Journal of the Marine Biological Association of the United Kingdom, 2012, 92(7), 1595 – 1601.
doi:10.1017/S0025315412000173
# Marine Biological Association of the United Kingdom, 2012
Impact of wave exposure on seasonal
morphological and reproductive responses
of the intertidal limpet Fissurella crassa
(Mollusca: Archaegastropoda)
jose’ pulgar1, marcos alvarez5, alejandro delgadillo1,4, ines herrera1,
samanta benitez1,2, juan pablo morales5, pilar molina3, marcela aldana1,3,6
and victor manuel pulgar7
1
Universidad Andres Bello, Departamento de Ecologı́a & Biodiversidad, República 470, Santiago Chile, 2Universidad Andres Bello
Escuela de Biologı́a Marina, República 440, Santiago, Chile, 3Pontificia Universidad Católica de Chile, Alameda 370, Santiago, Chile,
4
Escuela de Ingenierı́a en Acuicultura, Universidad Andres Bello, República 440, Santiago, Chile, 5Universidad Andres Bello,
Facultad de Ciencias Biológicas, República 217, Santiago, Chile, 6Escuela de Pedagogı́a en Biologı́a y Ciencias, Facultad de Ciencias
de la Educación, Universidad Central de Chile, Santa Isabel 1278, Santiago, 7Center for Research in Obstetrics & Gynecology,
Wake Forest School of Medicine and Biomedical Research Infrastructure Center, Winston-Salem State University, Winston-Salem
NC, USA
Intertidal organisms have long been considered an ideal system to quantify how physical variations determine differential
energy allocations in specimens inhabiting environmental gradients such as exposure to wave action. In habitats with differential intertidal wave exposure (sheltered, Sh; and exposed, E) seasonal gonadal and foot weight variations and their associations with exposure and food availability (algae abundance) were determined in the keyhole limpet Fissurella crassa.
Gonadal weight is used as a measure of reproduction allocation whereas foot weight is an indirect indicator of energy allocation to survival. RNA:DNA ratio in limpets obtained from Sh and E habitats during the two different seasons was used as
an indicator of biosynthetic capability. Our results indicate that algae abundance in E sites was higher in summer and lower
in winter compared to Sh sites. In E sites the muscular foot weight of limpet was higher in summer in contrast to Sh sites where
F. crassa muscular foot weight of limpet was higher in winter. Gonadal weight in Sh sites was higher in summer and remained
constant in winter; whereas in E sites gonadal weight was lower in summer and higher in winter. RNA:DNA ratios indicate
that regardless of intertidal wave exposure, F. crassa showed higher biosynthetic capability in summer. Energetic allocation in
animals that inhabit sheltered intertidal habitats would support constant allocation towards reproduction. In contrast,
animals that inhabit exposed habitats may favour seasonally reproduction allocation at expense of survival.
Keywords: shell morphology, RNA:DNA ratio, energetic trade-off
Submitted 17 January 2012; accepted 25 January 2012; first published online 28 March 2012
INTRODUCTION
Diversity and variability are key characteristics of animal life
(Spicer & Gaston, 1999). Environmental factors influence an
animal’s condition at several levels of biological organization,
including organismal (e.g. feeding rate and metabolic rate:
Sanford, 2002) subcellular levels (e.g. protein synthesis and
gene expression: Somero, 2002). To understand the effects
of climate change on biological phenomena throughout the
biosphere (Hofmann, 2005) is important to evaluate the
organism’s responses to environmental variations. As a
result, there has been increasing interest in determining the
variability in physiological condition and life-history traits
of organisms in their natural habitats (Colman, 1933;
Corresponding author:
J. Pulgar
Email: [email protected]
Wagner et al., 1998; Dahlhoff, 2004; Pulgar et al., 2011).
Physiological constraints are important determinants of the
distribution limits of species and populations (Gaston &
Spicer, 1998; Chown & Gaston, 2000); however, processes
associated with environmental tolerance explaining for
example differential habitat use at the local scale, or species
distribution patterns at the geographical scale, remain
poorly understood.
Rocky intertidal habitats experience a wide range of physical conditions, with daily and seasonal variability including:
degree of immersion; isolation; nutrient availability; and
exposure to different levels of wave action (Newell, 1970;
Truchot & Duhanel-Jouve, 1980). Organisms that inhabit
intertidal rocky shores are strongly influenced by a vertical
tidal emersion (Denny, 1988; Helmuth & Hofmann, 2001;
Somero, 2002) and a horizontal wave exposure gradient
(Jones & Demetropoulos, 1968; Dahlhoff et al., 2002). In intertidal organisms, biochemical and physiological processes and
1595
1596
jose’ pulgar et al.
ultimately organismal performance, are modified in response
to environmental conditions (Stickle & Bayne, 1987; Pulgar
et al., 2011). For instance wave action exerts great forces on
sessile or less mobile organisms, with the risk of dislodgement
being an important selective pressure (e.g. Carrington, 1990;
Gaylord et al., 1994; Denny, 1999). Under these conditions
animals face an energetic trade-off between survival (ability
to attach to the substrate) and reproduction (reproductive
tissue) (Brown & Quinn, 1988; Sibly, 1991). In intertidal molluscs, evidences also indicate that morphological features such
as shell morphology and body size are associated with differences in wave energy (Etter, 1988; Trussell et al., 1993).
Among the biochemical indicators used to determine
physiological condition and metabolic activity in situ the
RNA:DNA ratio is widely used as an index to determine condition of organisms in the field (Chı́charo & Chı́charo, 2008).
This index measures the protein biosynthetic capacity and is
usually correlated with the nutritional status under a given
set of environmental conditions (Buckley & Caldarone,
1999). Organisms in good condition, therefore tend to have
higher RNA:DNA ratios. This index has been used on a
wide range of marine organisms such as those constituting
the phytoplankton (Dortch et al., 1983) and zooplankton
(Ikeda et al., 2007), larval fish (Caldarone et al., 2003), juvenile
and adult fish (Thorpe et al., 1982), bivalves (Chı́charo et al.,
2001), crustaceans (Lemos et al., 2002) and intertidal fish
(Pulgar et al., 2011).
To address physiological responses to wave exposure in a
sessile organism we studied morphometric, reproductive and
in situ physiological variables in the intertidal limpet
Fissurella crassa (Lamarck, 1882) sampled at exposed and
sheltered intertidal sites. Fissurella crassa is distributed from
Peru to Chile (McLean, 1984; Oliva & Castilla, 1992), inhabiting sheltered and exposed intertidal zones (Pino et al.,
1994). These limpets are dioecious without external sexual
dimorphism, bearing a single gonad. Individuals may release
gametes during two major spawning events (Bretos et al.,
1983, 1988; Bretos & Chihuailaf, 1993). Fissurella crassa
shows seasonal differences in growth rate: in late summer
and autumn, there is an accelerated growth, which declines
again in late autumn and winter. This decrease in growth
rate may coincide with the spawning period (Mclean, 1984;
Oliva & Castilla, 1986).
MATERIALS AND METHODS
Quantification of wave action and collection
of F. crassa specimens
Wave action was measured in winter 2009 in intertidal sheltered (Sh) and exposed (E) sectors in central Chile (Quintay
(33811′ S 71841′ W)), using the methodology described by
Doty (1971) and Gerard & Mann (1979). This method
involves the measurement of the rate at which plaster
shapes dissolve assuming their diffusion rate is proportional
to the mass flow of water in motion (Guiñez & Pacheco,
1999). For these measurements ten wear units were used;
these units were dried to constant weight, and during a complete tidal cycle the units were removed and dried at 408C
until achieving constant weight. The decrease in weight of
the units (measured as the difference in weight, D) is
considered an estimation of wave action intensity in each
site (Guiñez & Pacheco, 1999).
Individuals of F. crassa were sampled from exposed
(summer E-S, N ¼ 22, winter E-W, N ¼ 22), sheltered
(summer Sh-S, N ¼ 22, winter Sh-W, N ¼ 22) sectors
during 2008 and winter 2009. Limpets sampled at both
seasons from each sector were deposited in labelled plastic
bags and then transported to the laboratory. Limpet foot
weight (g), gonad weight (g), and individual total weight (g)
were measured using an analytic balance (+/ – 0.01 g precision). We considered gonadal weight as an indirect estimator of reproductive tissue investment and foot weight as a
direct estimator of substrate attaching capability of F. crassa.
Total limpet shell length (cm), shell width (cm) and shell
height (cm) were measured using a digital caliper
(Mitutoyo) (+/ – 0.01mm) and analytic balance (0.01 g).
Limpet sexual condition was determined by direct observation
of gonad colour; male gonads are yellow and female gonads
are green (Olivares et al., 2009).
F. crassa food availability
Food availability for F. crassa in low intertidal of both E and
Sh study sectors, was evaluated using 100-m long transects
parallel to the coast. For each season and degree of exposure
considered, 50 × 50 cm quadrats randomly chosen were surveyed (E-S, N ¼ 12 quadrat; E-W, N ¼ 15, Sh-S, N ¼ 13; and
Sh-W, N ¼ 13). In each quadrat, macroalgae cover (as percentage of total) of Rodophyta (Mazzaella spp.) and Chlorophyta
(Ulva spp.) were considered as indirect estimator of food
F. crassa availability.
Molecular analyses
For molecular analyses, limpets were collected both in winter
(E sectors N ¼ 8 limpets, Sh, N ¼ 9) and summer (E, N ¼ 10
and Sh, N ¼ 10). The extraction of RNA and DNA was performed using TRIZOLw Reagent for the isolation of total
RNA from cells and tissues (Chomczynski & Sacchi, 1987).
We extracted 200 mg of foot tissue from each individual.
During the homogenization of the sample previously
extracted, TRIZOLw Reagent maintains the integrity of the
RNA, while disrupting cells and dissolving cell components.
Addition of chloroform followed by centrifugation separates
the solution into an aqueous phase and an organic phase.
RNA remains exclusively in the aqueous phase. After transfer
of the aqueous phase, the RNA is recovered by precipitation
with isopropyl alcohol. After removal of the aqueous phase,
the DNA in the supernatant can be recovered by sequential
precipitation (Chomczynski, 1993). After extracting, the
RNA and DNA were reconstituted in 50 and 900 ml of
nuclease-free water respectively. Both RNA and DNA were
quantified spectrophotometrically to 260/280 nm (Perkin
Elmer Lambda Bio L7110184) and expressed as mg/ml, corrected for body and sample size.
Statistical analyses
One-way analysis of variance (ANOVA) (general linear
models) was used to compare loss of weight of waste units
among sheltered and exposed sectors. Two-way ANOVA
(general linear models) was used to compare seasonal variations of the morphological and reproductive characteristics
morphological and reproductive response of f. crassa
of F. crassa, and algae richness and abundance between
exposed and sheltered intertidal sampled sites. Season and
wave exposure represent extreme intertidal ecological conditions and were considered fixed factors. The Tukey
a-posteriori test was used to assess specific differences
between factor levels.
Residual analysis was used to evaluate the effect of season
and wave exposure, on foot and gonad weight; results were
expressed with respect to total individual weight. Residual
analysis was also used to compare RNA:DNA ratios between
exposed limpets and sheltered shore limpets, in relation to
fresh body limpet weight. A significance level of P , 0.05
was selected for rejection of a null hypothesis of no significant
differences (Zar, 1996).
Fig. 1. Habitat variability: wave action and food availability. (A) Wave energy
exposition measured as the decrease in weight of wear units (D) in sheltered
(Sh) and exposed (E) sectors: (B) seasonal algae cover (%) in Sh and E
sampled sites. Vertical bars indicate + 1 SEM, ∗ P , 0.05.
Limpet morphology: soft structures
Data describing limpet morphological variables such as
gonadal weight, foot weight and limpet size, are indicated in
Table 1.
Limpets from the Sh sector showed greater gonadal weight in
summer than in winter. In contrast, individuals from the E
sector showed higher gonadal weight in winter (Figure 3A;
Table 5: Tukey a-posteriori test P , 0.05). Residual of foot
weight of F. crassa was higher in winter in the Sh sector
whereas in animals from the E sector the foot weight was
higher in summer (Figure 3B; Table 4: P , 0.05).
Habitat variability: wave action and food
availability
Molecular analysis
RESULTS
Wear units showed a greater weight loss in E than Sh sectors
(Figure 1A; Table 2), indicating that the former sectors are
subjected to greater wave energy. Algae cover was significantly
greater in winter in Sh, and in summer in E sites (Figure 1B;
Table 3), indicating differences in the amount of food availability to F. crassa depending on the season and wave
exposure.
Limpet morphology: shell structures
Analyses of shell morphometric characteristics of F. crassa
indicate that independent of wave exposure length, width
and height shell s were thinner in winter than summer
(Figure 2; Table 4).
Table 1. Basic morphological description of keyhole limpet in sampled
sectors (sheltered and exposed) during both seasons studied (summer, s;
winter, w). Results are expressed as mean + 1 SEM. E/s, exposed/
summer; E/w, exposed/winter; Sh/s, sheltered/summer; Sh/w, sheltered/
winter.
Gonadal weight (g)
Foot weight (g)
Limpet size (cm)
Sector/season
Mean
SEM
E/s
E/w
Sh/s
Sh/w
E/s
E/w
Sh/s
Sh/w
E/s
E/w
Sh/s
Sh/w
4.30
9.10
7.60
9.2
21.82
21.26
18.94
26.40
6.5
1.9
6.2
6.5
0.62
0.91
0.76
0.89
1.6
2.50
1.96
2.32
1.8
2.8
2.3
2.7
Residual of the relationship between RNA:DNA ratio to foot
and gonadal weight indicates that regardless of exposure,
animals present higher RNA:DNA ratios in summer compared to winter (Figure 4; Table 6).
DISCUSSION
Our results indicate that, in the intertidal limpets F. crassa,
traits associated with survival and reproduction, as well as biosynthetic capabilities present environmental and seasonal
variation. Whereas hard structures were thinner in winter,
soft structures showed both exposition and season-related
variations. Gonadal tissue weight was similar between
seasons and foot weight was greater in winter in sheltered
sectors. In the exposed sectors, gonadal weight was higher in
winter and foot weight higher in summer. At the molecular
level, a greater RNA:DNA ratio was observed in summer
regardless of intertidal exposure.
The balance between energy acquisition and expenditure is
critical for animal survival and reproductive success (Sibly,
1991). This balance depends on the interplay between food
intake, digestion, and the allocation of energy to various functions such as growth and reproduction (Karasov, 1986; Wiener,
1992). In animals inhabiting an environment with high physical variability, such as the intertidal system, traits related to
energy allocation dealing with survival (foot development),
Table 2. General linear model (analysis of variance) results comparing
the decrease in mean mass of the waste units from sheltered and
exposed zones. df, degrees of freedom; MS, mean square; F, F value;
P, probability value.
Effect
df
MS
F
P
Wave exposition
1
47.95
14.22
0.0011
1597
1598
jose’ pulgar et al.
Table 3. General linear model (analysis of variance) results comparing
algae abundance of sheltered and exposed zones during summer and
winter. df, degrees of freedom; MS, mean square; F, F value; P, probability
value.
Table 4. General linear model (analysis of variance) results comparing
Fissurella crassa shell length (cm), shell width (cm), shell height (cm) of
sheltered and exposed zones during summer and winter. df, degrees of
freedom; MS, mean square; F, F value; P, probability value.
Effect
df
MS
F
P
Effect
df
MS
F
P
Wave exposure (WE)
Season (S)
WE∗ S
Error
1
1
1
194
1598.90
1109.90
7305.7
2.72
1.89
12.48
0.10
0.17
0.00004
Shell length
Wave exposure (WE)
Season (S)
WE∗ S
Error
1
1
1
84
67.1
60720
24.2
136.8
0.49
443.84
0.17
0.48
0.0001
0.67
Shell width
Wave exposure (WE)
Season (S)
WE∗ S
1
1
1
14.34
19065.93
8.17
0.28
377.78
0.16
0.59
0.0002
0.68
84
50.47
1
1
1
84
0.14
2564.89
2.76
7.29
0.018
323.55
0.34
0.89
0.0003
0.55
and reproduction (reproductive tissue), are under strong selective pressure (Sibly & Calow, 1986; Stearns, 1992; Weiner,
1992). Intertidal invertebrates inhabit a wide range of variation
in a number of physical–chemical variables (Moore & Seed,
1986; Raffaelli & Hawkins, 1996). In this heterogeneous
habitat (e.g. substrate characteristics and physical–chemical
variables), wave action can affect energy intake, the time for
or success in feeding, and some predator species may benefit
from wave prey dislodgement (Sebens, 2002). We characterized
two sectors as sheltered and exposed depending upon the
energy of the wave action observed (Figure 1A). Our results
indicated seasonal variability in algae abundance in these
two sites, with the sheltered sector displaying higher abundance in winter and exposed sites displaying higher algae
abundance in summer (Figure 1B). To understand how the
variable environment in these two sites may affect energy allocations, we determined a number of presumably related morphological parameters in F. crassa. We observed thinner
keyhole shell in winter (Figure 2), probably associated with
wave action, which reportedly affects animal shell mineral
deposition (Moore & Seed, 1986; Raffaelli & Hawkins, 1996).
The effect of wave exposure has been described as a modulator
of body shape, corporal position, movements and thread production in intertidal organisms (Denny & Blanchette, 2000;
Astorga et al., 2002; Moeser et al., 2006). The foot weight
and F. crassa shell variability found in Sh animals and E sites
may be interpreted as wave exposure action on our sampled
population (Figure 2).
The most important energetic trade-off reported occurs
between reproduction and growth energy allocation, where
compensatory responses to habitat variability reveal the
action of natural selection on reproduction and survival
(Warner, 1984; Spicer & Gaston, 1999; Zera & Harshman,
2001). In limpets, gonadal weight is associated with reproductive potential whereas muscular foot weight, responsible for
Error
Shell height
Wave exposure (WE)
Season (S)
WE∗ S
Error
mobility and attachment to the substrate, could be associated
with individual survival (Serra et al., 2001). Our results indicate that gonadal weight as well as foot weight development
showed a seasonal response to environmental variability
(Figure 3). Limpet gonadal weight in sheltered habitats did
not show seasonal changes whereas in exposed habitats
gonadal tissue weight increased from summer to winter
(Figure 3A). On the other hand, foot weight in sheltered habitats increased from summer to winter, whereas in the exposed
sector foot weight decreased from summer to winter
(Figure 3B). The increased foot weight in winter would
enable individuals to remain associated with substrate and
the decrease in foot weight in exposed sites would evidence
the costs of inhabiting a physically stressful habitat in comparison to animals from the most benign sheltered habitat.
We interpret the opposite variation of gonadal weight and
foot tissue weight, i.e. increase in winter in exposed sites
and in summer in sheltered sites (Figure 3), as a strategy to
differentially allocate energy resources associated with different intertidal wave exposure. The change in allocated energy
to growth or reproduction processes is the principal trade-off
described for animals that inhabit environmental gradients
(Chown & Gaston, 1999; Smith et al., 2008).
Table 5. General linear model (analysis of variance) results comparing
Fissurella crassa gonadal and foot weight of sheltered and exposed zones
during summer and winter. df, degrees of freedom; MS, mean square;
F, F value; P, probability value.
Fig. 2. Limpet morphology: hard structures. Shell morphometric variability in
summer and winter. Vertical bars indicate + 1 SEM, ∗ P , 0.05.
Effect
df
MS
F
Gonadal weight
Wave exposure (WE)
Season (S)
WE∗ S
1
1
1
4.37
172.34
108.35
0.79
31.39
19.74
0.37
0.0001
0.0002
0.08
5.83
30.94
0,44
0.002
0.0001
Error
Foot weight
Wave exposure (WE)
Season (S)
WE∗ S
Error
84
5.48
1
1
1
84
0.43
29.60
157.02
5.07
P
morphological and reproductive response of f. crassa
Table 6. General linear model (analysis of variance) results comparing
Fissurella crassa RNA:DNA ratio from sheltered and exposed zones
during summer and winter. df, degrees of freedom; MS, mean square;
F, F value; P, probability value.
Fig. 3. Limpet morphology: soft structures. Seasonal Fissurella crassa gonadal
(A) and foot (B) weight residual in sheltered (Sh) and exposed (E) sectors.
Vertical bars indicate + 1 SEM.
Environmental variability associated with the sheltered
habitat is related to foot weight increase from summer to
winter, without affecting reproduction investment
(Figure 3B). However, in exposed habitats energetic restrictions are evident, and thus drastic decline in foot weight
from summer to winter is necessary to offset the summer to
winter increase in gonadal tissue weight (Figure 3A).
Our results indicate that in sheltered habitats, limpets may
have low maintenance costs compared with limpets inhabiting
exposed sites, and this fact would allow the former to experience an increase in foot weight during winter with no effects
on reproduction. In opposition, animals from stressful habitats may show physiological compensation (Hernandez
et al., 2002; Tomanek & Helmuth, 2002), that would result
in allocation of resources in limpets to reproduction at the
expense of survival (Stearns, 1992; Roff, 2002). Our evidences
indicate that animals in exposed habitats reallocate energy
Effect
df
MS
F
P
Wave exposure (WE)
Season (S)
WE∗ S
Error
1
1
1
41
84.137
4695.73
399.46
0.41
22.96
1.65
0.52
0.00001
0.20
among competing energy functions (Stearns, 1989; Ricklefs
& Wikelski, 2002).
Energetic allocation for the herbivorous F. crassa, is dependent on season, intertidal wave exposure and food availability.
The greater food availability in sheltered sites in winter as well
as the greater food availability in exposed sites in summer is
associated with increased limpet foot weight in both habitats
indicating that the energy budget is mostly allocated to
enhance survival (Figures 1 & 3).
Understanding the mechanisms by which environmental
variability modifies physiological performance of organisms
in nature is of great interest when considering the foundations
of community dynamics (Parmesan & Yohe, 2003; Dahlhoff,
2004). The RNA:DNA ratio is considered as an in situ indicator of the physiological status, because of its association
with the nutritional condition and growth in several marine
organisms (Buckley & Caldarone, 1999; Chı́charo &
Chı́charo, 2008). We observed a higher RNA:DNA ratio in
summer animals, regardless of their intertidal location
(Figure 4); in this context summer limpets would be increasingly well-off nutritionally (Palumbi, 2003).
A higher RNA:DNA ratio in F. crassa in summer suggests
greater protein synthetic activity producing greater foot
weight gain in exposed sites in summer, and greater gonadal
weight gain in sheltered habitats. At the molecular level
our evidences indicate a dynamic seasonal change in the
F. crassa biosynthetic capability (Figure 4), and no effect of
intertidal exposure. A higher summer limpet RNA:DNA
ratio may be associated with an accelerated growth
(McLean, 1984; Oliva & Castilla, 1986) and greater gonadal
tissue weight in summer than in winter (Bretos et al., 1988);
this evidence may help to understand dynamic reproductive
cycles in an important intertidal herbivore. To our knowledge
this represents the first evidence of adjustments in the rates of
energy acquisition and/or energy expenditure in the commercially important herbivorous mollusc F. crassa. This species is
also an important component in the control of the algal community (Aguilera, 2011). These allocations are thought to be
ultimately responsible for positive energy budgets in animals
(Hammond & Wunder, 1991; Piersma & Lindstrom, 1997)
associated with organism ‘decisions’ regarding energy allocation into maintenance, growth and reproduction (Wiener,
1992).
ACKNOWLEDGEMENTS
Fig. 4. Molecular analysis: RNA:DNA ratio. Seasonal RNA:DNA ratio in
limpets from sheltered (Sh) and exposed (E) sectors. Vertical bars
indicate + 1 SEM.
This study was funded by grants DI0508 and DI 17-10/R and
DI-16-12/R to J.P., Universidad Andres Bello. We thank the
Molecular Biology Laboratory staff of Universidad Andres
1599
1600
jose’ pulgar et al.
Bello for their strong support with the molecular biology
experiments.
REFERENCES
Aguilera M. (2011) The functional roles of the hervibores in the rocky
intertidal systems in Chile: a review of food preferences and consumptive effects. Revista Chilena de Historia Natural 84, 241 –261.
Astorga M.P., Guiñez R., Ortiz J.C. and Castilla J.C. (2002) Variación
fenotı́pica y genética en el tunicado Pyura praeputialis (Heller, 1878)
en el área Norte de la Bahı́a de Antofagasta, Chile. Revista Chilena
de Historia Natural 75, 515–526.
Bretos M. and Chihuailaf R. (1993) Studies on the reproduction and
gonadal parasites of Fissurella pulchra (Gastropoda: Prosobranchia).
Veliger 36, 245–251.
Bretos M., Tesorieri I. and Alvarez L. (1983) The biology of Fissurella
maxima Sowerby (Mollusca: Archaeogastropoda) in northern Chile. 2.
Notes on its reproduction. Biological Bulletin. Marine Biological
Laboratory, Woods Hole 165, 559–568.
Bretos M., Gutiérrez J. and Espinoza Z. (1988) Estudios biológicos para
el manejo de Fissurella picta. Medio Ambiente 9, 28–34.
Brown M.K. and Quinn J.F. (1988) The effect of wave action on growth
in three species of intertidal gastropods. Oecologia 75, 420–425.
Buckley L.J. and Caldarone E. (1999) RNA–DNA ratio and other nucleic
acid-based indicators for growth and condition of marine fishes.
Hydrobiologia 401, 265 –277.
Caldarone E.M., Onge-Burns J.M. and Buckley L.J. (2003) Relationship
of RNA/DNA ratio and temperature to growth in larvae of Atlantic
cod, Gadus morhua. Marine Ecology Progress Series 262, 229–421.
Carrington E. (1990) Drag and dislodgment of an intertidal macroalga:
consequences of morphological variation in Mastocarpus papillatus
Kützing. Journal of Experimental Marine Biology and Ecology 139,
185–200.
Chı́charo M. and Chı́charo L. (2008) RNA:DNA ratio on other nucleic
acid derived indices in marine ecology. International Journal of
Molecular Science 9, 1453–1471.
Chı́charo L., Chı́charo M.A., Alves F., Amaral A., Pereira A. and
Regala J. (2001) Diel variation of the RNA:DNA ratios in
Crassostrea angulata (Lamarck) and Ruditapes decussates (Linnaeus
1758) (Mollusca, Bivalvia). Journal of Experimental Marine Biology
and Ecology 259, 121–129.
Chomczynski P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.
Bio-Techniques 15, 532–537.
Chomczynski P. and Sacchi N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Analytic Biochemistry 162, 156 –159.
Chown S.L. and Gaston K.J. (1999) Exploring links between physiology
and ecology at macro scales: the role of respiratory metabolism in
insects. Biological Reviews 74, 87–120.
Chown S.L. and Gaston K.J. (2000) Areas, cradles and museums: the latitudinal gradient in species richness. Trends in Ecology and Evolution 8,
310–315.
Colman J.S. (1933) The nature of the intertidal zonation of plants and
animals. Journal of the Marine Biological Association of the United
Kingdom 18, 435–476.
Dahlhoff E.P. (2004) Biochemical indicators of stress and metabolism:
applications for marine ecological studies. Annual Review of
Physiology 66, 183–207.
Dahlhoff E.P., Stillman J. and Menge B. (2002) Physiological community
ecology: variation in metabolic activity of ecologically important rocky
intertidal invertebrates along environmental gradients. Integrative and
Comparative Biology 42, 862–871.
Denny M.W. (1988) Biology and the mechanics of the wave-swept environment. Princeton, NJ: Princeton University Press 329 pp.
Denny M.W. (1999) Are there mechanical limits to size in wave-swept
organisms? Journal of Experimental Biology 202, 3463–3467.
Denny M.W. and Blanchette C.A. (2000) Hydrodynamics, shall shape,
behavior, and survivorship in the owl limpet, Lottia gigantea.
Journal of Experimental Biology 203, 2623–2639.
Dortch Q., Roberts T.L., Clayton J.R. and Ahmed S.I. (1983) RNA/DNA
ratios and DNA concentrations as indicators of growth rate and
biomass in planktonic organisms. Marine Ecology Progress Series 13,
61–71.
Doty M.S. (1971) Measurement of water movement in reference to
benthic algal growth. Botánica Marina 14, 32–35.
Etter R.J. (1988) Physiological stress and color polymorphism in the
intertidal snail Nucella lapillus. Evolution 42, 660–680.
Gaston K. and Spicer J. (1998) Do upper thermal tolerances differ in geographically separated populations of the beachflea Orchestia gammarellus (Crustacea: Amphipoda)? Journal of Experimental Marine Biology
and Ecology 229, 256–276.
Gaylord B., Blanchette C.A. and Denny M.W. (1994) Mechanical consequences of size in wave-swept algae. Ecological Monographs 64, 287–
313.
Gerard V. and Mann K.H. (1979) Growth and production of Laminaria
longicruris (Phaeophyta) populations exposed to different intensities
of water movement. Phycology 15, 33–41.
Guiñez R. and Pacheco C. (1999) Estimaciones de la velocidad máxima
del oleaje en el intermareal rocoso de Chile Central, utilizando un
dinamómetro prototipo. Revista Chilena de Historia Natural 72,
251–260.
Hammond K.A. and Wunder B.A. (1991) The role of diet quality and
energy need in the nutritional ecology of a small herbivore: Microtus
ochrogaster. Physiological Zoology 64, 541 –567.
Helmuth B. and Hofmann G.E. (2001) Microhabitats, thermal heterogeneity, and patterns of physiological stress in the rocky intertidal
zone. Biological Bulletin. Marine Biological Laboratory, Woods Hole
201, 374 –384.
Hernandez C.E., Neill P.A., Pulgar J.M., Ojeda F.P. and Bozinovic F.
(2002) Water temperature fluctuations and territoriality in the intertidal zone: two possible explanations for the elevational distribution of
body size in Graus nigra (Kyphosidae). Journal of Fish Biology 61,
472–488.
Hofmann G.E. (2005) Patterns of Hsp gene expression in ectothermic
marine organisms on small to large biogeographic scales. Integrative
and Comparative Biology 45, 247–255.
Ikeda T., San F., Yamaguchi A. and Matsuishi T. (2007) RNA:DNA
ratios of calanoid copepods from the epipelagic through abyssopelagic
zones of the North Pacific Ocean. Aquatic Biology 1, 99–108.
Jones W.E. and Demetropoulos A. (1968) Exposure to wave action:
measurements of an important ecological parameter on rocky shores
on Anglesey. Journal of Experimental Marine Biology and Ecology 2,
46–63.
Karasov W.H. (1986) Energetics, physiology and vertebrate ecology.
Trends in Ecology and Evolution 1, 101 –104.
Lemos D., Garcia-Carren F.L., Hernández P. and Toro A.N. (2002)
Ontogenetic variation in digestive proteinase activity, RNA and
morphological and reproductive response of f. crassa
DNA content of larval and postlarval white shrimp Litopenaeus
schmitti. Aquaculture 214, 363–380.
Gastropoda) in the eastern Pacific. Journal of the Marine Biological
Association of the United Kingdom 81, 485 –490.
McLean J.H. (1984) Systematics of Fissurella in the Peruvian and
Magellanic faunal provinces (Gastropoda: Prosobranchia).
Contributions in Science: Natural History Museum of Los Angeles
County 354, 1–70.
Sibly R.M. (1991) The life-history approach to physiological ecology.
Functional Ecology 5, 184–191.
Moeser G.M., Leba H. and Carrington E. (2006) Seasonal influence of
wave action on thread production in Mytilus edulis. Journal of
Experimental Biology 209, 881–890.
Moore P.G. and Seed R. (1986) The ecology of rocky coasts. New York:
Columbia University Press, 467 pp.
Newell R.C. (1970) Biology of intertidal animals. Faversham, UK: Marine
Ecological Survey, 556 pp.
Sibly R.M. and Calow P. (1986) Physiological ecology of animals: an evolutionary approach. Oxford: Blackwell Scientific Publications, 190 pp.
Smith J.M., Green S.J., Kelley C.A., Prufert-Bebout L. and Bebout B.M.
(2008) Shifts in methanogen community structure and function
associated with long-term manipulation of sulfate and salinity in a
hypersaline microbial mat. Environmental Microbiology 10, 386–394.
Somero G.N. (2002) Thermal physiology of intertidal animals: optima,
limits, and adaptive plasticity. Integrative and Comparative Biology
42, 780–789.
Oliva D. and Castilla J. (1986) The effects of human exclosure on the
population structure of key-hole limpets Fissurella crassa and
Fissurella limbata in the coast of Central Chile. Marine Ecology 7,
201–217.
Spicer J. and Gaston K. (1999) Physiological diversity and its ecological
implications. Oxford: Blackwell Scientific Publications, 240 pp.
Oliva D. and Castilla J. (1992) Guı́a para el reconocimiento y
morfometrı́a de diez especies del género Fissurella Bruguière, 1789
(Mollusca:Gastropoda) comunes en la pesquerı́a y conchales
indı́genas de Chile central y sur. Gayana Zoologı́a 56, 77–108.
Stearns S.C. (1992) The evolution of life histories. Oxford: Oxford
University Press, 249 pp.
Olivares A., Jofré D., Alvarez C. and Bustos C. (2009). Hermafroditismo
funcional de la gónada de Fissurella crassa (Mollusca: Fissurellidae).
International Journal of Morphology 27, 509–514.
Palumbi S.R. (2003) Ecological subsidies alter the structure of marine
communities. Proceedings of the National Academy of Sciences of the
United States of America 21, 11927–11928.
Parmesan C. and Yohe G. (2003) A globally coherent fingerprint of
climate change impacts across natural systems. Nature 421, 37–42.
Pino C., Oliva D.P. and Castilla J. (1994) Ritmos de actividad en lapas
Fissurella crassa Lamarck 1822 y F. latimarginata Sowerby 1835:
efectos del ciclo de marea y fotoperiodo. Revista de Biologı́a Marina
29, 89–99.
Piersma T. and Lindstrom A. (1997) Rapid reversible changes in organ
size as a component of adaptive behaviour. Trends in Ecology and
Evolution 12, 134–138.
Pulgar J., Alvarez M., Morales J., Garcia-Huidobro M., Aldana M.,
Ojeda F.P. and Pulgar V.M. (2011) Impact of oceanic upwelling on
morphometric and molecular indices of an intertidal fish
Scarthichthys viridis (Blennidae). Marine and Freshwater Behaviour
and Physiology 44, 33–42.
Raffaelli D. and Hawkins S. (1996) Intertidal ecology. London: Chapman
and Hall, 356 pp.
Ricklefs R. and Wikelski M. (2002) The physiology/life-history nexus.
Trends in Ecology and Evolution 17, 462–468.
Roff D.A. (2002) Life history evolution. Sunderland, MA: Sinauer
Associates, 465 pp.
Sanford E. (2002) Community responses to climate change: links between
temperature and keystone predation in a rocky intertidal system. In
Schneider S.H. and Root T.L. (eds) Wildlife responses to climate
change: North American case studies. Covelo, CA: Island Press, pp.
165–200.
Sebens K.P. (2002) Energetic constraint, size gradient, and size limits in
benthic marine invertebrates. Integrative and Comparative Biology
142, 853 –861.
Serra G., Chelazzi G. and Castilla J.C. (2001) Temporal and spatial
activity of the key-hole limpet Fissurella crassa (Mollusca:
Stearns S.C. (1989) Trade-offs in life-history. Evolution 3 259–268.
Stickle W.B. and Bayne B.L. (1987) Energetics of the muricid gastropod
Thais (Nucella) lapillus. Journal of Experimental Marine Biology and
Ecology 107, 263–278.
Thorpe J.E., Talbot C. and Villarreal C. (1982) Bimodality of growth and
smolting in Atlantic salmon, Salmo salar L. Aquaculture 28, 123–132.
Tomanek L. and Helmuth B. (2002) Physiological ecology of rocky intertidal organisms: a synergy of concepts. Integrative and Comparative
Biology 42, 771–775.
Truchot J.P. and Duhanel-Jouve A. (1980) Oxygen and carbon dioxide in
the marine intertidal environments: diurnal and tide changes in rockpools. Respiration Physiology 39, 241–254.
Trussell G.C., Johnson A.S., Rudolph S.G. and Gilfillan E.S. (1993)
Resistance to dislodgment: habitat and size-specific differences in morphology and tenacity in an intertidal snail. Marine Ecology Progress
Series 100, 135–144.
Wagner M., Durbin E. and Buckley L. (1998) RNA:DNA ratios as indicators of nutritional condition in the copepod Calanus finmarchicus.
Marine Ecology Progress Series 162, 173–181.
Warner R.R. (1984) Deferred reproduction as a response to sexual selection in a coral reef fish: a test of the life historical consequences.
Evolution 38, 148–162.
Wiener J. (1992) Physiological limits to sustainable energy budgets in
birds and mammals: ecological implications. Trends in Ecology and
Evolution 7, 384–388.
Zar J.H. (1996) Biostatistical analysis. 5th edition. Englewood Cliffs, NJ:
Prentice-Hall, 622 pp.
and
Zera A.J. and Harshman L.G. (2001) Physiology of life history trade-offs
in animals. Annual Review of Ecology, Evolutions, and Systematics 32,
95–106.
Correspondence should be addressed to:
J. Pulgar
Universidad Andres Bello
Departamento de Ecologı́a & Biodiversidad
República 470, Santiago Chile
email: [email protected]
1601