/or RNA on individual N. spinipes were integrated with
individual development time, body lengths and growth rates. Further, in paper III,
investigations were performed on a multiple organizational level, were individual
cephalothorax lengths and RNA contents of A. crassa were integrated with
population dynamics and genetic variations. In Paper V, ecdysteroid responses
were linked with other biochemical parameters (i.e. CYP450 enzymes) as well as
life history traits of C. finmarchicus, which increased the understanding of its
development and reproduction.
1. RNA content and somatic growth
In paper I – IV, I have analysed the non-specific biomarker of individual RNA
content as a measurement of the instantaneous growth (Vrede et al., 2002). How
then, is it possible to estimate if an amplified RNA content is related to somatic
growth, or linked to increased e.g. stress related protein synthesis? For an increased
RNA content, the causality may be well-being of the organism, i.e. a positive effect
(Dahlhoff, 2004), or a defending system, i.e. a negative effect (Ibiam and Grant,
2005).
If the test organisms are of the same species, developmental stage, and incubated
at same conditions (e.g. feeding, temperature), comparing the RNA content with
the somatic growth of the test organisms may give some enlightening answers
(Figure 3). An organism that is large and has a high RNA content presumably may
be of good fitness and growing accordingly. A small-sized organism on the other
hand, with high RNA content, may be investing its energy in something else than
maintenance and somatic growth, e.g. stress-related proteins (Korsloot et al., 2004).
28
1.1. Partial life cycle exposure
Paper I and IV were based on investigations of the juvenile growth and
development of N. spinipes exposed to the pharmaceutical Simvastatin (paper
I) and the insecticide Lindane (paper IV). The results from paper I indicate
that Simvastatin may have impaired the endocrine system of N. spinipes. This is
interesting since during the last decade there have been discussions about
whether or not “classical” endocrine disrupters, such as estrogens, are able to
interact with the hormonal system of small crustaceans (e.g. Baldwin et al., 1995;
1997; Zou and Fingerman, 1997; Andersen et al., 2001), but the answers are
somewhat contradictory (Breitholtz and Bengtsson, 2001; Lafont and Mathieu,
2007; LeBlanc, 2007). Both Paper I and IV are based on partial life cycle tests
where N. spinipes were followed individually to stage CIII. The RNA contents
in the copepods differ however between the two different tests. Since statins
have been shown to induce growth-related hormones of crustaceans (Li et al.,
2003), the copepods in paper I responded in an expected way when exposed
to Simvastatin. The high RNA content together with decreased development
times in the lowest treatments may have reflected positive growth stimulation,
but a decreasing trend in body length with decreasing concentration of
Simvastatin indicates that the high RNA content may alternatively be due to
stress-related protein induction, i.e. a way to cope with toxicant stress
(Korsloot et al., 2004). The Simvastatin-stimulated developmental growth was
interrupted at higher concentrations, presumably by an overall toxic stress.
Hence, the animals invested their energy in developing fast (due to the
mechanistic effects of Simvastatin), but since there are energetic costs
involved, to both growth and survival in a toxic environment (confirmed by
amplified mortality in higher treatments), the animals have less energy left for
metabolic maintenance (Smit and Van Gestel, 1997).
It is interesting to note that the RNA content as endpoint in paper I was the most
rapid growth-related response to a possible energy-mediated effect, which indicates
that if the energy required for survival is high, there is less energy left for
maintenance and growth. This was seen in a significantly elevated RNA content in
the lowest concentration due to presumed stress-related protein induction, which
quickly dropped at the second lowest concentration of Simvastatin. Meanwhile the
endpoints of body length and development time responded only when the
copepods actually started to die off, and the growth rate did not show any such
response except in the highest concentration.
29
a
b
vs.
copepod
RNA
content
copepod
RNA
content
Figure 3. Schematic figure of coupling between RNA content and copepod somatic size. Black
bars indicate total RNA content. If the copepod is large with a high RNA content (a), it may
be presumed that the copepod has a high fitness and is growing accordingly. If the copepod is
small with a high RNA content (b), it may be presumed that the copepod is using its energy to
e.g. stress-related protein production, rather than somatic maintenance and growth.
On the other hand, the RNA contents in paper IV were not as sensitive as an
endpoint. The trend of RNA contents followed the same decreasing pattern as
the growth rates, and there was increased mortality with increasing
concentrations of Lindane. Since the intentional mode of action of Lindane is
to interfere with the vital GABAA receptors (Ogata et al., 1988), it may be
difficult to reveal if the growth-related results are due to the mode of action of
Lindane, or due to an energy-mediated effect. The indirect mode of action (i.e.
Lindane being an ecdysteroid antagonist; Sarker et al., 1999; Dinan et al., 2001),
complicates this issue further.
30
1.2. Full life cycle exposure
In paper II, the RNA levels were investigated in two generations in a life cycle
test. Regardless of treatment, the RNA content increased significantly in the
second generation of copepods as compared to the first generation. Normally,
I would consider the increased RNA content as a potential stress-related
protein induction, since the RNA content was significantly higher as compared
to the first generation, while stage-specific body lengths were relatively
invariant over the generations. As, however, both the development times and
juvenile survivals were high, and comparable between the two generations, and
the length-weight regressions for harpacticoid copepods are rather weak
(Satapoomin, 1999) (meaning that increased somatic weight does not
necessarily relate to increased somatic length), I however conclude that the
results do not indicate stress, even though further studies should be conducted
in this area.
1.3. Multigenerational exposure
In paper III, A. crassa was exposed to natural sediments (i.e. sediments
contaminated by anthropogenic actions, and clean sediments were used as control),
for several generations. As positive control we also tested natural control
sediments spiked with copper at low concentrations. The sediments were sampled
at two occasions (after 60 and 120 days). In this paper, we studied the usefulness of
body length and RNA content in multigenerational experiments, and these growthrelated measurements were placed in a larger perspective of population dynamics
and genetic diversity. RNA contents have been shown useful in long time exposure
with other harpacticoids earlier (Gardeström et al., 2006), but at exposure times up
to 5 times shorter than in the experiment in paper III (i.e. 24 days compared to
120 days).
Besides the rather invariant results of growth-related variables (i.e. RNA contents
and body lengths) from the naturally contaminated sediments, we found that the
copper treatment resulted in a significant decrease in genetic diversity after 120
days. Since the total population abundance remained unchanged, this was
indicating a selection in favour of copper-adapted copepods in the treatment.
Unfortunately, these interesting results were not integrated on the individual
growth-related level, since the copepods in the copper treatment were not analysed
31
for RNA content, due to time constrains. For the naturally contaminated
sediments, the invariant cephalothorax lengths after 60 and 120 days, and RNA
contents in sampling after 120 days, I suggest that the long time exposure may
have made surviving copepods adapt to the environment and that they were no
longer in need of e.g. synthesising stress-related proteins. The stress protein
production is a rapid response to a stressor (within minutes; Korsloot et al., 2004)
and over generations it is possible that the copepods would either die from the
toxic burden (shown in the mean abundance) or create a tolerance towards it.
There are two types of tolerance development in organisms exposed to
environmental stress: i) an inheritable adaptation through natural selection, which
is genetically based on inter-individual variations in tolerance (Korsloot et al., 2004)
and ii) an non-inheritable physiological adjustment in response to sublethal
concentrations of a stressor (Posthuma and van Straalen, 1993). It is, however,
often not clear which type of tolerance is observed. Although the ecological
realism may be high when assessing harmful effects of natural sediments
(Matthiessen et al., 1998), visual examinations of copepod life stages (with body
lengths ranging from 0.1 mm to 1 mm) in such matrix is both time consuming and
tricky.
2. Ecdysteroid content
The ecdysteroid contents in the copepods in paper IV, showed a quite clear
concentration dependent pattern as a response to the insecticide Lindane, with the
lowest amount of ecdysteroids in the control, and increasing amount with
increasing Lindane concentration until the second highest treatment. In the highest
treatment, the ecdysteroid content dropped. Of the five concentrations of Lindane,
the middle concentration showed a significantly increased ecdysteroid content,
compared to the control. In the second highest concentration of Lindane, the
copepods had the peak of ecdysteroids. The inter-variability of the ecdysteroid
contents in this treatment was however high and thereby probably masking the
effect statistically, indicating that the number of replicates used for this method in
the future perhaps should be higher than was used.
In paper V, the juveniles showed the highest amount of ecdysteroids, but at the
same time the highest variability between the individuals. The lowest amounts of
ecdysteroids were found in post-spawning females and in males. The ecdysteroid
32
involvement, together with cytochrome P450, in reproduction and lipid storage
consumption of C. finmarchicus, were investigated in paper V. This was performed
due to the importance of lipid storage for the reproductive success of C.
finmarchicus (almost the entire lipid resources are used for production of eggs and
spermatophores [Marshall and Orr, 1972; Jonasdottir, 1999]). The lipid storage in
the copepods dropped between the stage CV and in the youngest females,
indicating that it was additionally used for the stage development process and
moulting. Further, the lipid storage was continuously diminished during the egg
production of the females, as shown earlier (Marshall and Orr, 1972; Hygum et al.,
2000). The high variability of the ecdysteroid content in juvenile stage CV,
compared to the adult stages, may indicate an increased ecdysteroid production
closer to adult stage. It could also be assumed that the lipid storage increases while
the copepodite is maturing to adult stage. This hypothesis was supported by a
positive correlation between the size of the lipid storage and ecdysteroid levels in
stage CV. Indeed, it seems like ecdysteroid production is related to lipid levels of
the copepods.
In paper V, ecdysteroid levels were also linked with expression of three different
P450 enzymes, since these enzymes have been shown to modify cholesterol for
ecdysteroid production in insects (Rewitz et al., 2006) by the CYP300 gene family
(Grieneisen et al., 1993; Sieglaff et al., 2005) in the insect fat body (LeBlanc et al.,
1999). The hypothesis is that even though the insect fat body and the crustacean
lipid sack probably serve different functions, the gene expression of the P450
enzymes would be higher in copepods with large lipid storage. This hypothesis was
also confirmed by a correlation between the CYP330A1 mRNA levels and lipid
levels in CV and adult females. The highest levels of CYP330A1 mRNA and lipids
were found in young CV; thereafter - the older the female, the more both the
CYP330A1 mRNA and the lipids decreased. Further, the results in paper V
showed that CYP305A1 and CYP330A1 were more expressed during CV than at
the adult stages, which may indicate an involvement in ecdysteroid regulation, since
ecdysteroid levels showed the same pattern. The CYP301A1 levels were highest in
post spawning females and males, where the ecdysteroid levels at the same time
were lowest. This may indicate an involvement of CYP301A1 in ecdysteroid
degradation.
As mentioned in the introduction, the crustacean endocrinology is a complex issue,
with many gaps in our knowledge. On top of this, endocrinology of micro33
crustaceans, as compared to macro-crustaceans, is much less investigated, due to
diminutive biomass. For example, dissectional operations that can be made in
macro-crustaceans (i.e. performed to reveal effects on certain organs/tissues (e.g.
Wainwright et al., 1996; Barki et al., 2006; Reddy and Pushpalatha, 2007), are yet not
achievable in such small organisms. Since the micro-crustaceans are one of the
absolute foundations in the aquatic ecosystems (Fryer 1986, reviewed in Hopcroft
and Roff, 1998; Miller and Harley, 1999), they are important to protect, and this
can be done by collecting information about which stressors may influence e.g.
their physiology or behavioural patterns. But to do this, the basic science of the
micro-crustaceans needs to be mapped.
There are a number of facts to consider before starting an immunoassay in the
laboratory and additionally many facts to consider when evaluating the results.
With the use of the established ecdysteroid content analysis on individual microcrustaceans, I have so far both managed to integrate the ecdysteroids as an
endpoint with other ecotoxicological endpoints for stressor investigations (paper
IV) and used the endpoint as a tool for increasing the understanding of some of
the mechanisms behind basic life history traits of copepods (paper V).
3. A balanced ecotoxicological test
In this thesis, I have tried to find a compromise between reliability, repeatability,
sensitivity and relevance, features which ecotoxicological tests should be founded on.
For example, a full life cycle test may be costly, time consuming and difficult to
repeat. Hence, it is important to find a balance between test adequacy and costs,
something I believe was achieved by using partial life cycle tests based on the
juvenile developmental stages of copepods, instead of the much more elaborate
full life cycle studies and multi-generation tests. However, if the test is to be used
at high tier assessment or if there are suspicions that a chemical may specifically
disrupt reproduction, it is recommended that full life cycle tests be used.
4. Mean development time
In this thesis, the mean individual development time was used in paper I, II, and
IV, where it has been shown to be a very sensitive tool for investigations of
34
toxicant exposure. In paper I, it was not even possible to find the so called no
observed effect concentration (NOEC) for this endpoint. This means that the endpoint
was significantly different from the control even in the lowest treatment of
Simvastatin. The same sensitivity of the endpoint was seen when copepods were
exposed to Lindane in paper IV.
In paper IV, I decided to investigate the mean individual development time more
closely, by calculating the mean number of days, not only from NI - CIII, but also
to CI and to CII. It is generally stated in ecotoxicology that the juvenile life stages
are the most sensitive ones (Forget et al., 1998; Barata et al., 2002; Medina et al.,
2002), but I found that it may not be the younger the organism, the more sensitive.
In paper IV, the mean individual development time between NI to CI stages was
much less sensitive than the development time for copepods between the CI to
CIII stages, when exposed to Lindane. Further, the NOEC-value was not found
for the mean individual development times between CI to CIII, indicating that the
sensitivity of this endpoint may be even greater. The development time is indeed a
common and useful tool in ecotoxicological research with copepods. While cohorts
are commonly used (e.g. Medina and Barata, 2004; Wollenberg et al., 2005; Huang et
al., 2006), investigations on the mean individual development time has additionally
been proven useful, as shown in the current thesis.
5. Suboptimal conditions
In order to assure high reliability of the ecotoxicological tests using N. spinipes
different food regimes were tested by offering six different micro-algae in 25
different combinations in two full life cycle tests and in several partial life cycle
tests. The results in paper II show that N. spinipes indeed is sensitive to the food
regime, with mortality, malformations, as well as alterations in growth,
development time, and reproduction as a result of inadequate food. Even though
the adverse results may be species-specific for N. spinipes, it is most likely that
similar effects could occur in other copepods used for toxicity testing. Food quality
is one of the important reliability variables to consider when developing a test,
since the quality may affect the copepods well-being and thereby alter the results in
the bioassays (EPA, 1994).
In order to clearly reveal effects of a chemical, toxicity tests are normally used
in which the organisms are exposed to a chemical under otherwise constant
35
and optimal conditions. If the copepods condition is suboptimal, it means that
they may invest their energy into combating metabolic stress (such as stressrelated protein synthesis, Korsloot et al., 2004). Many organisms have the
ability to regulate an internal toxicant concentration by elimination,
detoxification, and/or storage in the body. These processes cost energy, and
thus, less energy is available for other crucial physiological processes (Smit and
Van Gestel, 1997). This means that organisms living under optimal conditions
may be able to better handle stressors, such as hazardous chemicals, than
organisms living under suboptimal conditions (van der Geest et al., 2002).
36
CONCLUSIONS
The present doctoral thesis demonstrates the accuracy and usefulness of two
established biochemical assays (resulting in endpoints of RNA and ecdysteroid
contents) for evaluation of toxicity on copepods. Further, both biochemical
endpoints were shown to integrate well with traditional life history traits in
partial or full life cycle tests with the copepods, resulting in investigations at
two levels of biological organisation in those tests. The ecdysteroid assay has
additionally been proven a useful tool for covering some of the knowledge
gaps in the physiological understanding of C. finmarchicus development and
reproduction. In tests with longer exposure time, the RNA content was shown
to be an insensitive tool for assessment of individual growth, and did not
reflect the results of differences in mean abundance of A. crassa.
FUTURE WORK AND PERSPECTIVES
An ecotoxicological investigation on two levels of biological organisation, i.e.
cellular and individual level, most likely increases the understanding and
relevance of ecotoxicological data (Heckmann et al., 2008). However, studies
on the population level would increase the relevance even further (Forbes and
Calow, 1999). To some extent, population level changes were investigated in
paper III. However, because of the immense work load at start and sampling
occasions, the use of this method for regulatory purposes is somewhat
questionable. A promising short cut is therefore to use individual life history
traits, such as those used in paper I, II and IV, in biologically structured
models to estimate population dynamic data (Menzie et al., 2008). If possible, it
would additionally be interesting to incorporate biochemical endpoints, such as
those used in the current thesis into such models. This work has already been
started by a new PhD student, to whom I am an assistant supervisor.
It would indeed be interesting and useful to make the biochemical assays used
in the present thesis even more sensitive. Today, I use the unspecific
biomarker, individual RNA, content as an indicator of growth or potential
stress related protein synthesis, compared to life-history traits of the copepods.
In the future, I would like to be able to perform assays on stress protein
37
contents in individual test specimens. Further, regarding the ecdysteroid assay,
it would be preferable to analyse not only ecdysteroids as a group, but rather
the different ecdysteroids individually. In that way it may be possible to link
specific chemical-induced responses on individual level to responses in specific
ecdysteroids and thereby amplify the mechanistic understanding of stressrelated responses.
It would additionally be interesting to gain deeper knowledge about what
exactly happens when the copepods are exposed to a toxic substance. This
could be made by e.g. 2D-proteomic or micro arrays. In such experiments,
pollutant induced up/down-regulation of genes/proteins is identified. This
would be a sensitive and useful tool in the way that hazardous effects may be
revealed at an early stage of toxicant exposure. This work has already been
initialized by using a proteomic pilot assay. Copepods were exposed to low
concentrations of the pharmaceutical Simvastatin. After merely 24 hours of
exposure, there was a significant difference between the treatment and the
control.
Finally, the subject of suboptimal conditions was raised when performing the
food regime experiment in paper II. However, since the environment is a
fluctuating matrix, and since the fluctuations may increase even more (due to
e.g. climate changes or release of harmful chemicals), the aspect of suboptimal
conditions would indeed be interesting to investigate further in combination
with toxicant exposure.
38
ACKNOWLEDGEMENTS
39
40
REFERENCES
Abou-Waly H., Abou-Setta M.M., Nigg H.N., Mallory L.L. (1991). Growth
response of freshwater algae, Anabaena flos-aquae and Selenastrum capricornutum to
atrazine and hexazinone herbicides. Bull. Environ. Contam. Toxicol. 46: 223229.
Abraham S., Gopalan U.K. (1975). Growth of an estuarine harpacticoid
copepod Nitocra spinipes Boeck cultured in the laboratory. Bull. Dep. Mar. Sci.,
University of Cochin. Volume VII, 2, pp. 309-378.
Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J.D. (1983).
Molecular biology of the cell. Garland Publishing, Inc., New York, pp. 223-246.
Andersen H.R., Wollenberger L., Halling-Sørensen B., Kusk K.O. (2001).
Development of copepod nauplii to copepodites – a parameter for chronic
toxicity including endocrine disruption. Environ. Toxicol. Chem. 20: 28212829.
Aneer G. (1975). Composition of the food of the Baltic herring (Clupea harengus
v. membras L.), fourhorn sculpin (Myoxocephalus quadricornis L.) and eel-pout
(Zoarces viviparous L.) from deep soft bottom trawling in the Askö-Landsort area
during two consecutive years. Merentutkimuslait. Julk./Havsforskningsints. Skr.
239: 146-154.
Baldwin W.S., Graham S.E., Shea D., LeBlanc G.A. (1997). Metabolic
androgenization of female Daphnia magna by the xenoestrogen 4-nonylphenol.
Environ. Toxicol. Chem. 16: 1905-1911.
Baldwin W.S., Milam D.L., LeBlanc G.A. (1995). Physiological and
biochemical perturbations in Daphnia magna following exposure to the model
environmental estrogen diethylstilbestrol. Environ. Toxicol. Chem. 14: 945-952.
Ban S., Minonda T. (1994). Induction of diapause egg-production in Eurytemora
affinis by their own metabolites. Hydrobiologia. 293: 185-189.
Barata C., Medina M., Telfer T., Baird D.J. (2002). Determining demographic
effects of cypermethrin in the marine copepod Acartia tonsa: stage-specific short
tests versus life-table tests. Arch. Environ. Contam. Toxicol. 43: 373-378.
Barki A., Karplus I., Manor R., Sagi A. (2006). Intersexuality and behavior in
crayfish: the de-masculinization effects of androgenic gland ablation. Hormones
and Behav. 50: 322–331.
41
Barnes H., Stanbury F.A. (1948). The toxic action of copper and mercury salts
both separately and when mixed on the harpacticoid copepod, Nitocra spinipes
(Boeck). J. Exp. Biol. 25: 270-275.
Bechmann R.K. (1994). Use of life tables and LC50 tests to evaluate chronic
and acute toxicity effects of copper on the marine copepod Tisbe furcata (Baird).
Environ. Toxicol. Chem. 13: 1509-1517.
Becker W.M., Kleinsmith L.J., Hardin J. (2000). The world of the cell – 4th ed.
In: The Benjamin/Cummings Publishing Company, San Francisco, pp. 376404.
Bejarano A.C., Chandler G.T., He L., Coull B.C. (2006). Individual to
population level effects of South Louisiana crude oil water accommodated
hydrocarbon fraction (WAF) on a marine meiobenthic copepod. J. Exp. Mar.
Biol. Ecol. 332: 49–59.
Bengtsson B.-E. (1978). Use of a harpacticoid copepod in toxicity tests. Mar.
Pollut. Bull. 9: 238-241.
Blankenship, D.W., Larson R.A. (1978). Plant growth inhibition by the water
extract of a crude oil. Water Air, Soil Pollut. 10: 471-476.
Block S.D., Bejarano A.C., Chandler G.T. (2003). Ecdysteroid concentrations
through various life-stages of the meiobenthic harpacticoid copepod,
Amphiascus tenuiremis and the benthic estuarine amphipod, Leptocheirus plumulosus.
Gen. Comp. Endocrinology. 132: 151-160.
Borst D.W., Laufer H., Landau M., Chang E.S., Hertz W.A., Baker F.C.,
Schooley D.A. (1987). Methyl farnesoate and its role in crustacean reproduction
and development. Insect Biochem. 17: 1123-127.
Breitholtz M., Bengtsson B.-E. (2001). Oestrogens have no hormonal effect on
the development and reproduction of the harpacticoid copepod Nitocra spinipes.
Mar. Pollut. Bull. 42: 879-886.
Breitholtz M., Rattfelt Nyholm J., Karlsson J., Andersson P. (in press). Are
individual NOEC levels safe for mixtures? A study on mixture toxicity of
brominated flame-retardants in the copepod Nitocra spinipes. Chemosphere
(2008).
Breitholtz M., Rudén C., Hansson S.O., Bengtsson B.-E. (2006). Ten
challenges for improved ecotoxicological testing in environmental risk
assessment. Ecotoxicol. Environ. Saf. 63: 324-335.
42
Breitholtz M., Wollenberger L., Dinan L. (2003). Effects of four synthetic
musks on the life cycle of the harpacticoid copepod Nitocra spinipes. Aquat.
Toxicol. 63: 103-118.
Brown J.H., Gillooly J.F., Allen A.P., Savage V.M., West G.B. (2004). Toward a
metabolic theory of ecology. Ecol. 85: 1771–1789.
Calow P. (1998). Ecological risk assessment: risk for what? How do we decide?
Ecotoxicol. Environ. Saf. 40: 15-18.
Calow P., Sibly R.M., Forbes V. (1997). Risk assessment on the basis of
simplified life-history scenarios. Environ. Toxicol. Chem. 16: 1983-1989.
Calow P. (1992). The three Rs of ecotoxicology. Funct. Ecol. 6: 617-619.
Calow P., Forbes V. (2003). Does ecotoxicology inform ecological risk
assessment? Environ. Sci. Technol. A, 146-151.
Chang E.S. (1993). Comparative endocrinology of molting and reproduction:
insects and crustaceans. Annu. Rev. Entomoll. 38: 161-180.
Chapman P.M. (2002). Integrating toxicology and ecology: putting the "eco"
into ecotoxicology. Mar. Pollut. Bull. 44: 7-15.
Carlisle D.B., Dohrn P.F.R. (1953). Studies on Lysmata seticaudata Risso
(Crustacea Decapoda). II. Experimental evidence for a growth – and moultaccelerating factor obtainable from eyestalks. Pubblicazioni della Stazione
Zoologica di Napoli. 24: 69-83.
Coast G.M., Webster S.G. (1998). Recent advances in arthropod
endocrinology. Cambridge University Press, Cambridge, U.K. ISBN: 0-52159113-9.
Coico R., Sunshine G., Benjamini E. (2003). Immunology: a short course. 5th
ed. Johan Wiley & Sons, Inc., New Jersey, USA. ISBN 0-471-22689-0.
Coleman R.N., Qureshi, A.A. (1985). Microtox and Spirillum volutans tests for
assessing toxicity of environmental samples. Bull. Environ. Contam. Toxicol.
35: 443-451.
Dahlhoff E.P. (2004). Biochemical indicators of stress and metabolism:
applications for marine ecological studies. Annu. Rev. Physiol. 66: 183-207.
deFur P.L. Crane M., Tattersfield L.J., Ingersoll C.G., Stahl R.G. Jr.,
Matthessen P., LeBlanc G.A. (1999). Workshop on endocrine disruption in
invertebrates: endocrinology, testing, and assessment (EDIETA). In: deFur
P.L., Crane M., Ingersoll C., Tattersfield L. (1999). Endocrine disruption in
43
invertebrates: endocrinology, testing, and assessment. Soc. Environ. Toxicol.
Chem. (SETAC), SA. ISBN 1-880611-27-9.
Delbecque J.P., Weidner K., Hoffmann K.H. (1990). Alternative sites for
ecdysteroid production in insects. Invertebr. Reprod. Dev. 18: 29-42.
Dinan L., Bourne P., Whiting P., Dhadialla T.S., Hutchinson T. H. (2001).
Screening of environmental contaminants for ecdysteroid agonist and
antagonist activity using the Drosophila melangoster B-II cell in vitro assay.
Environ. Toxicol. Chem. 20: 2038-2046.
Dole-Olivier M.J., Galassi D.M.P., Marmonier P., Creuzé des Châtelliers M.
(2000). The biology and ecology of lotic microcrustaceans. Freshw. Biol. 44:
63-91.
Downward J. (2004). RNA interference. Br. Med. J. 328: 1245-1248.
Dreyfuss G. (1986). Structure and function of nuclear and cytoplasmic
ribonucleoprotein particles. Ann. Rev. Cell. Biol. 2: 459-498.
Eales L.-J. (2003). Immunology for life scientists, 2nd ed. John Wiley & Sons
Ldt, West Sussex, England. ISBN 0470 84524 4.
Ek H., Nilsson E., Birgersson G., Dave G. (2007). TNT leakage through
sediment to water and toxicity to Nitocra spinipes. Ecotoxicol. Environ. Saf. 67:
341-348.
Elefant F., Palter K.B. (1999). Tissue-specific expression of dominant negative
mutant Drosophila HSC70 causes developmental defects and lethality. Mol.
Biol. Cell. 10: 2101-2117.
Ellis R.J., van der Vies S.M. (1991). Molecular chaperones. Annu. Rev.
Biochem. 60: 321-347.
Elser J.J., Sterner R.W., Gorokhova E., Fagan W.F., Markow T.A., Cotner J.B.,
Harrison J.F., Hobbie S.E., Odell G.M., Weider L.J. (2000). Biological
stoichiometry from gene to ecosystems. Ecol. Lett. 3: 540-550.
Enckell P.H. (1980). Kräftdjur (fältfauna). Signum, Lund, Sweden. ISBN 9185330-27-2.
Environment Canada (1999). EPS 1/RM/34 – December 1999. Guidance
document on application of single-species tests in environmental toxicology.
Method development and application section. Environmental technology
centre.
44
EPA/600/4-91/002 (1994). Short-term methods for estimating the chronic
toxicity of effluents and receiving waters to freshwater organisms. 3rd ed.
Environmental Protection Agency.
EPA/630/R-92/001 (1992). Framework for ecological risk assessment. Risk
Assessment Forum, U.S. Environmental Protection Agency, Washington, DC
20460.
European Commission (2003). Technical guidance document in support of
commission directive 93/67/ EEC on risk assessment for new notified
substances and commission regulation (EC) No 1488/94 on risk assessment for
existing substances. Luxemburg.
Feder M.E., Hoffman G.E. (1999). Heat-shock proteins, molecular
chaperones, and the stress response: evolutionary and ecological physiology.
Ann. Rev. Physiol. 61: 243-282.
Fingerman M. (1987). The endocrine mechanisms of crustaceans. J. Crust.
Biol. 7: 1-24.
Forbes V.E., Calow P. (1999). Is the per capita rate of increase a good measure
of population-level effects in ecotoxicology? Environ. Toxicol. Chem. 18: 15441556.
Forbes V.E., Calow P., Sibly R.M. (2001). Are current species extrapolation
models a good basis for ecological risk assessment? Environ. Toxicol. Chem.
20: 442–447.
Forget J., Pavillon J.F., Menasria M.R., Bocquené G. (1998). Mortality and
LC50 values for several stages of the marine copepod Tigriopus brevicornis
(Müller) exposed to the metals arsenic and cadmium and the pesticides atrazine,
carbofuran, dichlorvos, and malathion. Ecotoxicol. Eniron. Saf. 40: 239-244.
Fryer G. (1986). Structure, function and behaviour, and the elucidation of
evolution in copepods and other crustaceans. In: Schriever G., Schmike H.K.,
Shih C-t (eds). Proceedings of the 2nd international conference on Copepoda,
Ottawa, Canada. Syllogeus Series 58. National Museums of Canada, Ottawa, pp.
150-157.
Gardeström J., Gorokhova E., Gilek M., Grahn M., Bengtsson B.-E.,
Breitholtz M. (2006). A multilevel approach to predict toxicity in copepod
populations: assessment of growth, genetics, and population structure. Aquat.
Toxicol. 79: 41-48.
Gilbert J.J., Williamson C.E. (1983). Sexual dimorphism in zooplankton
(Copepoda, Cladocera, and Rotifera). Ann. Rev. Ecol. Syst. 14: 1-33.
45
Goad L.J. (1981). Sterol biosynthesis and metabolism in marine invertebrates.
Pure Appl. Chem. 53: 837-852.
Gorokhova E. (2003). Relationships between nucleic acid levels and egg
production rates in Acartia bifilosa: implications for growth assessment of
copepods in situ. Mar. Ecol. Prog. Ser. 262: 163-172.
Green A.S., Chandler G.T., Piegorsch W.W. (1996). Life-stage-specific toxicity
of sediment-associated chlorpyriphos to a marine, infaunal copepod. Envrion.
Toxicol. Chem. 7: 1182-1188.
Grennan A.K. (2005). High impact. Plant Physiol. 139: 536-565.
Grieneisen M.L., Warren J.T., Gilbert L.I. (1993). Early steps in ecdysteroid
biosynthesis: evidence for the involvement of cytochrome P-450 enzymes.
Insect Biochem. Mol. Biol. 23: 13-23.
Hansson S.O., Rudén C. (2006a). Priority setting in the REACH system.
Toxicol. Sci. 90: 304-30.
Hansson S.O., Rudén C. (2006b). Evaluating the risk decision process.
Toxicol. 218: 100-111.
Harlow E., Lane D. (1999). Using antibodies: a laboratory manual. Cold Spring
Harbor Laboratory Press, NY, USA. ISBN 0-87969-543-9.
Heckmann L.-H., Sibly R.M., Connon R., Hooper H.L., Hutchinson T.H.,
Maund S.J., Hill C.J., Bouetard A., Callaghan A. (2008). Systems biology meets
stress ecology: linking molecular and organismal stress responses in Dahpnia
magna. Genome Biol. 9: R40.1-R40.14.
Hirche H.-J., Kosobokova K. (2007). Distribution of Calanus finmarchicus in the
northern North Atlantic and Arctic Ocean – expatriation and potential
colonization. Deep-Sea Res. II, 54, 2729-2747.
Homola E., Chang E.S. (1997). Methyl farnesoate: crustacean juvenile
hormone in search of functions. Comp. Biochem. Physiol. 117B: 347-356.
Hopcroft R.R., Roff J.C. (1998). Zooplankton growth rates: the influence of
size in nauplii of tropical marine copepods. Mar. Biol. 132: 87-96.
Huang Y., Zhu L., Liu G. (2006). The effects of bis(tributyltin) oxide on the
development, reproduction and sex ratio of calanoid copepod Pseudodiaptomus
marinus. Estuar., Coast. Shelf Sci. 69: 147-152.
Huberman A. (2000). Shrimp endocrinology. A review. Aquacult. 191: 191208.
46
Huys R., Gee J.M., Moore C.G., Hamond R. (1996). Marine and brackish water
harpacticoid copepods (Part I). (Huys R., Gee J.M., Moore C.G., Hamond R.
Eds.). Synopsis of the British Fauna (New Series). The Dorset Press, Great
Britain, ISBN: 1-85153-256-0.
Hygum B.H., Rey C., Hansen B.W., Tande K.S. (2000). Importance of food
quantity to structure growth rate and neutral lipid reserves accumulated in
Calanus finmarchicus. Mar. Biol. 136: 1057-1073.
Ibiam U., Grant A. (2005). RNA/DNA ratios as a sublethal endpoint for largescale toxicity tests with the nematode Caenorhabditis elegans. Environ. Toxicol.
Chem. 24: 1155-1159.
Ingersoll C.G., Hutchinson T., Crane M., Dodson S., DeWitt T., Geis A., Huet
M.-C., McKenny Jr. C.L., Oberdörster E., Pascoe D., Versteeg D.J., Warwick
O. (1999). Laboratory toxicity tests for evaluating potential effects of
endocrine-disrupting compounds. In: Endocrine disruption in invertebrates:
SETAC Technical Publication (deFur P.J., Crane M., Ingersoll C.G.,
Tattersfield L.J. Eds), Brussels, Belgium, ISBN 1-880611-27-9.
ISO (1997). International Organization for Standardization. Water quality –
determination of acute lethal toxicity to marine copepods (Copepoda,
Crustacea). Draft International Standard ISO/DIS 14669. Geneve, Switzerland.
Jonasdottir S.H. (1999). Lipid content of Calanus finmarchicus during
overwintering in the Faroe-Shetland Channel. Fish. Oceanogr. 8: 61-72.
Karlsson J., Breitholtz M., Eklund B. (2006). A practical ranking system to
compare toxicity of anti-fouling paints. Mar. Poll. Bull. 52: 1661-1667.
Korsloot A., Van Gestel, C.A.M., van Straalen, N.M. (2004). Environmental
stress and cellular response in arthropods. CRC Press, Florida. ISBN: 0-41532886-1.
Lachaise F., Carpentier G., Somme G., Colardeau J., Beydon P. (1989).
Ecdysteroid synthesis by crab Y-organs. J. Exp. Zool. 252: 283–292.
Lafont R. (2000). The endocrinology of invertebrates. Ecotoxicology 9: 41-57.
Lafont R., Harmatha J., Marion-Poll F., Dinan L., Wilson I.D. (2008). The
ecdysone handbook. 3rd ed. – continuously updated.
http://ecdybase.org/.
Lafont R., Mathieu M. (2007). Steroids in aquatic invertebrates. Ecotoxicology
16: 109-130.
47
Lang K. (1948). Monographie der Harpacticiden. II. Nordiska bokhandeln,
Stockholm, p. 1682.
LeBlanc G.A. (2007). Crustacean endocrine toxicology: a review.
Ecotoxicology 16: 61-81.
LeBlanc G.A., Campbell P.M., den Besten P., Brown R.P., Chang E.S., Coats
J.R., deFur P.L. Dhadialla T., Edwards J., Riddiford L.M., Simpson M.G., Snell
T.W., Thorndyke M., Matsumara F. (1999). The endocrinology of invertebrates.
In: deFur P.L., Crane M., Ingersoll C., Tattersfield L. (1999). Endocrine
disruption in invertebrates: endocrinology, testing, and assessment. Soc.
Environ. Toxicol. Chem. (SETAC), SA. ISBN 1-880611-27-9.
Lee J.K., Kim H.-W., Gomez A.M., Chang E.S., Covi J.A., Mykles D.L. (2007).
Molt-inhibiting hormone from the tropical land crab, Gecarcinus lateralis: cloning,
tissue expression, and expression of biologically active recombinant peptide in
yeast. Gen. Comp. Endocrinology 150: 505-513.
Li S., Wagner C.A., Friesen J.A., Bors, D.W. (2003). 3-hydroxy-3methylglutaryl-coenzyme A reductase in the lobster mandibular organ:
regulation by the eyestalk. Gen. Comp. Endocrinology 134: 147-155.
Lindquist S. (1986). The heat shock response. Annu. Rev. Biochem. 55: 11511191.
Liu L., Laufer H., Wang Y., Hayes T. (1997). A neurohormone regulating both
methyl farnesoate synthesis and glucose metabolism in a crustacean. Biochem.
Biophys. Res. Commun. 237: 694-701.
Lomas L.O., Rees H.H. (1998). Endocrine regulation of development and
reproduction in acarines. In: Coast G.M., Webster S.G. (1998). Recent advances
in arthropod endocrinology. Cambridge University Press, Cambridge, U.K.
ISBN: 0-521-59113-9.
Marcus N.H., Lutz R., Burnett W., Cable P. (1994). Age, viability, and vertical
distribution of zooplankton resting eggs from an anoxic basin: evidence of an
egg bank. Limnol. Oceanogr. 39: 154-158.
Marshall S.M., Orr A.P. (1972). The biology of a marine copepod, Calanus
finmarchicus (Gunnerus). Springer Verlag, Berlin, Heidelberg, New York.
Matthiessen P., Bifield S., Jarrett F., Kirby M.F., Law R.J., McMinn W.R.,
Sheahan D.A., Thain J.E., Whale G.F. (1998). An assessment of sediment
toxicity in the river Tyne estuary, UK by means of bioassays. Mar. Environ.
Res. 45: 1-15.
48
Matzke M.A., Birchler J.A. (2005). RNAi-mediated pathways in the nucleus.
Nature 6: 24-35.
Mauchline J. (1998). The biology of calanoid copepods. Advances in marine
biology. Academic Press. WBC Book Manufacturers, Bridgend, Mid
Glamorgan. ISBN 0-12-026133-2.
McCarthy J.F. (1982). Ecdysone metabolism in premolt land crabs (Gecarcinus
lateralis). Gen. Comp. Endocrinology 47: 323-332.
Medina M., Barata C., Telfer T., Baird D.J. (2002). Age- and sex-related
variation in sensitivity to the pyrethroid cypermethrin in the marine copepod
Acartia tonsa Dana. Arch. Environ. Contam. Toxicol. 42: 17-22.
Medina M., Barata C. (2004). Static-renewal culture of Acartia tonsa (Copepoda
: Calanoida) for ecotoxicological testing. Aquacult. 229: 203-213.
Menzie C., Bettinger N., Fritz A., Kapustka L., Regan H., Moller V., Noel H.
(2008). Population protection goals. In: Population-level ecological risk
assessment. Barnthouse L.W., Munns Jr W. R., Sorensen M. T. Society of
Environmental Toxicology and Chemistry (SETAC Press). Florida, USA.
ISBN: 978-1-880611-93-7.
Miller S.A., Harley K.B. (1999). Zoology 4th ed. WCB, McGraw-Hill. ISBN 0697-34555-6.
Mu X., LeBlanc G.A. (2004). Cross communication between signaling
pathways: juvenoid hormones modulate ecdysteroid activity in a crustacean. J.
Exp. Zool. 301A: 793-801.
Noodt W. (1970). Zur ökologie der Copepoda Harpacticoidea des
Küstengebietes von Tvärminne (Finland). Aca Zoologica Fennica 128: 1-35.
Ogata N., Vogel S.M., Narahashi T. (1988). Lindane but not deltamethrin
blocks a component of GABA-activated chloride channels. Fed. Am. Soc. Exp.
Biol. J. 2: 2895-2900.
Owre H.B., Foyo M. (1967). Copepods of the Florida Current. Fauna Caribaea
1, Crustacea, Part 1. Copepoda, pp. 1-137.
Penttinen O.-P., Kilpi-Koski J., Jokela M., Toivainen K., Väisänen A. (2008).
Importance of dose metrics for lethal and sublethal sediment metal toxicity in
the oligochaete worm Lumbriculus variegatus. J. Soils Sed. 8: 59-66.
Pielou D.P. (1946). Lethal effects of DDT on young fish. Nature 158: 378.
49
Planque B. Batten S.D. (2000). Calanus finmarchicus in the North Atlantic: the
year of Calanus in the context of interdecadal change. ICES J. Mar. Sci. 57:
1528–1535.
Posthuma L., van Straalen N.M. (1993). Heavy-metal adaptation in terrestrial
invertebrates – a review of occurrence, genetics, physiology and ecological
consequences. Comp. Biochem. Phys. C-pharmacology Toxicol. Endocrinology
106: 11-38.
Poulet S.A. (1983). Factors controlling utilization of non-algal diets by particlegrazing copepods. A review. Oceanol. Acta 6: 221-234.
Reddy P.S., Pushpalatha T. (2007). Effect of serotonin on heamolymph
glucose regulation in the fresh water edible crab Oziotelphusa senex senex.
Aquacult. 266: 274-278.
Rewitz K.F., Styrishave B., Løbner-Olesen A., Andersen O. (2006). Marine
invertebrate cytochrome P450: emerging insights from vertebrate and insect
analogies. Comp. Biochem. Physiol. 143C: 363-381.
Ruppert E.E., Fox R.S., Barnes R.D. (2004). Invertebrate zoology – a
functional evolutionary approach. 7th ed. Thomson Brooks/Cole, CA USA.
ISBN 0-03-025982-7.
Saiz E., Calbet A., Fara A., Berdalet E. (1998). RNA content of copepods as a
tool for determining adult growth rates in the field. Limnol. Oceanogr. 43: 465470.
Sakshaug E., Bjørge A., Gulliksen B., Loeng H., Mehlum F. (1994).
Økosystem Barentshavet. Norwegian Research Program for Marine Arctic
Ecology (Promare). Universitetsforlaget, Oslo.
Sargent J.R., Henderson R.J. (1986). Lipids. In: The biological chemistry of
marine Copepods (E.D.S. Corner and S.C.M. O’Hara, eds), pp. 58-108.
Clarendon Press, Oxford.
Sarker S.D., Whiting P., Dinan L., Sik V., Rees H.H. (1999). Identification and
ecdysteroid antagonist activity of three resveratrol trimers (suffruticosols A, B
and C) from Paeonia suffruticosa. Tetrahedon 55: 513-524.
Sarvala J. (1977). The naupliar development of six species of freshwater
harpacticoid Copepoda. Ann. Zool. Fennici. 14: 135-161.
Satapoomin S. (1999). Carbon content of some common tropical Andaman
Sea copepods. J. Plankton Res. 21: 2117–2123.
50
Schindler D.W. (1987). Detecting ecosystem response to anthropogenic stress.
Can. J. Fish Aquat. Sci. 44: 6-25.
Schots A., De Boer J., Schouten A., Roosien J., Zilverentant J.F., Pomp H.,
Bouwman-Smits L., Overmars H., Gommers F.J., Visser B., Stiekema W.J.,
Bakker J. (1992). 'Plantibodies': a flexible approach to design resistance against
pathogens. Neth. J. P1. Path. 98, Supplement 2: 183-191.
Sieglaff D.H., Duncan K.A., Brown M.R. (2005). Expression of genes
encoding proteins involved in ecdysteroidogenesis in the female mosquito,
Aedes aegypti. Insect Biochem. Mol. Biol. 35: 471-490.
SIS (1991). Determination of acute lethal toxicity of chemical substances and
effluents to Nitocra spinipes Boeck – static procedure (in Swedish). Swedish
Standard SS 02 81 06. SIS – Standardiseringskommissionen i Sverige,
Stockholm, Sweden, pp 17.
Skreslet S., Borja A., Bugliaro L., Hansen G., Meerkötter R., Olsen K.,
Verdebout J. (2005). Some effects of ultraviolet radiation and climate on the
reproduction of Calanus finmarchicus (Copepoda) and year class formation in
Arcto-Norwegian cod (Gadus morhua). ICES J. Mar. Sci. 62: 1293-1300.
Smit C.E., Van Gestel C.A.M. (1997). Influence of temperature on the
regulation and toxicity of zinc in Folsomia candida (Collembola). Ecotox.
Environ. Saf. 37: 213-222.
Solomon K.R., Baker D.B., Richards R.P., Dixon K.R., Klaine S.J., La Point
T.W., Kendall R.J., Weisskopf C.P., Giddings J.M., Giesy J.P., Hall L.W. Jr.,
Williams M.W. (1996). Ecological risk assessment of atrazine in North
American surface waters. Environ .Toxicol. Chem. 15: 31–76.
Spaziani E., Rees H.H., Wang W.L., Watson R.D. (1989). Evidences that the
Y-organs of the crab Cancer antennarius secrete 3-dehydroecdysone. Mol. Cell.
Endocrinology 66: 17-25.
Spindler K.-D., Dinan L., Londershausen M. (1984). On the mode of action of
ecdysteroids in crustaceans. In: Hoffmann J., Porchet M. (Eds.). Biosynthesis,
metabolism and mode of action of invertebrate hormones. Springer, Berlin, pp.
255–264.
Subramoniam T. (2000). Crustacean ecdysteroids in reproduction and
embryogenesis. Comp. Biochem. Physiol. C-Toxicol. Pharmacol. 125: 135-156.
Sundby S. (2000). Recruitment of Atlantic cod stocks in relation to
temperature and advection of copepod populations. Sarisa 85: 277-298.
51
Sutcliffe W.H. (1965). Growth estimates form ribonucleic acid content in some
small organisms. Limnol. Oceanogr. 10: 253-258.
Tamone S.H., Chang E.S. (1993). Methyl farnesoate stimulates ecdysteroid
secretion from crab Y-organs in vitro. Gen. Comp. Endocrinology 89: 425-432.
Timbrell J. (2001). Principles of biochemical toxicology, 3rd ed. CRC Press,
Taylor and Francis Ltd., London, UK. ISBN 0-7484-0736-7.
Tomanek L., Somero G.N. (2002). Interspecific- and acclimation-induced
variation in levels of heat-shock proteins 70 (hsp70) and 90 (hsp90) and heatshock transcription factor-1 (HSF1) in congeneric marine snails (genus Tegula):
implications for regulation of hsp gene expression. J. Exp. Biol. 205: 677–685.
US EPA (1992). Framework for ecological risk assessment. EPA/630/R92/100. Washington DC.
van der Geest H.G., Soppe W.J., Greve G.D., Kroon A., Kraak M.H.S. (2002).
Combined effects of lowered oxygen and toxicants (copper and diazinon) on
the marine mayfly Ephoron vrigo. Environ. Toxicol. Chem. 21: 431-436.
van Straalen N.M. (2003). Ecotoxicology becomes stress ecology. Environ.
Sci. Technol. 37: 324A-330A.
Vogt C., Nowak C., Barateiro Diogo J., Oetken M., Schwenk K., Oehlmann J.
(2007). Multi-generation studies with Chironomus riparius – Effects of low
tributyltin concentrations on life history parameters and genetic diversity.
Chemosphere 67: 2192-2200.
Vrede T., Persson J., Aronsen G. (2002). The influence of food quality (P:C
ratio) on RNA:DNA ratio an somatic growth rate of Daphnia. Limnol.
Oceanogr. 47: 487-494.
Wainwright G., Webster S.G., Wilkinson M.C., Chung J.S., Rees H.H. (1996).
Structure and significance of mandibular organ-inhibiting hormone in the crab,
Cancer pagurus. J. Biol. Chem. 271: 12749-12754.
Westin L. (1970). The food ecology and the annual food cycle in the Baltic
population of fourhorn sculpin, Myoxocephalus quadricornis (L.). Pisces. Rep. Inst.
Freshw. Res., Drottningholm 50:168-210
Wild D. (2001). The immunoassay handbook. 2nd ed. Nature Publishing Group.
London. ISBN 0-333-72306-6.
Wilson K., Walker J. (2000). Principles and techniques of practical
biochemistry, 5th ed. Cambridge University Press. Cambrigde, UK. ISBN 0 521
65873X. pp. 206-262.
52
Winberg G.G. (1971). Methods for the estimation of the production of aquatic
animals. Academic Press, London, New York, 175 pp.
Winkaler E.U., Santos T.R.M., Machado-Neto J.G., Martinez C.B.R. (2007).
Acute lethal and sublethal effects of neem leaf extract on the neotropical
freshwater fish Prochilodus lineatus. Comp. Biochem. Physiol. Part C 145: 236–
244.
Wulff F. (1972). Experimental studies on physiological and behavioral response
mechanism of Nitocra spinipes (Crustacea: Harpacticoidea) from brackish water
rockpools. Int. J. Life Oceans Coast. Waters 13: 325-329.
Yang S., Wu R.S.S., Kong R.Y.C. (2002). Physiological and cytological
responses of the marine diatom Skeletonema costatum to 2,4-dichlorophenol.
Aquat. Toxicol. 60: 33-41.
Zou E., Fingerman M. (1997). Effects of estrogenic xenobiotics on moulting of
the water flea Daphnia magna. Ecotoxicol. Environ. Saf. 38: 281-285.
53