Received: 14 February 2017 | Revised: 4 August 2017 | Accepted: 19 September 2017

DOI: 10.1111/jfd.12747

SPECIAL ISSUE

Study of the hooking behaviour of Lepeophtheirus salmonis

(Krøyer, 1837) copepodids on Atlantic salmon, Salmo salar L.,
using a novel in vivo test system

C Delfosse1,2
le
| C Lafont-Lecuelle1 | H Barthe
my1 | C Chabaud1 | E Teruel1 |
C Bienboire-Frosini1 | P Pageat1,2

1
Research Institute in Semiochemistry and
Applied Ethology (IRSEA) Route du Che ^ne,
Quartier Salignan, Apt, France
2
Research Institute in Semiochemistry and
Applied Ethology – Aquaculture Research
Centre (IRSEA-ARC), Vikebukt, Norway
Correspondence
C Delfosse, Research Institute in
Semiochemistry and Applied Ethology
^ne, Quartier Salignan,
(IRSEA) Route du Che
Apt, France.
Email: c.delfosse@group-irsea.com
Abstract
The screening of putative semiochemicals involved in the interaction between
Lepeophtheirus salmonis copepodid and the Atlantic salmon could be used to develop
methods to prevent infestation. A bioassay was designed to evaluate the attractive-
ness of Atlantic salmon for L. salmonis copepodids by counting the number of
hooked parasites. A salmon was bathed in a tank with 60 copepodids during
45 min. The salmon was humanely killed and its body was scrubbed above a plastic
bag. The content of the bag was filtered to count the number of hooked parasites.
This procedure was tested at different water temperatures and for different body
mass. The temperature significantly influenced the number of hooked copepodids
(p < .05). Smolts presented significantly more hooked copepodids at a higher water
temperature (24.6 at 13.8°C) compared to smolts at a lower temperature (18.6 at
6.1°C; p = .011). No correlation was found between the body mass and the number
of hooked parasites (p = .65). This bioassay was used to successfully measure the
attractiveness of Atlantic salmon for L. salmonis copepodids, making it an interesting
tool to screen putative semiochemicals designed to prevent the infestation.

KEYWORDS
Atlantic salmon, attractiveness, copepodid, hooking behaviour, Lepeophtheirus salmonis

1 | INTRODUCTION
Lepeophtheirus salmonis (Krøyer, 1837) is a crustacean ectoparasite
that feeds on many salmonids and more specifically on Atlantic sal-
mon, Salmo salar L. (Boxaspen, 2006; Pike & Wadsworth, 1999; Tully
& Nolan, 2002). In salmon production, this parasite is responsible for
significant impairments in both productivity and welfare of infected
fish (Costello, 2009; Johnson, Treasurer, Bravo, & Nagasawa, 2004;
Liu & Bjelland, 2014). The life cycle of L. salmonis is composed of
eight stages: two free-swimming stages (nauplius I & II), one infesta-
tion stage (copepodid) and five parasitic stages (chalimus I & II, pre-
adult I & II and adult; Hamre et al., 2013). Regarding the life cycle,
the most critical stage is the copepodid, where the parasite has to
settle on a host to continue its development (Pike & Wadsworth,
1999). Copepodids are able to detect vibrations in the water created
by the fish swimming (Heuch & Karlsen, 1997). Upon detection of
these signals, the copepodid propels to the fish, hooks on it and
analyses the mucus to determine whether the fish is a suitable host
(Pike & Wadsworth, 1999). Some authors suggest that this hooking
behaviour is also triggered by the detection of semiochemicals from
the fish (Mordue Luntz & Birkett, 2009). Two days after the hooking
on a suitable host, the copepodid starts to produce a frontal filament
allowing the settlement (i.e., attachment) on the fish (Gonzalez-Ala-
nis, 2000).
Among the novel methods developed to fight against the para-
site, the use of semiochemicals to repel or to attract copepodids into

ttir et al.,
a trap appears to be a promising approach (Ingvarsdo
2002). This is why it was important to develop a bioassay to

J Fish Dis. 2017;1–4. wileyonlinelibrary.com/journal/jfd © 2017 John Wiley & Sons Ltd | 1
2 |
evaluate the attractiveness of Atlantic salmon for copepodids. Such
a test could be used to screen different putative semiochemicals
designed to prevent the hooking behaviour of the copepodid and so
the infestation.
The aim of this study was to develop a novel in vivo test system
in order to assess the attractiveness of Atlantic salmon for
L. salmonis by counting the number of copepodids hooking on the
fish. Body mass of the tested fish and water temperature were
included as factors in this bioassay.
2 | MATERIALS AND METHODS
2.1 | Experimental animals
This study was conducted under the approval of the Ethical Commit-
tee of IRSEA (no 125), and in accordance with the European Direc-
tive 2010/63/UE for the protection of laboratory animals. Salmon
smolts (SALMARâ, Vikebukt, Norway), obtained 3 weeks before the
beginning of the experimentation, were kept in 180-L holding tanks
(maximal density = 20 kg/m3) continuously supplied with filtered sea
water (5 L/min) pumped from Tresfjorden (62°320 25.5″N 7°080 18.1″
E, Vikebukt, Norway). Salmon were fed daily ad libitum with com-
mercial dry pellets (Skretting AS©, Norway).
Lepeophtheirus salmonis copepodids (Ilab©, Norway) were
obtained 2 days after the nauplius II/copepodid transformation and
used for the experiments in the next 2 days. The parasites were held
in a net-bucket system continuously supplied with filtered and oxy-
genated sea water (0.1 L/min).
2.2 | Design of the bioassay
First, copepodids were counted by splitting up the water containing
them in the wells of a clean ELISA plate thanks to a Pasteur pipette,
and under a stereoscopic microscope (Figure 1). Thus, 60 copepodids
were caught one by one and introduced in a beaker. This method
F I G U R E 1 Picture showing the accurate counting of parasites.
Lepeophtheirus salmonis copepodids were counted one by one under
stereoscopic microscope using a clean ELISA plate
DELFOSSE ET AL.
allows to exclude any copepodid showing poor fitness or any mor-
phological abnormality.
The fish was manually transferred into a 3.5-L flat beaker sup-
plied with oxygenated sea water. Ten minutes later, the 60 copepo-
dids were injected into the flat beaker. Then, 45 min after the
introduction of the copepodids, the fish was humanely killed by
injecting 2 ml of anaesthetic (Benzoakâ) into the flat beaker. The
entire body of the fish, from the front of the head down to the tail,
was scrubbed with a spoon, and rinsed above a plastic bag. To har-
vest all of the hooked copepodids, the procedure was carried out
three times on each fish (front of the fish + pectoral and dorsal
fins/rinse/back of the fish + pelvic, adipose, caudal and anal fins/
rinse/entire body + all fins/rinse). The content of the plastic bag was
then filtered, and the number of previously hooked copepodids was
counted using a magnifying glass.
2.3 | Experiment
A total of 82 Atlantic salmon smolts (60–153 g,
Mean  SD = 101  18) were individually tested using the previ-
ously described bioassay. The influence of the water temperature in
the flat beaker on the number of hooked copepodids was investi-
gated at 6.1, 9.2, 10.3, 12.3 and 13.8°C. Then, the influence of the
body mass of the fish on the number of hooked copepodids was
examined by studying the correlation between the two parameters.
2.4 | Statistical analysis
Data were analysed using SAS 9.4 software (2002–2012 by SAS
Institute Inc., Cary, NC, USA). The significance threshold was con-
ventionally set at 5%.
To assess the effect of water temperature on copepodid hook-
ing, the assumption of homogeneity of variances between groups of
water temperature was verified using the Levene test. Comparisons
were carried out using a one-way ANOVA model. Normality of
residuals was checked by observing residual diagnostic plots and
Shapiro–Wilk test. Post hoc multiple means comparisons were car-
ried out with the help of the Tukey test.
As the data for the number of copepodids hooked did not follow
a normal model, the Spearman rank-order correlation coefficient was
computed and tested to explore the correlation between the body
mass and the number of hooked copepodids.
3 | RESULTS
This bioassay highlighted significant differences in the number of
hooked copepodids according to water temperature (One-way
ANOVA, F-value = 6.68, df = 81, p < .001; Figure 2). Smolts pre-
sented significantly more hooked copepodids at a higher water tem-
perature (13.8°C, mean  SEM = 24.6  1.3) compared to smolts at
a lower temperature (6.1°C, mean  SEM = 18.6  0.7; Tukey test,
p = .011). The correlation between body mass and the number of
DELFOSSE ET AL.
F I G U R E 2 Mean  SEM of the number of hooked copepodids
for the water temperatures 6.1°C (n = 8), 9.2°C (n = 8), 10.3°C
(n = 8), 12.3°C (n = 12) and 13.8°C (n = 46). Different lowercase
letters (a, b, c and d) indicate statistical significant differences (Tukey
test, p < .05) between water temperature groups
F I G U R E 3 Scatter plot (n = 82) representing the number of
hooked copepodids according to the body mass of Atlantic salmon
smolts
hooked copepodids was not significant (Spearman coefficient corre-
lation, q = .051, p = .65; Figure 3).
4 | DISCUSSION
This new bioassay developed for the experiment shows promise as a
tool for evaluating attractiveness of Atlantic salmon for L. salmonis
copepodids. The in vivo test allows copepodids to hook to salmon.
However, it should be noted that some factors in the bioassay may
influence the test quality.
In our study, the water temperature was shown to have a signifi-
cant influence on the hooking behaviour of copepodids: there were
more hooked parasites at high temperatures compare to low temper-
atures. Tucker, Sommerville, and Wootten (2000) have also demon-
strated that the settlement of L. salmonis copepodids on Atlantic
salmon was higher at 12 than 7°C. In the present study, this signifi-
cant difference also occurred between 10 and 12°C. The reasons for
the increased hooking behaviour of L. salmonis copepodids at higher
temperature are not well known, but water temperature certainly
| 3
has a direct effect on the metabolic rate of copepodids and thus
affects the activity of the parasite (Tucker et al., 2000).
Salmon body mass does not seem to influence the number of
copepodids able to hook to it. Other studies describe a positive cor-
relation between body weight and lice burden (Gjerde, Ødeg
ard, &
Thorland, 2011; Glover, Nilsen, & Skaala, 2004). On the contrary,
the study of Kolstad, Heuch, Gjerde, Gjedrem, and Salte (2005)
describes no high correlation between the body weight of salmons
and lice burden.
This bioassay can be used to successfully evaluate the hooking
behaviour of L. salmonis copepodids on Atlantic salmon, making it an
interesting and easy tool to screen several putative semiochemicals
contributing to the communication between the parasite and the
host. In this case, solutions and controls would need to be tested at
the same water temperature to avoid any bias in the bioassay. By
scrubbing the fish to collect hooked copepodids, we make them
more visible and easier to count than directly on the fish, and spe-
cially on dark body parts (dorsal area and fins). Apart from testing
candidate semiochemicals, this bioassay can also be used to investi-
gate the influence of the physiological state of the host on copepo-
did hooking behaviour. This bioassay might be applicable to other
species of sea lice (Caligus elongatus, Caligus rogercresseyi, Argulus
japonicus) as well as other species of fish (Oncorhynchus mykiss,
Scophthalmus maximus).
In conclusion, this bioassay provides an efficient and simple test
for investigating the attractiveness of Atlantic salmon for L. salmonis
copepodids, and for subsequently screening candidate semiochemi-
cals involved in the host–parasite interaction, or any treatment aim-
ing to prevent the infestation.
ACKNOWLEDGEMENTS
We would like to thank B-V. Løvik and E-M. Sætre for their techni-
cal help in maintaining the fish and sea lice. We would like to thank
E. Landen, a professional translator, Dr. M. Mengoli and Dr. P. Asproni
for reviewing the manuscript. We are also grateful to IRSEA’s Ethical
Committee for their comments and the approval of the protocol.
ORCID
C Delfosse http://orcid.org/0000-0002-1725-1450
REFERENCES
Boxaspen, K. (2006). A review of the biology and genetics of sea lice.
ICES Journal of Marine Science, 63, 1304–1316.
Costello, M. J. (2009). The global economic cost of sea lice to the salmo-
nid farming industry. Journal of Fish Diseases, 32, 115–118.
Gjerde, B., Ødeg ard, J., & Thorland, I. (2011). Estimates of genetic varia-
tion in the susceptibility of Atlantic salmon (Salmo salar) to the sal-
mon louse Lepeophtheirus salmonis. Aquaculture, 314, 66–72.
Glover, K. A., Nilsen, F., & Skaala, O. (2004). Individual variation in sea
lice (Lepeophtheirus salmonis) infection on Atlantic salmon (Salmo
salar). Aquaculture, 241, 701–709.
4 |
Gonzalez-Alanis, P. (2000). Frontal filament morphogenesis in the salmon
louse Lepeophtheirus salmonis. Thesis, Department of Anatomy &
Physiology, Faculty of Veterinary Medicine, University of Prince
Edward Island.
Hamre, L. A., Eichner, C., Caipang, C. M. A., Dalvin, S. T., Bron, J. E., Nil-
sen, F., . . . Skern-Mauritzen, R. (2013). The salmon louse Lepeoph-
theirus salmonis (Copepoda: Caligidae) life cycle has only two
chalimus stages. PLoS One, 8, 1–9.
Heuch, P. A., & Karlsen, H. E. (1997). Detection of infrasonic water oscil-
lations by copepodids of Lepeophtheirus salmonis (Copepoda: Cali-
gida). Journal of Plankton Research, 19, 735–747.
Ingvarsdo
ttir, A., Birkett, M. A., Duce, I., Genna, R. L., Mordue, W., Pickett,
J. A., . . . Mordue, A. J. (2002). Semiochemical strategies for sea louse
control: Host location cues. Pest Management Science, 58, 537–545.
Johnson, S. C., Treasurer, J. W., Bravo, S., & Nagasawa, K. (2004). A
review of the impact of parasitic copepods on marine aquaculture.
Zoological Studies, 43, 229–243.
Kolstad, K., Heuch, P. A., Gjerde, B., Gjedrem, T., & Salte, R. (2005).
Genetic variation in resistance of Atlantic salmon (Salmo salar) to the
salmon louse Lepeophtheirus salmonis. Aquaculture, 247, 145–151.
Liu, Y., & Bjelland, H. V. (2014). Estimating costs of sea lice control strat-
egy in Norway. Preventive Veterinary Medicine, 117, 469–477.
DELFOSSE ET AL.
Mordue Luntz, A. J., & Birkett, M. A. (2009). A review of host finding
behaviour in the parasitic sea louse, Lepeophtheirus salmonis (Caligi-
dae: Copepoda). Journal of Fish Diseases, 32, 3–13.
Pike, A. W., & Wadsworth, S. L. (1999). Sealice on salmonids: Their biol-
ogy and control. Advances in Parasitology, 44, 233–337.
Tucker, C. S., Sommerville, C., & Wootten, R. (2000). The effect of tem-
perature and salinity on the settlement and survival of copepodids of
Lepeophtheirus salmonis (Kroyer, 1837) on Atlantic salmon, Salmo salar
L. Journal of Fish Diseases, 23, 309–320.
Tully, O., & Nolan, D. T. (2002). A review of the population biology and
host-parasite interactions of the sea louse Lepeophtheirus salmonis
(Copepoda: Caligidae). Parasitology, 124(Suppl), S165–S182.
How to cite this article: Delfosse C, Lafont-Lecuelle C,

le
Barthe
my H, et al. Study of the hooking behaviour of
Lepeophtheirus salmonis (Krøyer, 1837) copepodids on Atlantic
salmon, Salmo salar L., using a novel in vivo test system.
J Fish Dis. 2017;00:1–4. https://doi.org/10.1111/jfd.12747