17

Experimental study of the dependence of embryonic

development of Trachurus trachurus eggs on temperature
M. Emı́lia Cunha, Catarina Vendrell, and Patrı́cia Gonçalves

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Cunha, M. E., Vendrell, C., and Gonçalves, P. 2008. Experimental study of the dependence of embryonic development of Trachurus trachurus
eggs on temperature. – ICES Journal of Marine Science, 65: 17– 24.
To determine the effect of temperature on the development rates of artificially fertilized eggs of Trachurus trachurus, experiments
were carried out at temperatures ranging from 10.58C to 198C. Egg development through to hatching only took place at 11.7 –
198C. At lower temperature, eggs did not develop beyond the stage where the outline of the embryo was clearly discernible and a
defined median line of the embryonic shield (stage 4 in this study) was apparent. Development time took from 46 h at 198C to
126 h at 128C. A generalized linear model of the stage-dependent development time (age) as a function of incubation temperature
was developed. The data are also compared with those reported in the literature and related to sea temperature on the spawning
grounds.
Keywords: egg development, NE Atlantic, temperature, Trachurus trachurus.
Received 14 April 2007; accepted 17 October 2007; advance access publication 19 November 2007.
M. E. Cunha: Estação Piloto de Piscicultura de Olhão, L-IPIMAR/CRIPSul, Av. 5 de Outubro s/n, 8700-305 Olhão, Portugal. C. Vendrell and
P. Gonçalves: L-IPIMAR, Av. de Brası́lia, 1449-006 Lisboa, Portugal. Correspondence to M. E. Cunha: tel: þ351 289715346; fax: þ351 289715579;
e-mail: micunha@ipimar.pt

Introduction Different approaches have been used to model the time

Horse mackerel (Trachurus trachurus) are heavily exploited
throughout the Northeast (NE) Atlantic. Estimates of spawning-
stock biomass (SSB), and detailed knowledge of population age
structure and spawning characteristics, are used to produce an
appropriate total allowable catch (TAC) in Atlantic –Iberian
waters (Priede, 1994).
The daily egg production method (DEPM) (Lasker, 1985) is an
appropriate method to estimate the SSB of fish with indeterminate
fecundity and pelagic eggs, such as horse mackerel. An important
requirement of this approach is the need to know the age-
dependent abundance of eggs on the spawning grounds, assuming
constant mortality. Accurate staging of the eggs is important
because each stage reflects a possible age range, based on the esti-
mated time of day they were spawned and local water temperature
(Lo, 1985). Previous models of temperature-dependent egg devel-
opment rates for T. trachurus were developed by King et al. (1977)
for South Africa, and by Pipe and Walker (1987) for the southwest
coast of Ireland. Although the work of Pipe and Walker (1987)
dealt with a population of T. trachurus from the NE Atlantic, the
stock in Atlantic – Iberian waters is considered to be a discrete,
more southerly population (Murta, 2003), and it may be differ-
ently adapted to the local environment. Spawning of the
Atlantic –Iberian stock takes place mainly in waters ranging from
158C to 178C (mean 15.78C), some 38C warmer than the stock
studied by Pipe and Walker (1987) in the Celtic Sea. Moreover,
the stages described by Pipe and Walker (1987) do not provide
sufficient differentiation to provide accurate age estimates at temp-
eratures ,178C.
taken for fish eggs to develop at various temperatures. Lo (1985)
fitted the development time for each stage separately and used
non-linear fitting algorithms to estimate parameters, assuming a
log-normal error distribution. Chambers and Leggett (1989)
proposed the use of event analysis to quantify stage transition in
early life history studies. Using the latter approach, Pepin et al.
(1997) found that a Weibull error distribution was more appropri-
ate. A multinomial approach could have been appropriate to
describe stage-classified populations (Ibaibarriaga et al., 2007).
Generalized linear models (GLMs) are recommended by
Jiao and Chen (2004) to identify error structures in sequential
population analysis.
In this study, horse mackerel eggs from Atlantic – Iberian waters
were fertilized in vitro and allowed to develop to hatching under
controlled temperature conditions. We present a revised descrip-
tion of easily identifiable embryonic development stages that is
intended to provide greater resolution for age determination.
We developed a GLM of the age-structured sequential egg popu-
lation (with multiple stages per observation) as a function of
water temperature. GLMs offer a maximum-likelihood-based
method that provides a systematic framework by which par-
ameters can be estimated when the model error belongs to the
exponential family. One of the advantages of GLMs is that they
can readily deal with many types of error structures (Jiao et al.,
2004). We paid specific attention to identifying the appropriate
error structure of the model, and considered log-normal,
gamma, and Poisson distributions in modelling and diagnostic
analysis.

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18 M. E. Cunha et al.

Material and methods

During a survey aboard RV “Capricórnio” in February 2004, and
aboard RV “Noruega” in March 2005 and February 2006, hydrated
oocytes and semen from several mature horse mackerel were
stripped by gentle pressure on their abdomen. Two experiments
were performed during the 2004 cruise and one during each of
the other two cruises (Table 1). The oocytes and the semen were
stirred together with a small amount of seawater, then transferred

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into 1-l glass beakers filled with seawater. After 30 min, the floating
(fertilized) eggs were carefully collected and washed with local fil-
tered seawater, and distributed into several 1-l glass beakers filled
with filtered local seawater and incubated in baths at different con-
trolled temperature. Two replicate experiments were performed
for each temperature.
Incubations were performed at six temperatures, four lower and
two higher than ambient. The lower temperatures were maintained
by four recirculation reservoirs, each containing titanium evapor-
ators and temperature probes connected to a control thermostat.
The higher temperatures were maintained with two commercially
available aquarium heaters. The overall system (Figure 1) consists
of six 60-l tanks, each containing six 1-l beakers that float when
supported by Styrofoam plates. Gentle aeration of filtered seawater
in the beakers was accomplished with an aquarium pump.
Temperatures were set from 108C to 208C (see Table 1 for initial
settings). The choice of these temperatures was based on Portuguese
oceanic and coastal water temperatures during the horse mackerel
spawning season, which range from 138C to 188C. The temperature
in each tank was slightly different from the initial settings, as indi-
cated in Table 1, and varied within +0.58C.
Time 0 was defined as the time when the fertilized eggs were
placed in each beaker. From then on, in the first experiment,
eggs were sampled every hour during the first 18 h, and Figure 1. Schematic representation of the temperature-controlled
egg incubator.

Table 1. Basic information for the four egg incubation experiments

performed in this study. subsequently every 2 h until hatching. In the second experiment,
hourly sampling was only carried out during the first 12 h.
Date Collection Bath temperature (88 C) Sampling
During the third experiment, sampling was random, with eggs col-
time programme
Initial Measured lected at intervals that varied between 1 and 12 h. During the
settings mean fourth experiment, sampling was carried out every 2 h during
temperature the first 18 h and every 4 h thereafter. At each egg collection, the
(CV) time and the temperature of each incubation tank were recorded.
22 11:50 10; 10.6 (0.04); Every hour Eggs were sampled randomly and immediately fixed in 4% formal-
February 12; 12.5 (0.04); during the first dehyde for later staging. Hatched larvae were allowed to grow but
2004 14; 14.9 (0.03); 18 h; every 2 h not to feed. Eggs were examined under a binocular dissecting
16; 16.0 (0.03); until hatching microscope, and classified into one of the 11 stages shown in
18; 17.4 (0.04);
Figure 2, each lasting ,24 h.
20 19.0 (0.03)
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .
Embryonic development stages were adapted from the sardine
26 18:17 10; 10.6 (0.04); Every hour
egg classification of Gamulin and Hure (1955):
February 12; 12.9 (0.05); during the first
2004 14; 14.5 (0.03); 12 h; every 2 h
16; 15.8 (0.03); until hatching Stage 1: First segmentation, which, under dim reflected light, is
18; 17.1 (0.03); easily visible (Figure 2a). The stage lasts until individual cells
20 18.6 (0.03)
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . are easily distinguishable from each other, and counting them
13 March 16:30 20 18.6 (0.01) Variable is possible (64 cells). Equivalent to stages IA of Pipe and
2005 intervals
. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . Walker (1987), and 1 of King et al. (1977).
12 17:40 10; 11.0 (0.06); Every 2 h
February 12; 11.7 (0.02); during the first Stage 2: Cleavage proceeds until a blastodermal cap is formed,
2006 14; 13.3 (0.02); 18 h; every 4 h making individual cells indiscernible (Figure 2b). Equivalent to
16 15.2 (0.03) until hatching stages IA of Pipe and Walker (1987), and 1 of King et al. (1977).
Variation of temperature in the baths is represented as the coefficient of Stage 3: Development of the segmentation cavity. First appear-
variation (CV) of the mean. ance of a germinal ring, with a small peak at one pole (the
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Figure 2. The developmental egg stages of horse mackerel used in this study.

embryonic shield) (Figure 2c). Equivalent to stages IB of Pipe
and Walker (1987), and 1 of King et al. (1977).
Stage 4: First appearance of an embryonic axis. The outline
of the embryo is clearly defined in the median line of the
embryonic shield. The embryo develops, but the head and
tail are not yet discernible (Figure 2d). Equivalent to stages
II of Pipe and Walker (1987), and 1 and 2 of King et al.
(1977).
Stage 5: The cephalic region becomes apparent and an outline of
the optic vesicles can be discerned. Germinal ring development
20
proceeds around the yolk (Figure 2e). Equivalent to stages II of
Pipe and Walker (1987), and 2 of King et al. (1977).
Stage 6: This is marked by the closure of the blastopore. The tail
end develops and is swollen at its tip. Abdominal somites
take shape. The angle formed by the tail and yolk is 908
(Figure 2f). Equivalent to stages II of Pipe and Walker
(1987), and 2 and 3 of King et al. (1977).
Stage 7: The embryo tail begins to separate from the yolk mass.
The angle formed by the tail and yolk is ,908. Pupils can be
discerned in the eyes. Pigment spots appear in two rows
along the dorsal body contour and especially around the oil
globule (Figure 2g). Equivalent to stages III of Pipe and
Walker (1987), and 3 and 4 of King et al. (1977).
Stage 8: Growth of the tail still short of three-quarters of the egg
circumference (Figure 2h). Equivalent to stages III of Pipe and
Walker (1987), and 4 of King et al. (1977).
Stage 9: Embryo length is three-quarters of egg circumference.
Pigment spots develop in the caudal region (Figure 2i).
Equivalent to stages III of Pipe and Walker (1987), and 4 of
King et al. (1977).
Stage 10: Embryo length exceeds three-quarters of egg circum-
ference until the tail reaches the head (Figure 2j). Equivalent to
stages IV of Pipe and Walker (1987), and 5 of King et al. (1977).
Stage 11: Tail grows past the embryo head (Figure 2k), and
hatching takes place. Equivalent to stages IV of Pipe and
Walker (1987), and 5 of King et al. (1977).
Data analysis
The data are available from the senior author on request. Because the
initial numbers of eggs in the beakers were unknown, it was not poss-
ible to determine either total abundance or mortality. Instead, the
abundance of each stage in the samples was converted into ratios in
relation to the total number of eggs in that sample, and these were
used as weighting factors. Ages were calculated as the time elapsed
from when the fertilized eggs were placed in each of the beakers
(time 0) to sampling. The unexpected presence of egg stages whose
probabilities were ,0.05 was considered as zero, because they
could represent dead eggs, and experiments where no eggs developed
past a certain stage were not considered for the model.
The abundance of eggs at age was the outcome of an exper-
iment involving the random sampling of fixed time intervals
followed by observation of the number of discrete events per
sampling unit, which often have the characteristics of a Poisson
process (Dowdy and Wearden, 1990). We assumed that the appro-
priate probability model for the number of occurrences of egg
stages in the specified time interval and temperature is a Poisson
distribution, as described by Dowdy and Wearden (1990).
We started by modelling the dependence of development time
on temperature for each stage separately. Because the relationship
between the mean and the variance of the age of each egg stage
was exponential, we loge-transformed the development time. The
model was fitted using ordinary least squares, and tested for equal-
ity of intercepts and slopes within stages, using analysis of covari-
ance (Dowdy and Wearden, 1990). Because the differences
between slopes were not significant, we assumed a common trend
for all stages, and fitted a simpler generalized linear model (GLM;
McCullagh and Nelder, 1989; Zuur et al., 2007), with a Poisson dis-
tribution and log-link function to describe the exponential
M. E. Cunha et al.
relationship between eggs-at-age (with multiple stages per obser-
vation) with incubation temperature, using the egg stage as a categ-
orical variable. Age (Age_hoursi) was the response variable, and the
temperature of the incubation bath (Tempi) and egg stage (Stagei)
were explanatory variables. Ratios of the abundance of eggs of each
age at each stage were used as weighting factors.
The Poisson regression model can be summarized as
Ri  pðmi Þ and E½Age hoursi  ¼ mi
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¼ expða þ b Tempi þ Stagei Þ;
or loge ðE½Age hoursi Þ ¼ a þ bTempi þ Factor for Stagei :
The estimated slope (b) is the rate of ageing with temperature
and the intercept (a) the mean development time for all stages at
08C. Factor is a factorial measure for each stage and represents the
mean age at constant temperature.
Other than the Poisson error distribution, we explored two
other error structures commonly used in ontogenic development
of marine fish: log-normal and gamma. Of all the error assump-
tions used in a GLM, the one that gives the most homogeneous
deviance residuals is considered the most appropriate. To diagnose
whether the model errors are homogeneous, we used its residuals
(Zuur et al., 2007) plotted against the predicted age of the eggs
(given the model and estimates of its parameters), and the plots
were inspected visually. A distinct pattern indicated lack of hom-
ogeneity. We validated our choice of “best” model using quantile–
quantile plots (QQ–plots; Zuur et al., 2007). The residuals from
each particular error distribution were plotted against quantile
points (the difference between a quantile and percentile point is
a factor of 100), to determine how well the model fitted the
data. The best fit was considered the one where the resulting
points lay closer to a straight line.
The Brodgar (v 2.5.2) software package (# Highland Statistics
Ltd, 2000), which contains an interface to the statistics package
R (version 1.8.1), was used for the analysis.
Results
The dependence of development time of each egg stage on temp-
erature illustrates the temporal evolution of egg stages, with the
distribution of older stages displaced to the upper right corner
of each plot (Figure 3). The first two stages (1 and 2) displayed
great variability in age at lower temperature (Figure 3) because
of the very long development times. In fact, eggs did not hatch
at the coldest temperature of the incubation baths (108C), which
corresponded to mean incubating temperatures of 10.6– 118C
(Table 1). The embryos must have died in the early stages of clea-
vage, because the oldest stage attained by these eggs was Stage 4
(Figure 2d). At 128C (mean 11.78C), the second coldest tempera-
ture, the eggs were able to hatch and the total development time
was less than at 138C. However, this result was derived from a
single experiment performed in February 2006 (Table 1).
The temperature dependence of development time is strong, all
stages exhibiting an exponential relationship between age and
temperature (Table 2). All stages also share a common slope,
because the rate of development with temperature was the same
within the 11 stages, with no significant differences (F10,614 =
0.195, p . 0.999).
Intercepts and slopes obtained with a generalized linear model
with Poisson error distribution were highly significant (p . 0.001;
Dependence of embryonic development of Trachurus trachurus eggs on temperature 21

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Figure 3. Conditional scatterplots showing the relationship between age (h) and the incubation temperature for each of the 11 horse
mackerel egg stages. Each plot represents one stage (St.).

Table 3) and the deviance residuals of the model were homogeneous Discussion
(Figure 4). When log-normal and gamma error distributions were The experimental study presented here made it possible to describe
used, the residuals were heterogeneous (Figure 4). The standard for the Atlantic –Iberian horse mackerel stock the exponential
deviance of the residuals lay closer to a straight line in the QQ– increase in age of eggs with decreasing temperature using egg
plots (Figure 5), showing that the assumption of a Poisson distri- stage as a categorical variable. The results showed that all stages
bution of model errors is acceptable, but not ideal. The goodness exhibited the same semi-log relationship between age and temp-
of fit of the model is also demonstrated by the straight line with erature, with a common temperature factor of exp(kT), where
slope 1 of the plot of predicted against observed egg development Œk = 20.145. It is likely that the development of eggs is governed
time (Figure 6), although with slightly greater spread for the older by a single underlying process rather than by a succession of
ages, reflecting the greater variability at older age. different processes with differing temperature dependences. The
The result of fitting the model is shown in Figure 7. With this controlling processes are probably parts of the respiratory
model, the time-lag to 50% of hatching (Stage 11) at the lowest
and highest incubation temperatures (128C and 198C, respect-
ively) are 126 and 46 h. The slope obtained with the GLM
(Table 3; – 0.145) corresponds to an ageing rate 15% faster for Table 3. Results of the GLM with Poisson distribution and log-link
each degree rise in temperature. The average change in develop- function describing the development time of horse mackerel eggs
ment rate for a 108C change in temperature (Q10) was 4.3. as a function of temperature and egg stage.
Coefficients Estimate s.e. z-value
Intercept ( b ) 3.819 0.0788 48.44*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Table 2. Individual stage regression lines of age on temperature. Slope ( a ) 20.145 0.0037 239.01*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .

Group r2 n Slope s.e. t-value Factor for Stage 2 0.806 0.0690 11.68*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .

Stage 1 0.293 56 20.137 0.0284 24.82* Factor for Stage 3 1.258 0.0757 16.61*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 2 0.556 86 20.132 0.0127 210.36* Factor for Stage 4 1.557 0.0649 24.00*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 3 0.796 39 20.132 0.0107 212.34* Factor for Stage 5 1.839 0.0685 26.86*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 4 0.745 68 20.133 0.0094 214.09* Factor for Stage 6 2.061 0.0621 33.18*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 5 0.930 32 20.130 0.0063 220.62* Factor for Stage 7 2.260 0.0635 35.60*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 6 0.872 62 20.140 0.0068 220.57* Factor for Stage 8 2.380 0.0624 38.13*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 7 0.890 46 20.145 0.0075 219.26* Factor for Stage 9 2.500 0.0654 38.22*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 8 0.890 68 20.145 0.0063 223.08* Factor for Stage 10 2.626 0.0605 43.41*
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 9 0.893 36 20.135 0.0078 217.34* Factor for Stage 11 2.756 0.0673 40.92*
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
Stage 10 0.888 75 20.147 0.0060 224.43* The Factor for Stage 1 is 0. The Poisson regression model is: (E[Age_hoursi]) ¼
. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .
a þ b Tempi þ Factor for Stagei, where E[Age_hoursi]) ¼ mean age of stagei
Stage 11 0.855 20 20.152 0.0140 210.86* (in h) ¼ exp(a þ b Tempi þ Factor for Stagei). z-values are all significant at
Age is loge-transformed, and t-values are all significant at p , 0.001 (*). p , 0.001 (*).
22 M. E. Cunha et al.

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Figure 4. Homogeneity diagnostic plot of deviance residuals under three error assumptions using a GLM to describe the exponential
relationship between the age of eggs and incubation temperature, with egg stage used as a categorical variable for (a) the Poisson,
(b) the gamma, and (c) the log-normal error distributions.

Figure 5. Best fit diagnostic plot of the deviance residuals under three error assumptions in the GLM describing the exponential relationship
between age of the eggs and incubation temperature, using the egg stage as a categorical variable for (a) the Poisson, (b) the gamma, and
(c) the log-normal error distributions.

Figure 6. Relationship between predicted and observed age for all

developmental stages of horse mackerel off Portugal using a Poisson Figure 7. Stage-dependent predicted development times of horse
error distribution. mackerel eggs off Portugal in relation to temperature.
Dependence of embryonic development of Trachurus trachurus eggs on temperature
metabolism, and they prevent temperature from disorganizing
development. Temperature-dependence is quite strong and a
108C rise in temperature leads to an ageing rate 4.3 times faster.
It is interesting that the magnitude of the temperature effect
observed in this study is similar to that observed for species
living in very different temperature conditions, such as cod
(Gadus morhua) and haddock (Melanogrammus aeglefinus). Data
from Pepin et al. (1997) yielded a Q10 of 3.7 for cod, and from
Page and Frank (1989) a value of 4.6 for haddock. This suggests
that the process that drives the maturation of eggs evolved with
a compensation for temperature. The implication is that differ-
ences in embryonic development time in fish species may be
caused not by temperature but rather by the size of the embryo
(Clarke, 1983).
The use of this information, together with the new staging
scheme described here, permits better assignment of age, in
days, to field-collected eggs than previously possible. Although
earlier descriptions of the egg stages of horse mackerel (King
et al., 1977; Pipe and Walker, 1987) do exist, some stages lasted
considerably longer than 1 day at the range of temperatures
found in Atlantic surface waters of the Iberian Peninsula during
the horse mackerel spawning season (13– 188C), and therefore
may have limited the accuracy of assignment of age. The longest
period between stages using the egg staging methodology
suggested here (between stages 6 and 7) is 13 h at 138C, but this
still allowed good age resolution even at low temperature.
According to Berenbeim et al. (1973), the optimum temperature
for development of Trachurus eggs off the Iberian Peninsula is
168C. At that temperature, the total development time for
horse mackerel eggs in Atlantic waters of the Iberian Peninsula
(as determined here) and in the Celtic Sea (Pipe and Walker,
1987) are similar, 70 h. In contrast, the development time off
Namibia at 168C was much shorter, i.e. 51 h (King et al., 1977).
Pipe and Walker (1987) attribute this difference, at least in part,
to the fact that King et al. (1977) used eggs from the wild and con-
ducted their experiments on eggs already at the blastodisc stage
(stage 2 in the present work, and stage IA of Pipe and Walker,
1987).
In general, horse mackerel egg development off the Atlantic
coast of the Iberian Peninsula lasts from 46 h at 198C to 126 h at
128C. These results are similar to the values of 50% hatching
obtained by Pipe and Walker (1987) for the Celtic Sea. The main
difference is the temperature at which eggs developed through to
hatching. In Atlantic waters of the Iberian Peninsula, T. trachurus
did not hatch at a mean temperature ,11.78C, but in the Celtic
Sea (Pipe and Walker, 1987) some eggs hatched at 10.48C,
though with high mortality. These results suggest that off the
Iberian Peninsula, spawning to be successful must take place at
temperatures .128C, whereas in the Celtic Sea it can be at slightly
lower temperature, but still .118C, supporting the observations of
Letaconnoux (1951), who suggested that spawning would not com-
mence where surface water temperature was ,118C.
Acknowledgements
We thank editor Pierre Pepin and two anonymous reviewers for
constructive comments on the submitted manuscript, and
A. J. C. G. Murta for similar input to an earlier version. We also
thank the crew of RVs “Capricórnio” and “Noruega” for their
support in conducting the experiments. The work forms part of
the QCA-3/MARE/FEDER-NeoMAV project. PG was supported
23
through a grant from Plano Nacional de Recolha de Dados-
PNAB/EU DCR-Data Collection Regulation.
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