51

Using multinomial models to analyse data from Iberian

sardine egg incubation experiments: a comparison
with traditional techniques
Miguel Bernal, Leire Ibaibarriaga, Ana Lago de Lanzós, Mike E. Lonergan, Carmen Hernández,

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Concha Franco, Inmaculada Rasines, Luis Valdés, and David L. Borchers

Bernal, M., Ibaibarriaga, L., Lago de Lanzós, A., Lonergan, M. E., Hernández, C., Franco, C., Rasines, I., Valdés, L., and Borchers, D. L. 2008. Using
multinomial models to analyse data from Iberian sardine egg incubation experiments: a comparison with traditional techniques. – ICES
Journal of Marine Science, 65: 51– 59.
Multinomial rather than traditional models were applied to improve data analysis of incubation experiments for Iberian sardine
(Sardina pilchardus) eggs. Incubation experiments were carried out in 2002 in the Gulf of Cádiz, southwestern Spain, and data
were taken from the literature for the Cantabrian Sea area. Rigorous review of the traditional models revealed shortcomings in the
statistical framework for modelling the developmental progression of egg stages as well as a lack of transparency of the inherent
assumptions. Multinomial model results show that egg stage duration varies for all stages, with stages 3 and 4 having the shortest
duration. Comparison of the results between multinomial and traditional models shows that multinomial models provide improved
insight into developmental mechanisms than that can be achieved using traditional models.
Keywords: age determination, anchovy, daily egg production method, egg development, multinomial model, sardine.
Received 23 January 2007; accepted 8 October 2007; advance access publication 15 November 2007.
M. Bernal: Instituto Español de Oceanografı́a (IEO) Estación Pesquera de Cádiz, Centro Andaluz de Ciencia y Tecnologı́a CACYTMAR, República
Saharahui s/n, Campus Rı́o San Pedro, 11510 Puerto Real, Cádiz, Spain. L. Ibaibarriaga: AZTI Foundation, Fisheries and Food Technological
Institute, Herrera Kaia Portualdea z/g, 20110 Pasaia, Basque Country, Spain. A. Lago de Lanzós and C. Franco: IEO, Centro Oceanográfico de
Madrid, Corazón de Maria 8, 28002 Madrid, Spain. M. E. Lonergan: NERC Sea Mammal Research Unit, University of St Andrews, St Andrews,
Fife KY16 8LB, UK. C. Hernández: IEO, Centro Oceanográfico de Santander, Promontorio San Martı́n s/n 39004, Santander, Spain. I. Rasines
and L. Valdés: IEO, Centro Oceanográfico de Gijon, Avenida Prı́ncipe de Asturias, 70 bis 33212 Gijón, Spain. D. L. Borchers: CREEM, University
of St Andrews, The Observatory, Buchanan Gardens, St Andrews, Fife KY16 9LZ, UK. Correspondence to M. Bernal: tel: þ34 956 016290; fax:
þ34 956 016415; e-mail: miguel.bernal@cd.ieo.es

Introduction production method (DEPM; Lasker, 1985), in which a parametric

Incubation experiments describing the development of fish eggs
are the primary source of information for assigning ages to
sampled eggs. Unlike other life stages of fish, such as larvae, juven-
iles, or adults, ages cannot be assigned directly to eggs using hard
structures. Therefore, to obtain an estimate of age, an indirect
approach has to be applied. The egg phase is divided into as
many identifiable ordered stages as possible, using prominent
developmental features (Ahlstrom, 1943; Gamulin and Hure,
1955; Lockwood et al., 1977; Thompson and Riley, 1981; Moser
and Ahlstrom, 1985; Pipe and Walker, 1987). Once an egg is classi-
fied into a stage, its range of possible ages is reduced relative to the
total range for the egg phase. It is therefore possible to know
whether an egg is older or younger than another at a different
developmental stage, if they were reared under similar conditions.
If an absolute estimate of the age of sampled eggs is required,
then an estimate of the duration of each developmental stage
becomes necessary, and is usually obtained by an incubation
experiment. Absolute estimates of egg age are required in any
method that aims to estimate egg mortality, as well as in develop-
mental studies. An example of such a method is the daily egg
mortality curve is assumed. Rates of mortality and daily egg pro-
duction are estimated and used in combination with estimates
of adult population fecundity to obtain estimates of spawning–
stock biomass. As for developmental studies, comparative
studies are of importance in understanding differences in egg mor-
tality among species (Pepin, 1991) or ecosystems (Chambers,
1997), or between years with different environmental conditions.
In incubation experiments (Lo, 1985; Miranda et al., 1990;
Motos, 1994; Le Clus and Malan, 1995), eggs are reared in con-
trolled conditions, and the transition from one stage to the next
is monitored. Rates of egg development and stage duration are
temperature- and species-dependent (Ciechomski and Sanchez,
1984; Lo, 1985; Moser and Ahlstrom, 1985; Pepin, 1991;
Gunderson, 1993; Le Clus and Malan, 1995; Fox et al., 2003).
Therefore, incubation experiments on a given fish species are per-
formed at a temperature range plausible for age determination of
staged eggs from the field. As eggs are only observed at discrete
times, and the set of temperatures used in the experiment is
only a subset of the possible temperatures found at sea, it is
often necessary to obtain a continuous model that relates the

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52
succession of stages with age and temperature. Other environ-
mental factors, such as egg size (Pepin, 1991) or parental con-
ditions (Guisande et al., 1998), have been reported to influence
the rates of egg development, but the effect of these variables on
egg development is considered to be less important than tempera-
ture and up to now they have been neglected in egg incubation
experiments.
A number of different approaches to analyse data from egg
incubation experiments and to estimate stage duration have
been used. In the case of sardine and anchovy worldwide, the
most widely used method is that of Lo (1985), which includes
both a procedure to analyse data from incubation experiments
and an automatic method of age determination that uses external
data on spawning synchronicity to refine the age estimates of
sampled eggs. The original method of Lo (1985) is based on
certain assumptions, but some are not readily outlined for end
users (see Methods below), and have not always been tested in
the different populations to which the method has been applied.
An alternative method of age determination for these species
was proposed by Bernal et al. (2001), who treated the output of
the incubation experiments as a stochastic model with resampling,
and for which sampling probabilities were provided by a synchro-
nicity assumption similar to that of Lo (1985). However, no new
tools to analyse the egg incubation experiment and to fit appropri-
ate stochastic models to its outcome were provided. Within a
recent EU-funded project (see review in ICES, 2004), the resam-
pling procedure of Bernal et al. (2001) was redefined in a
Bayesian framework, opening the possibility to analyse the incu-
bation experiments by modelling the probability of being at a
given stage as a function of the age and the variables controlled
in the incubation experiment (e.g. temperature). Building from
these studies, Ibaibarriaga et al. (2007) proposed a multinomial
model as the appropriate way of modelling egg incubation experi-
ments of anchovy (Engraulis encrasicolus) in the Bay of Biscay.
In this study, the incubation model of Lo (1985) and its
implementation in the case of Iberian sardine (Sardina pilchardus)
by Miranda et al. (1990; hereafter referred to as the traditional
approach) as well as the first application of the multinomial
model developed by Ibaibarriaga et al. (2007) to the Iberian
sardine (hereafter referred to as the multinomial approach) are
described. Information on other methods of analysing sardine
and other small pelagic fish is also provided. The assumptions
needed to apply the different models and their appropriateness
for Iberian sardine are clearly outlined and tested. Finally, a com-
parison of the results from both approaches in two incubation
experiments is given, using data from Miranda et al. (1990) and
an independent incubation experiment performed in this work.
To compare the new multinomial method with traditional
methods, a common outline for the figures from both methods
was used. Output from multinomial experiments can be presented
in a traditional fashion, but not the other way round. Therefore,
although the advantages of visualizing multinomial models are
presented, comparative plots are made using the traditional
methods of visualization.
Methods
Incubation experiments
Two temperature-dependent incubation experiments were used to
study the progression of sardine egg stages through ages in the
Iberian Peninsula: (i) the incubation experiment carried out by
M. Bernal et al.
Miranda et al. (1990) in the Cantabrian Sea (north Spanish
Atlantic coast), and (ii) a new experiment carried out for this
work in Cádiz (south Spanish Atlantic coast) in 2002.
The methods used in the Miranda et al. (1990) incubation
experiment are described in detail by the authors, and the
methods used in the Cádiz experiments are described below.
Both experiments were carried out with similar methodology,
but with small differences. Miranda et al. (1990) used
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in-laboratory incubators and sampled fewer eggs, whereas for
the experiment described here, the incubation was carried out in
an onboard incubator specifically designed for egg-rearing experi-
ments on sardine and anchovy (Motos, 1994).
Fertilized eggs for the Cádiz incubation experiment were
obtained during a survey carried out on the RV “Vizconde de
Eza” in the Gulf of Cádiz (SW Spain), between 10 and 17
February 2002. Adult sardine were caught in a commercial trawl,
in water of surface temperature 16.58C. Eggs were obtained by
exerting abdominal pressure on ripe-and-running sardine
females, and placed in an open jar. Sperm was collected by strip-
ping ripe males after drying the genital papilla using absorbent
paper, the milt being collected carefully in a 1 ml syringe and
immediately deposited over the hydrated eggs. The jar was then
gently shaken and left for 15 min to allow fertilization. Filtered sea-
water was then added to separate the sperm and to allow the
chorion to harden. Viable eggs floated to the surface, and were
immediately extracted and placed in the incubator for the rest of
the experiment, without acclimatization to the different water
temperatures.
The incubator used was that of Motos (1994). The temperature
gradient was achieved by heat diffusion across an aluminium
plaque, with both heat and cold sources thermostatistically con-
trolled. The aluminium plaque consists of 20 columns, each repre-
senting a temperature within the gradient, and seven rows, each at
the same temperature within a given column. Five temperatures
within the range 9 –178C, representative of natural conditions in
the Cantabrian Sea during spawning (Solá et al., 2001), were
used for the experiment (Table 1). Five 50 ml glass tubes, filled
with filtered seawater from the sampling location, were placed in
each temperature row, each containing 200 eggs. Two additional
jars with filtered seawater were placed in each row to allow refilling
of the jars containing eggs with water of the same temperature.
The incubation experiment starting time was taken as the
moment at which all eggs were placed in the incubator (21:45
UTC). To monitor stage progression in relation to time and temp-
erature, eggs were taken randomly from one jar for each
Table 1. Mean temperatures, number of sampled eggs per jar, and
total incubation time (i.e. the time until all eggs sampled had
become larvae) for the different incubators, with the corresponding
coefficients of variation (CV).
Incubator Mean temperature Mean number of Time to
(88 C) and (CV) eggs sampled (CV) hatch (h)
A 9.31 (0.12) 29 ( –) –
. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .
B. . . . . . . . . . . . . . . . . . . . . . ..10.75 (0.10) 27 (39) 129.17
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .
C 13.00 (0.05) 28 (39) 96.08
. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .
D 15.46 (0.05) 33 (43) 84.03
. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .
E 16.98 (0.05) 33 (38) 63.00
Eggs at 98C did not hatch, and maximum and minimum hatching times
were 5 d (129 h, incubator B) and 3 d (63 h, incubator E).
Using multinomial models to analyse data from Iberian sardine egg incubation experiments
temperature and sampling event. All temperatures were sampled at
each sampling event, and sampling frequency was as follows:
samples were taken at 1-h intervals during the first 6 h of the
experiment, after which they were taken at 3-h intervals until
hatch. Following the classification of Moser and Ahlstrom
(1985), all eggs in the sample were classified into one of 11 devel-
opmental stages, based on their morphological features. After
staging, the eggs were returned to the jar from which they came,
and any loss of water from the jar was replaced from the filtered
seawater jars at the same temperature. At least twice a day, dead
eggs were siphoned from the bottom of the jar and the water
volume was replenished with the filtered seawater at the same temp-
erature. At least 30% of the water volume was replaced each day.
Traditional approach
Traditionally, the succession of stages through time has been
analysed by computing either stage duration or mean age at
each stage (see review by Lasker, 1985), and plotting a cumulative
egg-stage duration or directly the progression of mean age at each
stage through all stages. Using the latter method, Lo (1985) devel-
oped a model to describe the results of an incubation experiment
on anchovy eggs, and a method to assign ages to field-sampled
eggs depending on stage and temperature. Different modifications
of her method have until now been used routinely in sardine and
anchovy fishery experiments around the world (Lasker, 1985;
Miranda et al., 1990; Motos, 1994; see review in Stratoudakis
et al., 2006).
Although not clearly indicated in the original manuscript, the
incubation model of Lo (1985) consists of two different models:
(i) a model of mean age for all stages and temperatures, and
(ii) a model of stage duration for all stages and temperatures.
The combination of both models predicts the possible range of
ages of an egg at any stage, reared at any temperature within the
range used in the incubation analysis. The mean age model for
all stages and temperatures is defined by
E½yi;t  ¼ aeðbtciÞ id ; ð1Þ
where ȳi,t is the mean age of stage i at temperature t, and (a, b, c, d )
are the parameters to be estimated by fitting Equation (1) to
the data.
Assuming that the age distribution for each stage and temper-
ature is approximately normally distributed, stage duration is esti-
mated from
½yi;t  2sdi;t ; y i;t þ 2sdi;t ; ð2Þ
where sdi,t is the standard deviation of ages of stage i at tempera-
ture t. The problem in this case is that only a small set of discrete
temperatures is observed in the incubation experiment, so a model
of standard deviation as a function of stage and temperature is
needed to be able to age the eggs found in the field.
To fit Equation (1), estimates of mean age for each stage at the
observed temperatures are required. How to obtain those estimates
is not described by Lo (1985). Given an incubation experiment, in
which eggs are observed at j discrete times ( j = 1, . . . , m), the age
of an egg is only available at those discrete times, and it is com-
puted as the elapsed time between fertilization and each of the
observation times j. If exactly the same number of eggs are
sampled at all times, the mean age of an egg at stage i can be
53
estimated as
ni;t;1 agei;t;1 þ    þ ni;t;m agei;t;m
yi;t ¼ Pm ; ð3Þ
j¼1 ni;t;j
where ni,t,j is the number of eggs at stage i sampled at temperature t
and time j. This is the equation traditionally used to estimate mean
age for any stage and temperature for Iberian sardine (Miranda
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et al., 1990). Nevertheless, if the number of eggs sampled varies
between sampling events, Equation (3) provides a biased estimate,
and should be corrected for sample size. Bias correction, if done,
was not described by Miranda et al. (1990). For the Cádiz
experiment, the number of eggs sampled varies at different sampling
events (see CV of mean sampled eggs in Table 1), so Equation (3)
can lead to biased results. An alternative ad hoc unbiased estimator
to use when sampling size varies can be defined as
pi;t;1 agei;t;1 þ    þ pi;t;m agei;t;m
yi;t ¼ Pm ; ð3aÞ
j¼1 pi;t;j
in which pi,t,j represents the percentage of eggs at stage i and temp-
erature t sampled at time j from the total number of eggs sampled
at that temperature and sampling time.
Stage duration is described using the standard deviation of the
observed ages [as in Equation (2)]. Two different models for sdi,t
have been used: (i) a common standard deviation for each stage
through all temperatures (e.g. Lo, 1985; Motos, 1994) (sdi,t ;
sdi), and (ii) a continuous model of standard deviation as a func-
tion of stage and temperature (e.g. as currently used in Iberian
sardine egg age determination procedures) [sdi,t = f (i,t)]. In the
first case, the mean of the observed standard deviation through
the discrete incubation temperatures is often used as the estimate
of standard deviation for a given stage through all temperatures.
However, this may not be appropriate, because stage duration
decreases with increasing temperature (Pepin, 1991; Gunderson,
1993; Le Clus and Malan, 1995). Taking this into consideration,
a model of stage duration in relation to stage and temperature
has been used routinely to age Iberian sardine eggs:
E½sdi;t  ¼ aeðbtciÞ id : ð4Þ
In Equation (4), stage duration decreases exponentially with
increasing temperature, and shows a logistic increase from the
initial to the final egg stage. This model has not been described
in the literature, but it has been used routinely together with the
mean age model of Miranda et al. (1990) to assign ages to
Iberian sardine eggs.
Multinomial approach
The data from an incubation experiment are more appropriately
regarded as observations from a multinomial distribution, in
which the classes are the egg stages. Thus, any egg from the incu-
bation experiment is in a class i, out of some k given possible
classes (i = 1, . . . , k), with probability pi, which can be a function
of covariates such as age and temperature. Ibaibarriaga et al.
(2007) proposed a multinomial model of stages given age
54 M. Bernal et al.

(i.e. observed times) and temperature: Results

Cádiz incubation experiment
N! Eggs incubated at 9.318C did not hatch, and only progressed up to
f ðnjp; NÞ ¼ pn1 . . . pnk k ; ð5Þ
n1 ! . . . nk ! 1 stage 3. Also a large percentage of eggs at this temperature showed
malformation, so could not be effectively classified into stage. For
where n = (n1, . . . , nk) is the number of randomly sampled eggs these reasons, data from this temperature were not used in the
of stage i (i = 1, . . . , k), from a population of N eggs, and analysis.
p = (p1, . . . , pk) is the probability of belonging to stage i Figure 1 shows the range of ages observed for each stage, as

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(i = 1, . . . , k). Each probability depends on both the age of the
egg and the temperature at which it is reared [pi = f (age, temp)].
A model for pi can be fitted using different procedures and
assumptions. Here, generalized linear models (GLM; McCullagh
and Nelder, 1983) were used, and two different type of models
were tested (Ibaibarriaga et al., 2007): (a) a proportional odds
model (McCullagh, 1980) fitted using the MASS package
(Venables and Ripley, 2002), and (b) an extended continuation
ratio model (ICES, 2004; Stratoudakis et al., 2006).
All models were fitted using the software R (Ihaka and
Gentleman, 1996). Model (a) assumes that the effect of age and
temperature is identical for all stages, and only the intercept is
different for the different stages. As we use age and temperature
as the explanatory variables, the equation for the fitted model is
logitðP½iþÞ ¼ ai þ b1 age þ b2 temp þ b3 age:temp; ð6Þ
used traditionally to fit Lo’s (1985) incubation model. Stages 3
and 4 were observed on just a few sampling occasions for all
temperatures and in low numbers. At high temperatures, stage
3 was only observed once (at 158C), or even not observed (at
178C), and stage 4 was observed just once, at 178C. Observed
ages for each stage followed a monotonically increasing trend
with stage, as expected, although there was some degree of
overlap between stages. With increasing temperatures, or for
later stages, the degree of overlap between stages was larger
than for young stages and/or cold temperatures. Therefore, in
late sampling events through the incubation experiment,
samples contained up to four stages. Total incubation time to
hatching decreased with temperature (Table 1, Figure 1). The
range of observed ages for each stage did not follow a mono-
tonic trend, and only a decreasing range of observed ages for
any stage with increasing temperature could be extracted from
Figure 1.

where the response P[i+] represents the cumulative probabilities

Traditional approach
of being at stage i and above (P[i+] = pi +    + pk)
Figure 2 shows the results for the Cádiz incubation experiment,
(Ibaibarriaga et al., 2007). In Equation (6), age:temp represents
and the fit to Lo’s (1985) model. Although variation in the
the interaction between age and temperature, the parameters bj
number of randomly sampled eggs at different sampling inter-
(for j = 1, 2, 3) are independent of the stage i (i.e. they are
vals was large (see CVs in Table 1), the differences in mean
common to all stages), and only the intercept ai differs for differ-
age estimated using Equation (3) (assuming equal n) and four
ent stages. The model was fit using a logit link function and bino-
(correcting for unequal n) did not show striking differences.
mial error structure.
Model (b) is less restrictive and allows the effect of both age and
temperature to interact with stage. The general equation for model
(b) is

logitðP½i þ jði  1ÞþÞ ¼ hðage,tempÞ; ð7Þ

where h is a linear predictor. In this case, interactions between

temperature and/or age and stage can be included in the linear
predictor. Different models, including (i) all possible interactions
between stage, temperature, and age, (ii) only two-way inter-
actions, and (iii) no interactions, were fitted to the data. Model
selection was performed using a backward stepwise procedure,
and non-significant terms (using t-statistics) were dropped
sequentially until all terms were significant. As in Model (a),
logit link function and binomial error structure were used. Once
an appropriate model for (P[i + j(i21)+]) is obtained, estimates
of p̂i can be obtained easily (ICES, 2004; Ibaibarriaga et al., 2007).
To compare the results with those from the traditional model,
the probability density function (pdf) of ages for each stage at any
temperature was obtained from the multinomial model using
Bayes’ theorem, without taking into account the possible effect
Figure 1. Observed ages for each stage and temperature in the Cádiz
of mortality (ICES, 2004; Ibaibarriaga et al., 2007). Mean age for experiment. The colour gradient represents the number of eggs
any stage at any temperature was estimated as the mean of the counted in that stage at that temperature and sampling time.
pdf of ages, and stage duration was estimated as the age range The abundance scale is on the right of each graph. Panels represent
between the 5th and 95th percentiles of the cumulative density the temperatures used in the experiment with successful hatching:
function of age, given stage, and temperature. (a) 10.768C, (b) 138C, (c) 15.468C, and (d) 16.988C.
Using multinomial models to analyse data from Iberian sardine egg incubation experiments
Figure 2. Lo’s (1985) model for the Cádiz area incubation experiment.
Black dots represent the mean age computed using the model of
Miranda et al. (1990), horizontal lines represent the mean age computed
using the bias-corrected equation, vertical lines show the observed stage
duration (mean age+2 s.d.), and points in grey are the observations.
Panels represent the temperatures used in the experiment with
successful hatching: (a) 10.768C, (b) 138C, (c) 15.468C, and (d) 16.988C.
Both mean age and standard deviation were treated in the tra-
ditional method as the observations from the incubation exper-
iment, which was modelled using Equations (1) and (4),
respectively. Generally, Lo’s (1985) model for mean age fitted
the data well. However, the model underestimated mean age
at stage 3 and overestimated that at stage 5, at all temperatures.
For any temperature, stage duration did not show any clear
trend through stages, although stages 3 and 4 tended to have
the shorter stage duration at all temperatures. Nevertheless, a
clear decrease in stage duration was obvious for all stages with
increasing temperature.
Figure 3 presents the results for mean age and stage duration
from the Cantabrian Sea incubation experiment. Generally, the
model fitted the data well, except for stage 7 at lower temperature
(118C), where the observed mean age was nearly outside the model
range. This result is probably an indication that there were insuffi-
cient observations from which to estimate the mean age and to
predict the stage duration. Similar to the Cádiz incubation experi-
ment, the Cantabrian Sea incubation experiment showed no trend
in stage duration with increasing development for any tempera-
ture, although a decreasing stage duration for increasing tempera-
tures was observed for all stages. These results imply that Equation
(4), which assumes a monotonic increase in stage duration, is no
longer valid.
Parameter estimates for the Cantabrian Sea were generally
larger than those in the Cádiz area, except for parameter (d)
(Table 2). Predicted values for both areas show some differences,
especially at low temperature (11– 158C), where the Cantabrian
Sea model consistently overestimated mean age for all stages
(Figure 3). For higher temperatures (208C), the Cantabrian Sea
and Cádiz model overlapped, but the Cádiz model overestimated
mean age for late stages (9 –11).
55
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Figure 3. Lo’s (1985) model for the Cantabrian Sea incubation
experiment, with observed (dashed) and modelled confidence
interval (2 s.d.). The grey line represents the Cádiz model. Panels
represent the temperatures used in the experiment with successful
hatching: (a) 10.768C, (b) 138C, (c) 15.468C, and (d) 16.988C.
Multinomial approach
Continuation models explain a greater percentage of the variation
in the data for the Cádiz incubation experiment when modelling
mean age than proportional odds models (Table 3). Because not
all terms included in the initial continuation ratio model were sig-
nificant, the final model selected after backward elimination
included stage, age, and temperature as covariates, and the
two-way interaction of age with temperature. Validation of the
model using analysis of residuals was not carried out, because
residual analyses of binomial or multinomial models are not
very informative (McCullagh, 1980).
Results from the final model are shown in Table 4 and Figure 4.
The lines in Figure 4 represent the fitted probability of each stage,
for given ages and temperatures. The height of each curve for a
given age represents the probability that an egg of that age at
that temperature is at the stage represented by the curve. The
width of the bell-shaped curves depends on the stage duration.
Numbers in the figure (i.e. 1 –11) represent the observed pro-
gression of the relative frequency of stage occurrence through
time. Apart from stages 3 and 4, the transition from one stage to
the next is clear, with the percentage occurrence of a stage
quickly increasing up to a maximum, then decreasing rapidly as
the next stage starts to increase in number. As in Figure 1, for
Table 2. Parameter estimates for Lo’s (1985) model [Equation (1)]
for the Cantabrian Sea and Cádiz area, together with standard
errors (s.e.).
Parameter Cantabrian Sea Cádiz area (s.e.)
a. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..17.52 9.35 (1.98)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
b. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .0.14 0.12 (0.004)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
c. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .0.17 0.15 (0.03)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
d 2.22 2.30 (0.20)
56 M. Bernal et al.

Table 3. Different multinomial models used and their summary statistics.

Type of model Equation % dev d.f.
Proportional odds Age+Temp+Age:Temp 79 14
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .
Continuous ratio Age+Temp+Stage+Age:Temp+Age:Stage+Temp:Stage+Age:Temp:Stage 95 47
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .
(Final model) Stage+Age+Temp+Age:Temp 96 15
% dev is the percentage of deviance explained, and d.f. the model degrees of freedom. Variables included in the different models are elapsed time since the
beginning of the incubation experiment (Age), temperature (Temp), and egg stage (Stage). A colon between variables represents interaction between them
(i.e. Age:Temp is the age and temperature interaction, and Age:Temp:Stage is the three-way interaction between age, temperature, and stage).

Downloaded from https://academic.oup.com/icesjms/article-abstract/65/1/51/611825 by Universidad de Pamplona user on 17 April 2020

increasing temperatures, or for later stages, the degree of overlap moving to the next stage dominates. The individual effects of age
between stages was larger than for young stages and/or cool temp- and temperature modulate the fit, by decreasing the effects of the
eratures (Figure 4). interaction for older ages and higher temperatures (i.e. the tran-
The multinomial model fitted the observed percentages well, sitions are slower than expected from the linear effect of the
and the curves are capable of showing the low probability of age –temperature interaction for later stages, indicated by
finding stages 3 and 4, because of extensive overlap in age younger ages, and temperatures).
between stages (Figure 4). For older stages, the degree of overlap To compare the results from the multinomial model with those
between the curves is large, so the fitted probability of stage 6 from the traditional approach, Figure 5 shows the estimated mean
and older stages never reaches one. This adequately represents age and duration of each stage for the observed temperatures,
the variety of stages observed at each sampling time as hatching obtained from the multinomial model using the methods
approaches for each temperature (Figure 1). described above. The predicted values of mean age and stage dur-
The width of the fitted curves (i.e. the duration of the stage) is ation matched the observed data well, outperforming the tra-
narrower for higher temperatures, indicating a decrease in stage ditional model fitted to the Cádiz data. Stage duration for stages
duration with increasing temperature. Nevertheless, there is no 3 and 4 were the shortest at all temperatures, followed by stage 1.
clear monotonic trend in the width of the curves with increasing Stage duration did not show a monotonic trend with increasing
development, in contrast to what is assumed in the stage duration development, but there was a decreasing trend as temperature
model described in Equation (4) (Figure 5). increased.
Both age and temperature effects show significant negative par- Estimated hatching time (i.e. the time at which all eggs have left
ameters on P[i+j(i21)+], whereas the age – temperature inter- stage 11 and become larvae) from the multinomial model (data
action effect shows a significant positive estimate (Table 4). not shown, but see the decreasing size of the predicted proportion
These parameters are masked between them, because an increase of stage 11 at each temperature in Figure 4) shows a decrease with
associated with the age – temperature interaction indicates an temperature, consistent with the observed data (Table 1).
increase in the probability of passing from stage i21 to stage i
(therefore, as age and temperature increases, the probability of Discussion
passing from one stage to the next increases), whereas decreases This study has shown the advantages of using multinomial models
in the individual age and temperature effects indicate the opposite. to describe the development of the egg phase of Iberian sardine.
Values of the age –temperature interaction are larger, so the posi- The incubation experiment performed provided the usual infor-
tive effect of increasing age and temperature in the probability of mation required for studies of egg development. Total egg incu-
bation time ranged between 130 and 63 h for a range of
temperatures between 118C and 178C. These times are consistent
Table 4. Summary of the selected multinomial model. with studies made on similar species in other parts of the world
Stage and Estimate s.e. z-value Probability (Pepin, 1991; Le Clus and Malan, 1995; Gutierrez et al., 2002).
variable (>jzj) In comparison with the results provided by Miranda et al.
Stage 1 6.878e+00 6.533e+01 0.105 0.916 (1990) for the northern Iberian peninsula, eggs in the Cádiz area
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
Stage 2 1.196e+00 2.339e201 5.113 3.16e207 seem to develop faster at lower temperature, whereas development
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
Stage 3 24.170e+00 2.437e201 217.112 ,2e216 times were similar at higher temperature. No malformation or
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
aberrant developmental behaviour was observed at the higher
Stage 4 23.326e+00 2.623e201 212.682 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
temperatures used for both experiments. Hatching in the Cádiz
Stage 5 24.018e+00 2.595e201 215.482 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . experiment was not achieved at temperatures less than 108C,
Stage 6 28.335e+00 2.922e-01 228.528 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . but morphological malformation was clear when it was colder
Stage 7 21.307e+01 3.694e201 235.381 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . than this. Miranda et al. (1990) used temperatures of 118C,
Stage 8 21.546e+01 4.146e201 237.301 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
138C, 158C, 188C, and 208C, and showed that eggs at 118C took
Stage 9 21.756e+01 4.538e201 238.704 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
as long as 5 d to hatch. The results therefore suggest a lower phys-
Stage 10 21.895e+01 4.864e201 238.959 ,2e216 iological limit for Iberian sardine egg development of 108C,
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
Stage 11 22.079e+01 5.191e201 240.041 ,2e216 although the lower lethal limit may vary depending on the local
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
Stage 12 22.215e+01 5.471e201 240.483 ,2e216 range of temperatures in the spawning area.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
The rates of egg development rates in both incubation experi-
Age 21.843e201 5.693e203 232.378 ,2e216
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
ments increased with temperature, and stage duration therefore
Temp 29.383e202 1.625e202 25.775 7.67e209
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . decreased, as reported for other species and for other experiments
Age:Temp 3.474e202 8.236e204 42.177 ,2e216 with sardine (e.g. Pepin, 1991; Le Clus and Malan, 1995).
Using multinomial models to analyse data from Iberian sardine egg incubation experiments
Figure 4. Output of the multinomial model. The numerals 1– 11 represent the observed relative frequencies of stages 1– 11. Lines are
the predicted probability from the model. Panels represent the temperatures used in the experiment with successful hatching: (a) 10.768C,
(b) 138C, (c) 15.468C, and (d) 16.988C.
However, the duration of the different stages used for the
classification of egg development was not equal, nor was there evi-
dence of a monotonic trend with later development that could be
described by a simple continuous function. At all incubation
temperatures used during this study, stages 1, 3, and 4 were the
shortest. These results are supported by observations of a low
Figure 5. Mean age (open boxes) and stage duration (vertical lines)
estimated from the multinomial model. Observed ages (dots),
estimated mean age and stage duration from the model of Miranda
et al. (1990), and predicted mean age from the Lo (1985) model are
plotted for comparison. Panels represent the temperatures used
in the experiment with successful hatching: (a) 10.768C, (b) 138C,
(c) 15.468C, and (d) 16.988C.
57
Downloaded from https://academic.oup.com/icesjms/article-abstract/65/1/51/611825 by Universidad de Pamplona user on 17 April 2020
percentage of these stages in samples collected during Iberian
sardine ichthyoplankton surveys (MB and Y. Stratoudakis, unpub-
lished data). This feature was not clear when reviewing incubation
experiments based on the traditional method, because only the
model for mean age is clearly stated and tested in most papers
(e.g. Miranda et al., 1990; Motos, 1994). The cause for such discre-
pancies arises from the manner in which the results are presented:
point estimates for the traditional model vs. probability curves for
the multinomial model.
Generally, the traditional model explained the stage pro-
gression and mean ages of both incubation experiments well,
but failed to describe the rapid transition exhibited by stages 3
and 4, which was evident from the multinomial model results.
Multinomial models clearly illustrate the extensive overlap for
stages 2– 5, which is caused by the fast transition from stage 3 to
stage 4. In addition, the multinomial model clearly demonstrated
substantial overlap for later developmental stages (stages 9 –11),
particularly at higher temperatures. Therefore, the multinomial
model outperforms the traditional model in representing egg
development from incubation experiment data.
Apart from better representation of the data, there are other
advantages from using multinomial models to describe egg devel-
opment. Theoretically, age (i.e. sampling times) is fixed in most
incubation experiments, and stage is the observed random vari-
able, statistically defined as an ordered factor (i.e. an ordered dis-
crete variable). In the traditional approach, randomness is
allocated to the wrong variable, age, and stage is used as the inde-
pendent variable, which is treated as a continuous variable. Apart
from theoretical implications, the spacing of the observations of
the independent variable (i.e. stage) is unknown a priori, so the
statistical significance of the fit is meaningless. The significance
of the parameters is further masked by the fact that mean ages
are treated as observations, when in fact they are already estimates
obtained from the raw observed data using different equations
58
[e.g. Equations (3) or (4)]. Consequently, the statistical
significance of the parameter estimates obtained using the tra-
ditional approach is incorrect. Nevertheless, if the traditional func-
tional form is flexible enough, it can still provide a description of
the progression of mean age for the consecutive stages at different
temperatures.
Some of the shortcomings of the traditional methods revealed
in this work have been overcome by different methods developed
in other fields of science. For example, the age of planktonic cope-
pods, which can be divided into identifiable morphological stages,
has been analysed using different techniques, some of them not
very different from the multinomial models described here
(Klekowski and Fischer, 1975; Landry, 1975; Peterson and
Painting, 1990; Bonnet and Carlotti, 2001; Campbell et al.,
2001). Event analysis (see review in Cox and Oakes, 1984) has
also been used to analyse the progression of different ontogenic
events (Chambers and Leggett, 1989), and cod egg development
(Pepin et al., 1997). The analysis presented in Pepin et al. (1997)
provides a better statistical representation of the transition
between stages for a fish egg incubation experiment, but it does
so by treating stages separately. In general, although some of
these methods overcome part of the statistical shortcomings of
the traditional method, they do so by modelling each life stage
independently (i.e. they provide independent representations of
each stage). In comparison with multinomial models, this is a dis-
advantage, because multinomial models allow one to provide a
general representation of all stages into which a given process
has been divided.
In summary, the multinomial models presented here have a
number of advantages over traditional methods. First, multino-
mial models provide a better representation of egg development,
both visually and in relation to the accuracy of the fitting.
Second, multinomial models provide an adequate statistical treat-
ment of the age and stage variables, and therefore an adequate fra-
mework for statistical inference and for estimating the variance
associated with age determination. Finally, multinomial models
overcome the traditional requirement for the combination of
mean age and stage duration models, which has not always been
well documented in the literature. In relation to the DEPM, the
implementation of multinomial models for egg incubation data,
and the development of a Bayesian framework for determining
the ages of eggs of synchronously spawning fish (ICES, 2004),
allows us to: (i) simplify the age determination process,
(ii) improve the accuracy of age estimates, and (iii) introduce
the variance associated with age detrmination into the estimation
process of the DEPM. These improvements are expected to result
both in a more accurate DEPM-based estimates of spawning –
stock biomass and more realistic estimates of the associated
variance.
Acknowledgements
We thank AZTI and Lorenzo Motos for allowing access to the
incubator to carry out this experiment, the crew of RV
“Vizconde de Eza” for assistance, and the laboratory personnel
involved in the analysis of the data. Ana Miranda providing
some raw data from her incubation experiment, and Mbulelo
Dupolo a detailed review of an early version of this manuscript.
Both are sincerely acknowledged, as are editor Pierre Pepin and
two anonymous reviewers for their constructive and detailed
reviews. Most of the work was developed within a cooperative
M. Bernal et al.
EU project (EU 99/080), so we thank all participants in this
project, especially Yorgos Stratoudakis and Simon Wood, for
useful discussion, comments, and suggestions on how to present
the results presented here.
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