ICES Journal of Marine Science Advance Access published November 23, 2014

ICES Journal of

Marine Science
ICES Journal of Marine Science; doi:10.1093/icesjms/fsu205

Macrozooplankton predation impact on anchovy (Engraulis

encrasicolus) eggs mortality at the Bay of Biscay shelf break
spawning centre

Laboratory of Genetics, Department of Genetics, Physical Anthropology & Animal Physiology, University of the Basque Country UPV/EHU,
Leioa 48940, Spain
2
Red Sea Research Center, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
3
AZTI Tecnalia, Marine Research Division, Herrera Kaia Portualdea z/g, PO Box 20110, Pasaia, Gipuzkoa, Spain
*Corresponding author: tel: +34 946015503; fax: +34-946013145; e-mail: [email protected]
Albaina, A., Irigoien, X., Aldalur, U., Boyra, G., Santos, M., and Estonba, A. Macrozooplankton predation impact on anchovy
(Engraulis encrasicolus) eggs mortality at the Bay of Biscay shelf break spawning centre. ICES Journal of Marine Science,
doi: 10.1093/icesjms/fsu205.
Received 28 August 2014; revised 23 October 2014; accepted 24 October 2014.
A real-time PCR based method involving a species-specic probe was applied to detect Engraulis encrasicolus eggs predation by the macrozooplankton community during the 2011 spawning season. Three locations along the shelf break presenting contrasting but high prey densities were
sampled. A total of 840 individuals from 38 taxa of potential macrozooplankton predators were assayed for E. encrasicolus DNA presence and
27 presented at least one positive signal. Carnivorous copepods were responsible for the most predation events (66%) followed by euphausiids
(16%), chaetognaths (5%), and myctophid sh (4%). Macrozooplankton predation on anchovy eggs followed a type-I functional response with
daily mortalities ,4% of available prey abundance suggesting a negligible impact on the species recruitment at the shelf break spawning centre.
Keywords: Bay of Biscay, DNA, Engraulis encrasicolus, macrozooplankton, molecular assay, predator prey interactions.

Introduction
Disentangling predator/prey relationships with the aim of resolving
complete foodwebs is crucial to the desired Ecosystem Based
Fisheries Management (e.g. Gallego et al., 2012). Furthermore,
efforts to rebuild fisheries can be undermined by not incorporating
ecological interactions into fisheries models and management plans
(Richardson et al., 2011). In this context, methods capable of yielding a reliable, fast and cost-effective direct estimation of fish early life
stages (ELSs) mortality by predation are demanded as this factor has
been traditionally either ignored or grossly estimated, based in
indirect data, in fisheries management resulting in limited or null
value in standard fisheries recruitment models (Kenchington,
2013). The technical limitations related to traditional visual assessment of contents could explain the relative scarcity of field studies
devoted to predation of fish eggs (Heath, 1992; Houde, 2008).
# International

However, nowadays, molecular methods offer an alternative to

measure predation in the field (Symondson, 2002; King et al.,
2008; Pompanon et al., 2012).
In this sense, while predation by fish, including other clupeids
and cannibalism, is known to be responsible of a significant part
of anchovies ELS mortality (e.g. Szeinfeld, 1991), studies applying
traditional (visual) methods to invertebrate predators of anchovy
ELSs are scarce (e.g. Terazaki, 2005). Applying immunoassays,
two studies revealed the importance of invertebrate predation
on anchovy ELS mortality. While Krautz et al. (2007) showed that
predation by the euphausiid Euphausia mucronata accounted for
24 27% of eggs natural mortality in the Chilean anchoveta
(Engraulis ringens), Theilacker et al. (1993) reported that euphausiids accounted for between 47 and 78% of the natural mortality
on northern anchovy (Engraulis mordax) eggs and yolk-sac larvae.

Council for the Exploration of the Sea 2014. All rights reserved.
For Permissions, please email: [email protected]

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Aitor Albaina 1*, Xabier Irigoien 2, Unai Aldalur 1, Guillermo Boyra 3, Mara Santos3,
and Andone Estonba 1

Page 2 of 10
To characterize the range of predators of anchovy ELS in the Bay of
Biscay a DNA-based method was developed and applied to both invertebrate and vertebrate potential predators during the 2010 spawning
season (Albaina et al., Under Review). These authors reported that
,5% of the macrozooplankton predators presented anchovy DNA
remains within their gut contents when sampling two SE Biscay offshore stations. These results pointed to a reduced impact on
anchovy eggs mortality (respectively 1.3 and 3.6%) corresponding
to 250 eggs m22 prey abundances. However, to clarify the impact
of macrozooplankton predation on anchovy eggs survival at the
shelf break spawning centre a wider range of prey densities needs to
be assessed. Furthermore, ideally, the whole potential spawning area
of the species should be queried. It is known that Bay of Biscay
anchovy can spawning along the whole shelf break but this takes
place only at years of high species abundance (e.g. Motos et al.,
1996; ICES, 2011). In this sense, in 2011, for the first time after a
decade of low recruitments, the Bay of Biscay anchovy recovered to

A. Albaina et al.
historical maximum levels of both adults and egg production allowing collecting macrozooplankton predators at areas of high anchovy
egg abundances along the whole Bay shelf break area. By assaying the
presence of anchovy DNA in these specimens, we expect to give
insights on the role of macrozooplankton predation on anchovy
recruitment.

Material and methods

Prey and predators sampling
Macrozooplankton was collected during the BIOMAN 2011 survey
(627 May) on-board the research vessel Investigador. Briefly, the
BIOMAN survey applies the Daily Egg Production Method (Lasker,
1985) to estimate fishable anchovy biomass based in the amount of
eggs produced during the peak spawning period of the species and
adult anchovy information. In 2011, anchovy egg abundance was
measured for a grid of 699 stations by vertical hauls of a 150 mm
PairoVET net with 0.1 m2 of mouth opening area (Figure 1; ICES,
Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

Figure 1. Prey and predators spatial location in BIOMAN 2011 campaign. The three MIK hauls location (large stars) along with anchovy egg
abundance based in PairoVET net vertical hauls (small crosses) is shown. Egg abundance (log10ind. 1000 m23, scale superimposed) was interpolated
using kriging method (SURFER 10; Golden Software). Isobaths of 100, 200, 1000 and 2000 m are shown (bold lines) along with the spatial limits of
anchovy spawning area in the 2010 campaign (the two empty polygons).

Page 3 of 10

Macrozooplankton predation impact on anchovy

Sidmar) with a fluorescence sensor (Seapoint Chlorophyll Fluorometer; Seapoint Sensors, Inc.).

Egg predation detection assay

The DNA-based assay described and validated in Albaina et al.
(Under Review) was applied to the 38 macrozooplankton taxa
sorted in 2011 for anchovy predation detection. Briefly, this assay,
that includes an E. encrasicolus species-specific TaqMan probe
(15 bp long; located within an 87 bp amplicon of the cytochrome-b
gene), measures the amount of anchovy DNA within the stomach
contents of potential predators by the real-time PCR technique.
This assay was capable of detecting 0.005 ng of anchovy DNA
(roughly 1/100 of a single egg assay) in a reliable way and had a
90% success in detecting predation events occurred in the last 3 h
for an experiment performed with the megalopae stages of two
swimming crab (genus Liocarcinus) species. Anchovy DNA was
not detectable after .6 h of digestion.

Detection of anchovy DNA within predators

stomach contents
Both DNA extraction protocol and real-time PCR assay settings
followed Albaina et al. (Under Review). DNA was extracted in
1.5 ml Eppendorf tubes using a modified salt extraction protocol
(Aljanabi and Martinez, 1997) including a mechanical homogenization step, using a plastic pestle treated with bleach and UV radiation
after each use, for malacostracans. For every juvenile/adult myctophid fish and other large organisms, at least partial dissection of the
stomach contents was performed to facilitate the DNA extraction
process. Before extraction, individual organisms were placed over
a highly absorbent wiper and washed with distilled water using a
Pasteur pipette. Dissection tools were flamed with ethanol after
each sample. Two types of extraction blanks (EBs), negative controls
where no tissue is added to the extraction buffer before DNA extraction protocol, were included every 10 samples to prevent crosscontamination: including or not the introduction of a plastic
pestle. Following extraction, DNA was resuspended in 100 ml ultrapure H2O and stored at 2208C. The DNA yield (ng ml21) was determined using an ND-1000 Spectrophotometer (NanoDrop). Assays
were run on an Applied Biosystems 7900 real-time sequence detection system in 384-well reaction plates including 20 no template
controls (NTCs; another negative control) and 12 positive controls
(DNA extracted from anchovy muscle tissue) per plate. After 3 min
at 958C, the run comprised 40 cycles of 5 s at 958C followed by 15 s at
608C. Each 10 ml volume reaction contained 0.083 ml of 60 assay
(corresponding to 125 nM of anchovy probe and 450 nM of both
the F and R primers), 5 ml of Brilliant III Ultra-Fast QPCR Master
Mix (Agilent Technologies), 0.15 ml of ROX reference dye (1 mM;
Agilent Technologies), 1.25 ml BSA (#B9001S New England
Biolabs; 10 mg ml21), 2.517 ml of ultrapure H2O and 1 ml extracted
DNA.
After the real-time PCR run, each wells threshold cycle value
(Ct; the number of PCR cycles at which a significant exponential
increase in the signal is detected) was computed using the
Sequence Detection Software version 2.3 (Applied Biosystems).
The Ct value is directly correlated with the number of copies of
target DNA present in the reaction (see, e.g. Albaina et al., 2010).
The thresholds defined in Albaina et al. (Under Review) for the unambiguous detection of anchovy DNA within predators extracted
DNA were applied. While Ct values over 35.4 units were required
for calling a positive when ,50 ng of DNA extracted from
stomach contents was tested, for values between 50 500 and

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

2011). Sampled stations covered the whole species spawning area

from 47823 N to 3854 W. The net was lowered to 100 or 5 m
above the bottom at shallower stations. Apart from PairoVET
samples, the Continuous Underway Fish Egg Sampler (CUFES;
Checkley et al., 1997) was used to record the eggs found at 3 m
depth with a net mesh size of 350 mm. CUFES sampling device
collect eggs along 1.5 nm ship tracks at both sides of the PairoVET
location. Anchovy eggs were identified and counted on-board for
both sampling devices and abundances were computed.
Three MIK (Methot Isaac Kidd) net samples, with a mesh size of
1 mm and a mouth area of 1 m2, were collected along the shelf break
as to sort potential predators for assay testing (Figure 1). MIK hauls
were performed from 70 to 75 m depth to surface (ship at two knots,
cable retrieved at 6 m min21 speed), during the night and at areas
of high but contrasting anchovy eggs. Immediately after collection,
samples were preserved in 100% ethanol. This ethanol was changed
at least two times including one after 24 h (on-board). The qualitative and quantitative analysis of MIK net samples was carried out
under a stereoscopic microscope and identification was made to
genus or species level when possible (Table 1). Gelatinous organisms, mainly siphonophores and salps but also jellyfish and
ctenophores, were grouped together due to relatively damaged condition, caused by an inappropriate sampling device, preventing
identification. Because of this, potential predators did not include
gelatinous zooplankton. For the remaining groups, only taxa
reported as carnivorous or, at least, omnivorous in the literature
were sorted for assay testing. While every large animal was sorted
from the whole sample (mainly juvenile fish, salps .20 mm total
length and pteropods and malacostracans over 7 mm cephalothorax
length) the rest of the sample was aliquoted using a Motoda plankton splitter and aliquots were sorted until a minimum of 150 individuals for assay testing were sorted. Every individual to be
assayed was transferred to a 2 ml microtube (Sarstedt) with fresh
ethanol until DNA extraction.
Beside this, the acoustic data recorded on-board during the three
MIKS hauls were analysed. Acoustic data were recorded with a Simrad
EK60 split-beam scientific echosounder at 38 and 120 kHz frequencies (Kongsberg Simrad AS). The echosounder was calibrated in accordance with Foote et al. (1987). The acoustic data were selected,
classified, and analysed with Echoview Myriaxand MATLAB
(MathWorks) software. Data analysed were restricted to the depth
sampled by the net, from 10 m depth from surface to MIK
maximum depth as recorded by the mounted CTD. Data from the
first 10 m were discarded to avoid the near field of the 38 kHz transducer as it is usually recommended (Simmonds and Maclennan,
2005). Acoustic echoes were discriminated with a bifrequency acoustic method developed by Ballon et al. (2011); the method was applied
directly with few modifications as in Lezama-Ochoa et al. (2011). This
method uses the 38 and 120 kHz frequencies to split, based on their
scattering models, acoustic signals in three categories: (i) fish, (ii)
fluid-like zooplankton, and (iii) other plankton. According to
authors the fluid-like group includes euphausiids, copepods, salps,
siphonophores (without gas inclusion), and other large crustacean
zooplankton while the other plankton group included all targets
other than fluid-like zooplankton and fish. For each of these broad
taxonomic categories, the acoustic backscattering was integrated
to provide an acoustic abundance index, nautical area scattering coefficient (NASC; m2 nm22), an acoustic biomass index determined
according to MacLennan et al. (2002).
Finally, both PairoVET and MIK nets were fitted with an
RBR XR-420 CTD (Conductivity, Temperature, and Depth profiler;

Page 4 of 10

A. Albaina et al.

Table 1. Macrozooplankton species list.

Abundance (ind. 1000 m23)
I

II

III

1.3
15.3
10.2
12.7
20.4
0.0
15.3
40.8
76.5
0.0
20.4
5.1
20.4
5.1
15.3
117.2
0.0
0.0
0.0
15.3
20.4
40.8
0.0
193.7
0.0
71.4
66.3
10.2
5.1
20.4
5.1
0.0
103.2
20.4
0.0
25.5
56.1
6.4
5.1
10.2
0.0
66.3
0.0
5.1
25.5
25.5
71.4
5.1
10.2
1.3
3.8
15.3
0.0
20.4
20.4
45.9
0.0
91.7
1459.0
3747.4
5206.4

0.0
0.0
0.0
1.0
15.2
0.0
639.7
0.0
0.0
22.8
30.5
76.2
106.6
0.0
7.6
441.7
0.0
0.0
60.9
53.3
114.2
106.6
15.2
198.0
22.8
129.5
83.8
15.2
76.2
0.0
0.0
7.6
392.2
38.1
22.8
243.7
220.9
1.0
0.0
0.0
799.7
30.5
7.6
15.2
7.6
228.5
0.0
0.0
37.1
0.0
0.0
22.8
0.0
0.0
0.0
7.6
0.0
45.7
4345.7
4059.2
8405.0

8.3
0.0
0.0
0.0
33.4
16.7
83.5
8.3
0.0
0.0
58.4
41.7
58.4
0.0
0.0
559.4
16.7
8.3
33.4
217.1
217.1
33.4
0.0
200.4
0.0
175.3
91.8
16.7
25.0
8.3
0.0
0.0
229.6
179.5
0.0
451.9
108.5
3.1
0.0
0.0
8.3
25.0
0.0
0.0
33.4
50.1
0.0
0.0
57.4
1.0
0.0
41.7
2.1
0.0
0.0
8.3
83.5
16.7
3212.5
3719.8
6932.3

Assayed
+
+

Positive
+
+

+
+
+

+
+

+
+
+
+
+
+
+

+
+
+
+
+

+
+
+
+

+
+

+
+
+

+
+
+

+
+
+
+
+
+
+

+
+
+
+

+
+
+

+
+
+

+
+
+

+
+

+
+

+
+

+
+

Average taxa abundances (individuals 1000 m23) is shown for the three MIK hauls along with total relative abundance. Last two columns show, respectively, the
taxa selected for E. encrasicolus DNA assay testing and, those with at least one positive reaction.

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

Cephalopoda (paralarvae)
Tomopteris spp.
Polychaeta larvae
Cymbulia peroni
Clio spp.
Pteropod spp.
Calanus helgolandicus
Rhincalanus nasutus
Eucalanus elongatus
Centropages typicus
Candacia armata
Euchirella rostrata
Euchirella curticauda
Euchirella spp.
Metridia lucens
Pleuromamma robusta
Pleuromamma xiphias
Pleuromamma spp.
Euchaeta acuta
Euchaeta hebes
Euchaeta spp.
Paraeuchaeta gracilis
Paraeuchaeta norvegica
Paraeuchaeta tonsa
Paraeuchaeta spp.
Undeuchaeta major
Undeuchaeta plumosa
Undeuchaeta spp.
Other/damaged Copepods
Conchoecilla daphnoides
Parathemisto abyssorum
Diastylidae
Meganyctiphanes norvegica
Nematoscelis megalops
Euphausia krohnii
Damaged Euphausiacea (eye bilobed)
Euphausiacea spp. (eye simple)
Pasiphaea sivado
Pasiphaea spp.
Solenocera larvae
Zoea Porcellana
Brachyuran zoeae
Porcellana megalopa
Other brachyuran megalopae
Other decapod larvae
Chaetognatha
Echinodermata larvae
Oikopleura spp.
Benthosema glaciale
Myctophum punctatum
Damaged myctophid (juvenile/adult)
Myctophidae larvae
Saccopharyngiformes
E. encrasicolus larvae
Clupeid larvae damaged
Other sh larvae
Fish egg = Anchovy
Others (non-gelatinous)
Non-gelatinous
Gelatinous
Total

Real-time polymerase chain reaction

%
Average
0.05
0.10
0.07
0.09
0.35
0.08
3.04
0.30
0.49
0.09
0.53
0.54
0.83
0.03
0.13
5.19
0.08
0.04
0.40
1.35
1.63
0.84
0.06
2.99
0.09
1.81
1.20
0.21
0.46
0.17
0.03
0.03
3.32
1.14
0.09
3.30
1.76
0.06
0.03
0.07
3.21
0.67
0.03
0.09
0.35
1.31
0.46
0.03
0.49
0.01
0.02
0.39
0.01
0.13
0.13
0.36
0.40
0.85
42.02
57.98
100.00

Page 5 of 10

Macrozooplankton predation impact on anchovy

500 5000 ng, a threshold of, respectively, 32.4 and 29.4 Ct units
was applied. Finally, the percentage of positive signals was computed
per taxa and MIK haul.

Anchovy egg mortality estimations

NP = p DC ,

(1)

where NP is the number of anchovy eggs consumed over the previous

24 h per unit area, p is the proportion of positive TaqMan assay for a
certain taxon, and DC is the estimated density of the predators per
unit area. Then, for each sampled location taking into account
every assayed taxon:

MP =

DP +

N
P

NP

100,

(2)

where MP is the daily mortality at the sampling location exerted by

macrozooplankton predation and DP is the estimated abundance of
anchovy eggs per unit area. The variable DP was estimated based in
CUFES data due to the high discrepancy between CUFES and
PairoVET records (Table 2). While PairoVET hauls are more sensitive to patchiness due to the small area sampled (0.1 m2), CUFES
data integrate egg abundances along 1.5 nm at both sides of the
PairoVET location (where approximately the MIK net tow starts).
CUFES data were transformed to eggs per meter squared by applying
a CUFES/PairoVET ratio of 6 (SD 4 6; consistent along 2011
sampling depth and abundances ranges).

Prey and predator distribution

Anchovy eggs were distributed in two main areas in the BIOMAN
2011 campaign reaching up to 47.58N and 5.78W (Figure 1).
While spawning on the inner shelf (0100 m depth) was present
only along the French coast, the second spawning band, at shelf
break location, also included the Spanish area. In between, in
waters with 100200 m depth, the presence of anchovy eggs was
rare. The same patterns are kept when plotting CUFES device abundances (data not shown). Regardless of the discrepancy between
CUFES and PairoVET sampling devices (see Material and
methods), the three MIK samples were collected at areas of relatively
high anchovy egg abundances along the shelf break (Figure 1 and
Table 2). Samples were collected at the onset of the stratification
period and in waters with a primary production cline developed
at 30 m depth for MIK-II and -III stations and at 50 m for
MIK-I (Figure 2). The vertical distribution of pelagic biomass
during the haul is shown by acoustic biomass profiling.
Maximum acoustic biomasses corresponded to (swimbladder
bearing) fish category. Regarding distribution along the analysed
depth strata (1025, 25 45, and 45 70/75 m), while acoustic
signals corresponding to fish always peaked at shallower waters
(with values in MIK-I being one order of magnitude higher than
those in MIK-II and -III), both plankton categories presented
highest abundances in the shallowest strata (10 25 m depth) at
MIK-I location but at the deepest strata at MIK-II and -III ones
(Figure 2). Taxonomic identification of the net collected individuals
included 58 distinct taxa (Table 1) and abundances from 5.2 to
8.4 ind. m23. Apart from gelatinous organisms (58% of total abundance), the remaining taxa showing relative abundances 1%
included copepods (22%), euphausiids (10%), decapods larvae
(4%), and chaetognaths (1%). A total of 38 taxa, including molluscs,
annelids, crustaceans, chaetognaths, and fish, were sorted for assay
testing (Table 1). Considering only the assayed taxa their abundances were 1, 2.5, and 2.8 ind. m23 for, respectively, MIK-I, -II,
and -III hauls. The number of assayed specimens was related with
their field abundance and because of this, copepods and euphausiids
comprised 82% of the assayed organisms (respectively 56 and 26%;
Table 2).

Detection of anchovy DNA within macrozooplankton taxa

A total of 17% of the assayed organisms yield a positive signal for
anchovy DNA (140 of 840). Among these, the most positive reactions corresponded to copepods (66%) followed by euphausiids
(16%), chaetognaths (5%), and myctophids (4%). However, considering only abundant taxa, those with at least 25 assayed individuals (13 taxa; Table 2), only five presented a predation incidence
over 20% and four of them were copepods: Paraeuchaeta gracilis
(52%), P. tonsa (40%), Undeuchaeta plumosa (31%), and
U. major (24%), followed by chaetognaths (21%). For the abundant euphausiids and myctophids, only 10% of the assayed individuals presented anchovy DNA remains within their stomach
contents. When all the assayed taxa are considered together a
total of 48, 5, and 9% of positive signals corresponded to, respectively, MIK-I, -II, and -III hauls. Plotting these values against the
estimated anchovy egg densities a positive relationship between
prey abundance and predation incidence is shown (Figure 3).
Apart from this, none of the 190 negative controls tested positive
for anchovy DNA (respectively 102 EBs and 88 NTCs; see Material
and methods).

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

We made the following assumption: each assay positive signal

corresponded to one anchovy egg killed in the last 24 h. Although
the detectability experiment performed in Liocarcinus megalopae
showed that predation events were detectable during 3 h (Albaina
et al., Under Review) and, therefore, an individual continuously
feeding along the 24 h cycle could consume up to eight times the
amount detected in the last 3 h; however, the variety of taxa involved
and the lack of information about zooplankton feeding behaviour
and digestion times (e.g. Durbin et al., 2011) make us consider
the 1 positive assay 1 egg/larvae killed in the last 24 h as a reasonable conservative assumption representing minimum estimation of the predation impact of macrozooplankton on anchovy.
Beside this, the risk of positive signals arising from predation
events dated .24 h ago is discarded by the Liocarcinus digestion
experiment and the available literature on marine invertebrates
detectability experiments using real-time PCR assays targeting
short mtDNA regions (Albaina et al., 2010; Durbin et al., 2011).
Although the DNA-based assay cannot distinguish between the
anchovy egg and larval stages, we restrict to anchovy egg distribution
data to compute mortality as these are the only available prey
abundances. However, at this early stage of the species spawning
season anchovy eggs would undoubtedly represent the bulk of
anchovy ELS and thus, a significant bias due to the previous simplification is not to be expected (e.g. Motos et al., 1996). Furthermore,
due to the quantitative nature of real-time PCR, we can estimate
the number of anchovy eggs corresponding to a certain Ct value
(Albaina et al., Under Review); applying this we found only five
cases (of 140 positive assays) where measured Ct values could corresponded to the amount of DNA of .1 anchovy egg thus giving
further support to the 1 positive assay 1 egg killed in the last
24 h assumption. Then daily egg mortality at the sampled locations
was computed as the fraction of anchovy eggs eaten in the last 24 h
[Equations (1) and (2)]. For each assayed taxon,

Results

Page 6 of 10

A. Albaina et al.

Table 2. Detection of anchovy eggs/larvae predation by macrozooplankton taxa.

Date
Time of haul (local time)
Haul depth (m)
Bottom depth (m)
Anchovy eggs (PairoVET)
Anchovy eggs at 3 m depth (CUFES)

% + assays
100.0
66.7
11.1
25.0

MIK-II
19 May 2011
2:41
69.5
3000
7165.1
160 53.9
n assayed
1
3
9
4

0.0
100.0
50.0
100.0
50.0
36.4

4
1
4
1
2
22

0.0
0.0
100.0
68.4
50.0
76.9
50.0
56.5
0.0
0.0

3
3
8
38
14
13
2
115
4
1

50.0
50.0
0.0
0.0
44.4
50.0
100.0
100.0
0.0

20
4
3
3
27
2
1
2
1

100.0
0.0
100.0
50.0
18.2

5
8
1
2
11

0.0
48.2

7
193

% + assays

MIK-III
22 May 2011
4:20
75.6
2944
642.8
28 228.5
n assayed

0.0
0.0

1
2

0.0
0.0
7.1

4
10
14

0.0
2.1

1
47

0.0
0.0
0.0
15.4
4.0
12.5
0.0
0.0
4.2

7
7
9
13
25
16
11
3
167

100.0
5.0
0.0

1
60
2

5.0
4.7
0.0

20
85
1

0.0

9.1
5.6

22
18

0.0
4.8

3
21

5.0

302

% + assays
0.0

All

n assayed
1

0.0
0.0
0.0
0.0
0.0

4
1
7
5
7

5.0
0.0
0.0
0.0
8.0
75.0
33.3
15.0
9.1
0.0
10.4
0.0

60
1
4
23
25
4
24
20
11
2
193
1

3.2
0.0
0.0
10.4
5.8
33.3

31
25
3
48
104
3

66.7
0.0
10.0
100.0
0.0
11.5
0.0
0.0
9.3

3
6
20
1
5
26
2
1
345

% + assays
50.0
66.7
10.0
10.0
0.0
0.0
6.3
12.0
100.0
33.3
9.3
0.0
0.0
0.0
5.4
52.0
40.2
24.0
31.4
14.3
19.4
0.0
0.0
100.0
12.6
6.5

n assayed
2
3
10
10
1
15
16
25
1
3
129
1
11
33
37
25
87
50
35
7
475
5
1
1
111
31

8.5
10.2
33.3
100.0
100.0
0.0
66.7
21.2
6.5
100.0
10.0
10.3
0.0
0.0
16.7

71
216
6
1
2
3
3
33
46
2
10
58
2
8
840

MIK hauls data are shown along with the number of predators assayed per species and the percentage of the assays testing positive for E. encrasicolus DNA. Prey
abundance (egg 1000 m23) based in both PairoVET net and CUFES device are shown (see Material and methods).

Anchovy eggs mortality due to macrozooplankton

predation
Daily anchovy eggs mortality due to macrozooplankton predation
(MP; see Material and methods) was 1.6, 3, and 4% for, respectively,
MIK-I, -II and -III (Figure 3). The range of prey abundances was
2682122 eggs m22. No relationship between prey abundance and
MP was evident.

Discussion
Twenty-five years after the seminal paper of Bailey and Houde (1989)
on the fate of predation on fish ELSs mortality, detailed knowledge
and understanding of the sources and stage-specific rates mortality,
and of the relative roles of density-independent vs. density-dependent
processes, remains elusive (Browman and Skiftesvik, 2014).
However, nowadays, molecular identification of prey in the stomachs

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

Cephalopoda (paralarvae)
Tomopteris spp.
Cymbulia peroni
Clio spp.
Pteropod spp.
Candacia armata
Euchirella rostrata
Euchirella curticauda
Euchirella spp.
Metridia lucens
Pleuromamma robusta
Pleuromamma xiphias
Euchaeta acuta
Euchaeta hebes
Euchaeta spp.
Paraeuchaeta gracilis
Paraeuchaeta tonsa
Undeuchaeta major
Undeuchaeta plumosa
Undeuchaeta spp.
Total copepods
Conchoecilla daphnoides
Parathemisto abyssorum
Diastylidae
Meganyctiphanes norvegica
Nematoscelis megalops
Euphausia krohnii
Damaged Euphausiacea (eye bilobed)
Total euphausiids
Pasiphaea sivado
Pasiphaea spp.
Solenocera larvae
Other brachyuran megalopae
Other decapod larvae
Chaetognatha
Benthosema glaciale
Myctophum punctatum
Myctophidae larvae
Total myctophids
Saccopharyngiformes
Other sh larvae
Total

MIK-I
12 May 2011
3:56
75.1
1070
2589.3
127 312.6

Macrozooplankton predation impact on anchovy

Page 7 of 10

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Figure 2. MIK hauls acoustic and CTD vertical proles. Top row graphs show the acoustic biomasses corresponding to the MIK haul towed
distance (a, b, and c graphs for, respectively, MIK-I, -II, and -III), expressed as NASC values (log10 values; m2 nm22). The three different lines
correspond to the sh (solid line with full triangles; top axis), uid-like zooplankton (broken line with full squares, bottom axis) and other
plankton (broken line with empty squares; bottom axis) dened categories (see Material and methods for further information). Data are shown by
depth strata, from 10 m depth to 25, from 25 to 45 and, from 45 m to maximum MIK haul depth (left axis). Bottom row graphs show the vertical
(haul depth in meters; left axis) proles of density (sigma-t, top axis; solid bold line) and uorescence (relative units, bottom axis; broken line) from
the CTD data of the three MIK hauls (from left to right MIK-I, -II and -III). Sigma-t (kg m23) is the density anomaly of a water sample when the total
pressure on it has been reduced to atmospheric pressure (i.e. zero water pressure), but the temperature and salinity are in situ values.
of predators allows obtaining important information on trophic
interactions that may be difficult if not impossible to obtain in any
other way. In this sense, applying a real-time PCR-based assay
capable of detecting European anchovy (E. encrasicolus) DNA
traces, we have provided insights on the generally neglected role of

macrozooplankton predation on anchovy eggs mortality. The target

species spawns along two main areas in the Bay of Biscay: the shelf
around the Gironde river mouth and the shelf break, from a core
region at the SE edge of the Bay up to the whole shelf break area in
years of high anchovy abundance (e.g. Motos et al., 1996; ICES,

Page 8 of 10

A. Albaina et al.

2011). In 2011, for the first time in a decade, we were able to study
macrozooplankton predation along the whole shelf break spawning
area. The main results from the application of our molecular
method are that (i) macrozooplankton predation impact is low,
with daily egg mortalities (MP) ,4% for a broad range of prey abundances and that, (ii) both MP and predation incidence patterns
suggest macrozooplankton predation on anchovy ELSs following a
functional response I (Figure 3). Although a value up to 50% of
positive signals was recorded for the macrozooplankton predators
community in MIK-I station, this corresponded to the third
highest prey abundance record for the whole BIOMAN 2011 campaign (2122 eggs m22). Present results point to a low and densityindependent impact and, therefore, suggest that macrozooplankton
predation exert a negligible effect on anchovy egg survival at the
shelf break spawning centre. However, a range of factors potentially
affecting this conclusion need to be discussed.
On one hand, other factors, apart from prey abundance, could be
contributing to the observed patterns; these include vertical match/
mismatch of prey and predators, alternative prey availability, and
the relative abundance of competing predators (the amount of prey
available per predator; e.g. Arditi and Ginzburg, 2012). The bulk of
positive signals corresponded to large species of carnivorous calanoid
copepods (mainly Aetideidae and Euchaetidae families) characterized by performing relatively large amplitude diel vertical migrations
(DVM) and feeding at night in shallower waters (e.g. Hays et al., 1994;
Mauchline, 1998). Apart from these, only chaetognaths, myctophid
fish, and euphausiids exerted a significant impact in anchovy eggs
mortality. These organisms also perform large DVM (e.g. Kaartvedt
et al., 2002; Irigoien et al., 2004; Dypvik et al., 2012) and due to the
permanent shallow location of fish eggs (mainly in the first 20 m;
Boyra et al., 2003; Coombs et al., 2004) the putative predatory
impact of these species is limited both in the time and space. In this
sense, the higher percentage of animals having ingested anchovy
DNA at MIK-I could also be partially explained by the shallower
location of plankton as estimated acoustically (Figure 2). However,
the reduced taxonomic resolution of the existing algorithms prevents

further testing of this hypothesis and depth-stratified plankton sampling would be required. Interestingly, the location of the Chl-a cline
was deeper at the former station (50 m compared with 30 m for
MIK-II and -III). Although we lacked actual measurements of
alternative prey abundances, this cline generally coincides with the
centre of distribution for herbivorous plankton (e.g. Longhurst,
1976). A distant location regarding anchovy eggs strata could favour
a vertical mismatch for predation as small-medium-sized copepods
are typical foods of the above cited predators. As an example, switching
from carnivorous to herbivorous feeding modes during spring
phytoplankton bloom has been documented for the abundant
Meganyctiphanes norvegica (Kaartvedt et al., 2002). However, the
above commented higher predation incidence in MIK-I, including
the 71% of the M. norvegica positive assays in 2011, makes us reject
this hypothesis. Finally, the reported patterns could be affected by
the relative abundance of predators. The fact that assayed predator
abundance in MIK-I was around one-third of those measured for
the remaining hauls could implya reduced competence for the existing
prey resource. Nevertheless, this is confused by the fact that prey abundance at this particular location was five to eight times higher than in
the remaining hauls. Finally, while typically, predation studies are
focused in one or few predators, the high-throughput character of
the molecular method allows an holistic approach to the predation
impact on anchovy eggs reducing the bias potentially associated
with the omission of competing macrozooplankton predators to a
minimum. Beside this, the fate of false-positive signals in the reported
results is unlikely due to the included negative controls results.
However, false negatives can arise from the conservative nature of
the assay and thus results are to be considered as minimum values
(see Albaina et al., Under Review for further discussion).
On the other hand, other predators apart from the assayed ones
might be exerting a mortality pressure on anchovy eggs. For
example, gelatinous organisms were not sorted for assay testing,
but these organisms can be important predators of fish eggs worldwide (e.g. Purcell and Arai, 2001). However, to our knowledge, no
work addressing the role of gelatinous organism in anchovy eggs

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

Figure 3. Macrozooplankton predation on anchovy eggs. Full circles represent the relationship between the macrozooplankton predation
incidence (percentage of positive signals; left axis) and the abundance of anchovy eggs at the MIK haul location (as estimated from CUFES device,
see Material and methods). Empty circles correspond to the relationship between egg abundance and daily mortality due to macrozooplankton
(MP, see Material and methods; right axis).

Page 9 of 10

Macrozooplankton predation impact on anchovy

myctophid Myctophum punctatum and, a higher presence of the

copepod Pleuromamma robusta and the euphausiid Nematoscelis
megalops, corresponding to the northernmost located hauls, in
2011. However, regarding the other Bay of Biscay anchovy spawning
centre, the shelf between Gironde and Adour river mouths
(Figure 1), 2010 results indicated that macrozooplankton alone,
dominated mainly by mysids and decapods larvae instead of copepods and euphausiids, could control anchovy recruitment at low
abundances and that predation followed a functional response II
pattern (Albaina et al., Under Review). While 63 and 66% of the
positive assays in the shelf break area corresponded to copepods
in, respectively, 2010 and 2011 surveys (followed by euphausiids
with another 11 and 16% of the predation events, respectively), 23
and 70% corresponded to mysids and decapods in the 2010 shelf
one. A combination of feeding behaviour (shelf break vs. shelf
macrozooplankton community) and prey availability would
explain the reported patterns for anchovy egg predation in the Bay
of Biscay.
Finally, a reduced mortality due to low predation pressure and
enough food availability does not necessarily imply a higher survival
in the shelf break spawning centre. Along with disease, parasitism
and pollutants, a mortality source of special relevance at offshore
spawning areas is the advection of eggs and larvae to unsuitable
habitats. In this sense, models predicting minimum or no survival
off the shelf due to unfavourable winds/currents have been proposed for the Bay of Biscay anchovy eggs and larvae (Allain et al.,
2007) and this could counterbalance the reduced predation
impact at this domain. In this sense, based in otolith microchemistry
analyses for a reduced number (n 40) of anchovy juveniles collected along the Bay of Biscay, Aldanondo et al. (2010) reported
that all of those juveniles had been spawn at low salinity waters
suggesting low survival at the shelf break spawning area. Beside
this, both research groups reported the highest survival for
anchovy eggs laid after the peak spawning season (Allain et al.,
2007; Aldanondo et al., 2010) where BIOMAN campaigns take
place. Because of this, further analysis of a more anchovy juvenile
otoliths along with a broader temporal coverage of predation
studies is needed as to resolve the role of the shelf break spawning
centre in the Bay of Biscay anchovy recruitment.

Acknowledgements
We are grateful to the crews of the RV Investigador and the
BIOMAN 2011 campaign on-board scientists and analysts for
their support during sampling and, to D. Abad, O. Niembro, and
I. Mendia (UPV/EHU) for their technical assistance. SGIker technical and human support (UPV/EHU, MICINN, GV/EJ and
ESF) is gratefully acknowledged. This research was financially supported by the projects ECOGENBAY (MICINN CTM2009-13570C02-02), funded by the Ministry of Science and Research of the
Government of Spain, and BIOMAN, funded by the Department
of Economic Development and Competitiveness of the Basque
Government and by the European Commission.
Figure 4. Anchovy eggs daily mortality due to macrozooplankton (MP)
in the Bay of Biscay (2010 and 2011 data). Present work data (BIOMAN
2011 campaign) are plotted along with those in Albaina et al. (Under
Review; BIOMAN 2010 campaign). Bottom axis represents the
abundance of E. encrasicolus eggs at the MIK haul location. While empty
circles correspond to the stations sampled in 2011, squares refer to MIK
stations located at the two spawning centres in 2010 (see Figure 1),
respectively, shelf break (empty squares) and shelf (full squares)
stations. Note that the full square at the upper left has a different scale.

References
Albaina, A., Fox, C. J., Taylor, N., Hunter, E., Maillard, M., and Taylor,
M. I. 2010. A TaqMan real-time PCR based assay targeting plaice
(Pleuronectes platessa L.) DNA to detect predation by the brown
shrimp (Crangon crangon L.) and the shore crab (Carcinus
maenas L.)assay development and validation. Journal of
Experimental Marine Biology and Ecology, 391: 178 189.

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

mortality has been performed in the Bay of Biscay and thus this
question remains undetermined. Beside this, zooplanktivorous
fish are another important source of anchovy ELSs mortality worldwide (e.g. Szeinfeld, 1991; Krautz et al., 2007). Regarding the Bay of
Biscay, recently, two studies have measured the fish predation
impact on anchovy eggs mortality. While Bachiller (2013), using
visual identification of contents in eight fish species including cannibalism by anchovy, reported that zooplanktivorous fish were
responsible of 16 57% of the anchovy eggs mortality in the
whole Bay of Biscay (for respectively, the 2008 and 2009 BIOMAN
campaigns), an 7% was reported by Albaina et al. (Under
Review) when applying the present molecular method to sardines
in the BIOMAN 2010 campaign. The latter reduced to a mere 2%
when considering solely the shelf break spawning area (Albaina
et al., Under Review). Interestingly, based on the combination of
sufficient food fields for larvae and juveniles and the fact that fish
predators of anchovy ELSs are relatively scarce at Bay of Biscay offshore waters, Irigoien et al. (2007) proposed that anchovy could be
recruited through a spatial loophole (sensu Bakun and Broad, 2003).
In this sense, present results, regarding macrozooplankton predation on anchovy eggs, along with those on anchovy larvae growth
by Cotano et al. (2008), where higher survival was reported at offshore waters, support the consideration of shelf break spawning
area as a predation refuge for anchovy ELSs. Although present
data were based on three stations for a sole survey, data from
another two macrozooplankton hauls in the 2010 BIOMAN campaign (Albaina et al., Under Review) allow further testing of the
reported pattern. Figure 4 shows that 2010 MP data corresponded
well with 2011 ones where a broader density field and spatial area
were sampled. Shelf break macrozooplankton communities were
dominated by the same taxa in both campaigns with just the appearance, in small numbers, of the euphausiid Euphausia krohnii and the

Page 10 of 10

Houde, E. D. 2008. Emerging from Hjorts shadow. Journal of

Northwest Atlantic Fishery Science, 41: 53 70.
ICES. 2011. Report of the Working Group on Anchovy and Sardine
(WGANSA), 24 28 June 2011, Vigo, Spain. ICES CM 2011/
ACOM:16. 470 pp.
Irigoien, X., Conway, D. V. P., and Harris, R. P. 2004. Flexible diel vertical
migration behaviour of zooplankton in the Irish Sea. Marine Ecology
Progress Series, 267: 85 97.
Irigoien, X., Fiksen, O., Cotano, U., Uriarte, A., Alvarez, P., Arrizabalaga,
H., Boyra, G., et al. 2007. Could Biscay Bay Anchovy recruit through
a spatial loophole? Progress in Oceanography, 74: 132 148.
Kaartvedt, S., Larsen, T., Hjelmseth, K., and Onsrud, M. 2002. Is the omnivorous krill Meganyctiphanes norvegica primarily a selectively
feeding carnivore? Marine Ecology Progress Series, 228: 193 204.
Kenchington, T. J. 2013. Natural mortality estimators for informationlimited fisheries. Fish and Fisheries , doi: 10.1111/faf.12027.
King, R. A., Read, D. S., Traugott, M., and Symondson, W. O. C. 2008.
Molecular analysis of predation: a review of best practice for
DNA-based approaches. Molecular Ecology, 17: 947 963.
Krautz, M. C., Castro, L. R., and Gonzalez, M. 2007. Interaction of two
key pelagic species in the Humboldt Current: euphausiid predation
on anchoveta eggs estimated by immunoassays. Marine Ecology
Progress Series, 335: 175 185.
Lasker, R. (Ed.) 1985. An Egg Production Method for Estimating
Spawning Biomass of pelagic fish: Application to the Northern
Anchovy, Engraulis mordax. NOAA Technical report NMFS 36,
US Department of Commerce, Washington, DC. 100 pp.
Lezama-Ochoa, A., Ballon, M., Woillez, M., Grados, D., Irigoien, X., and
Bertrand, A. 2011. Spatial patterns and scale-dependent relationships between macrozooplankton and fish in the Bay of Biscay: an
acoustic study. Marine Ecology Progress Series, 439: 151 168.
Longhurst, A. R. 1976. Interactions between zooplankton and phytoplankton profiles in the eastern tropical Pacific Ocean. Deep-Sea
Research, 23: 729 754.
MacLennan, D. N., Fernandes, P. G., and Dalen, J. 2002. A consistent approach to definitions and symbols in fisheries acoustics. ICES
Journal of Marine Science, 59: 365 369.
Mauchline, J. 1998. The biology of calanoid copepods. Advances in
Marine Biology, 33:1 710.
Motos, L., Uriarte, A., and Valencia, V. 1996. The spawning environment of the Bay of Biscay anchovy (Engraulis encrasicolus L.).
Scientia Marina, 60: 117 140.
Pompanon, F., Deagle, B. E., Symondson, W. O. C., Brown, D. S., Jarman,
S. N., and Taberlet, P. 2012. Who is eating what: diet assessment using
next generation sequencing. Molecular Ecology, 21: 19311950.
Purcell, J. E., and Arai, M. N. 2001. Interactions of pelagic cnidarians and
ctenophores with fish: a review. Hydrobiologia, 451: 27 44.
Richardson, D. E., Hare, J. A., Fogarty, M. J., and Link, J. S. 2011. Role of
egg predation by haddock in the decline of an Atlantic herring population. Proceedings of the National Academy of Sciences of the
United States of America, 108: 13606 13611.
Simmonds, J., and Maclennan, D. 2005. Fisheries Acoustics: Theory and
Practice, 2nd edn. Blackwell Science. Fish and Aquatic Resources
Series 10, Oxford. 437 pp.
Symondson, W. O. C. 2002. Molecular identification of prey in predator
diets. Molecular Ecology, 11: 627 641.
Szeinfeld, E. 1991. Cannibalism and intraguild predation in clupeoids.
Marine Ecology Progress Series, 79: 17 26.
Terazaki, M. 2005. Predation on anchovy larvae by a pelagic chaetognatha, Sagitta nagae in the Sagami Bay, central Japan. Coastal
Marine Science, 29: 162 164.
Theilacker, G. H., Lo, N. C. H., and Townsend, A. W. 1993. An immunochemical approach to quantifying predation by euphausiids on the
early stages of anchovy. Marine Ecology Progress Series, 92: 3550.

Handling editor: Stephane Plourde

Downloaded from http://icesjms.oxfordjournals.org/ by guest on November 23, 2014

Aldanondo, N., Cotano, U., Tiepolo, M., Boyra, G., and Irigoien, X. 2010.
Growth and movement patterns of early juvenile European anchovy
(Engraulis encrasicolus) in the Bay of Biscay based on otolith microstructure and chemistry. Fisheries Oceanography, 19: 196208.
Aljanabi, S. M., and Martinez, I. 1997. Universal and rapid saltextraction of high quality genomic DNA for PCR based techniques.
Nucleic Acids Research, 25: 4692 4693.
Allain, G., Petitgas, P., and Lazure, P. 2007. The influence of environment
and spawning distribution on the survival of anchovy (Engraulis
encrasicolus) larvae in the Bay of Biscay (NE Atlantic) investigated
by biophysical simulations. Fisheries Oceanography, 16: 506514.
Arditi, R., and Ginzburg, L. R. 2012. How species interact. Altering
the standard view on trophic ecology. Oxford University Press,
New York. 192 pp.
Bachiller, E. 2013. Trophic ecology of small pelagic fish in the Bay of
Biscay: ecological effects of trophic interactions. Ph.D. Thesis,
University of the Basque Country, Leioa, Spain, unpublished.
Bailey, K. M., and Houde, E. D. 1989. Predation on eggs and larvae of
marine fishes and the recruitment problem. Advances in Marine
Biology, 25: l 83.
Bakun, A., and Broad, K. 2003. Environmental loopholes and fish
population dynamics: comparative pattern recognition with focus
on El Nino effects in the Pacific. Fisheries Oceanography, 12: 458473.
Ballon, M., Bertrand, A., Lebourges-Dhaussy, A., Gutierrez, M., Ayon,
P., Grados, D., and Gerlotto, F. 2011. Is there enough zooplankton
to feed forage fish population off Peru? An acoustic (positive)
answer. Progress in Oceanography, 91: 360 381.
Boyra, G., Rueda, L., Coombs, S. H., Sundby, S., Adlandsvik, B., Santos,
M., and Uriarte, A. 2003. Modelling the vertical distribution of eggs
of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus). Fisheries Oceanography, 12: 381 395.
Browman, H. I., and Skiftesvik, A. B. 2014. The early life history of fish
there is still a lot of work to do!. ICES Journal of Marine Science, 71:
907 908.
Checkley, D. M., Ortner, P. B., Settle, L. R., and Cummings, S. R. 1997. Acontinuous, underway fish egg sampler. Fisheries Oceanography, 6: 5873.
Coombs, S. H., Boyra, G., Rueda, L. D., Uriarte, A., Santos, M., Conway,
D. V. P., and Halliday, N. C. 2004. Buoyancy measurements and vertical distribution of eggs of sardine (Sardina pilchardus) and
anchovy (Engraulis encrasicolus). Marine Biology, 145: 959 970.
Cotano, U., Irigoien, X., Etxebeste, E., Alvarez, P., Zarauz, L., Mader, J.,
and Ferrer, L. 2008. Distribution, growth and survival of anchovy
larvae (Engraulis encrasicolus L.) in relation to hydrodynamic and
trophic environment in the Bay of Biscay. Journal of Plankton
Research, 30: 467 481.
Durbin, E. G., Casas, M. C., and Rynearson, T. A. 2011. Copepod feeding
and digestion rates using prey DNA and qPCR. Journal of Plankton
Research, 34: 72 82.
Dypvik, E., Rstad, A., and Kaartvedt, S. 2012. Seasonal variations in
vertical migration of glacier lanternfish, Benthosema glaciale.
Marine Biology, 159: 1673 1683.
Foote, K., Knudsen, H. P., and Vestnes, G. 1987. Calibration of Acoustic
Instruments for Fish Density Estimation: A Practical Guide.
International Council for the Exploration of the Sea, Copenhagen,
Denmark.
Gallego, A., North, E. W., and Houde, E. D. 2012. Understanding and quantifying mortality in pelagic, early life stages of marine organisms old
challenges and new perspectives. Journal of Marine Systems, 93: 13.
Hays, G. C., Proctor, C. A., John, A. W. G., and Warner, A. J. 1994.
Interspecific differences in the diel vertical migration of marine
copepods: the implications of size, color, and morphology.
Limnology and Oceanography, 39: 1621 1629.
Heath, M. R. 1992. Field investigations of the early life stages of marine
fish. Advances in Marine Biology, 28: 1 174.

A. Albaina et al.