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Differential infectivity of Caligus flexispina

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_{Copepoda, Caligidae in three farmed salmonids in}

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Chile

Laura Gonzalez

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a,)

_{, Juan Carvajal}

a

_{, Mario George-Nascimento}

b

a

Dept. de Recursos Naturales y Medio Ambiente, UniÕersidad de Los Lagos, Casilla 557, Puerto Montt, Chile

b

UniÕersidad Catolica de la Santısima Concepcion, Casilla 297, Concepcion, Chile´ ´ ´ ´

Accepted 4 August 1999

Abstract

The ectoparasitic copepod, Caligus flexispina, is causing increasing problems in farmed salmonids in southern Chile. Field and experimental approaches were used to assess whether any of the three host species, rainbow trout Oncorhynchus mykiss, atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch, is preferentially colonized. Prevalence, abundance, density and developmental stages attained by the parasite in three host species were compared. Results clearly revealed that rainbow trout is the most susceptible species: under field conditions, C.

flexispina is more prevalent and abundant and there is a higher proportion of adult stages. No

ovigerous females were found in coho salmon. In experimental infestations, rainbow trout were more heavily colonized by infective copepodids, and these were more likely to reach the adult stage. A mixture of factors inherent to each host-parasite relationship is considered to play a role in these observations because coho salmon is also colonized by copepodids but a low proportion of the parasites reach the adult stage. However, atlantic salmon is less suitable for colonizing larva.q_{2000 Elsevier Science B.V. All rights reserved.}

Keywords: Sea lice; Caligus flexispina; Farmed salmonids; Host susceptibility; Copepodid settlement

1. Introduction

Chilean sea lice, Caligus flexispina, ectoparasitic copepods belonging to the Caligi-dae family, have a life cycle consisting of the following stages: two nauplius, one

Ž

infectious copepodid, four attached chalimus stages, one pre-adult and the adult

unpub-)_{Corresponding author. Tel.:}_q_{56-65-255744; fax:}_q_{56-65-254788; e-mail: [email protected]}

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lished data . With the exception of the naupliar stages, sea lice feed on host mucus, skin and blood. Sea lice have been reported to reduced productivity and cause disease

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outbreaks in a variety of farmed fish species Pike, 1989; Berland, 1993; Grimnes and

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Jakobsen, 1996 .

Large scale aquaculture of introduced salmonids began in the early 1980’s in southern Chile. The first report of sea lice on farmed salmonids in Chile was Caligus

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teres found on coho salmon Reyes, 1983; Bravo, 1987 . Then Gonzalez and Carvajal

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Ž1994 and Gonzalez et al..

´

Ž1997 reported for the first time the presence of C..

flexispina on rainbow trout from sea pens. The latter was soon recognized to be a major

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problem on rainbow trout and atlantic salmon on sea sites Carvajal et al., 1998 .

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However, infections with this sea louse have not been reported as a problem on farms

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raising coho salmon Associacion Chilena de Productores de Salmon y Trucha, pers.

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.

com. .

C. teres and C. flexispina have low host specificity and are found on several species

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of wild hosts commonly present around salmon farms Carvajal et al., 1998 . In addition to both species, seven other species of Caligus have been reported from Chilean wild

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fish Fernandez and Villalba, 1986 . Seven of these species have been found in coastal

´

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waters and others in offshore areas. Nevertheless, Gonzalez and Carvajal 1994 first

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reported the presence of C. flexispina on farmed salmonids in 1992, even though the

species had been previously reported in offshore areas such as the Juan Fernandez

´

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archipelago and Easter Island, as parasites of non-salmonid fish Carvajal et al., 1998 . The aim of this study was to determine whether there are differences in the

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susceptibility of rainbow trout Oncorhynchus mykiss , atlantic salmon Salmo salar

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and coho salmon O. kisutch to C. flexispina infection under both field and controlled laboratory conditions. The development rate of the parasite in the three hosts was assessed to determine host preference.

2. Materials and methods

2.1. Farm obserÕations

Ž X Y X Y

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Sampling trips to a site located at Quinchao island 42831 00 S, 73827 40 W Chiloe

´

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archipelago, Chile Fig. 1 , were carried out in April, July, September and November 1996 and January 1997 to determine if under field conditions there were differences in

Table 1

Summary of parameters for the experimental infestation in tanks

Trial Tank Host species Number of fish Weight

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number number examined g

1 1 Rainbow trout 3 300

Coho salmon 14 80

Atlantic salmon 16 74

2 Rainbow trout 12 150

Coho salmon 2 138

Coho salmon 4 138

Coho salmon 17 34

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infection levels between the three host species. Rainbow trout, atlantic and coho salmon are farmed at this site. Each time 7 to 14 specimens of each host species were collected using a hand net, anesthetized with BZ20 and placed in individual plastic bags. Samples were kept cool during transport to the laboratory, in Puerto Montt where parasite counts were taken.

Caligids were collected from fish skins using a dissecting microscope and stored in 70% alcohol prior to microscope determination of ontogenic and reproductive stages. Taxonomic identification was done according to the descriptions and keys of Wilson

Ž1905 , Lewis 1964 and Fernandez and Villalba 1986 .. Ž .

´

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2.2. Experimental studies

Ovigerous C. flexispina were obtained from farmed rainbow trout. Eggs were hatched and reared at the laboratory, to the infectious copepodid stage, in 1 l bottles with

Table 2

Infection of the three salmonids species farmed at the same site in southern Chile with adults, pre-adults and juvenile C. flexispina. Samples were collected in autumn, winter and spring 1996 and summer 1997. Std: standard deviation

Host species Number of Prevalence Mean

Žnumber examined. copepods Ž .% Abundance"

S.D.

4r96

Ž .

Rainbow trout 10 94 100 9.4"2.1

Ž .

Atlantic salmon 9 16 67 1.8"0.5

Ž .

Coho salmon 15 30 47 2.0"0.8

7r96

Ž .

Rainbow trout 10 104 100 10.4"1.8

Ž .

Coho salmon 10 2 20 0.2"0.1

9r96

Ž .

Rainbow trout 10 28 80 2.8"0.7

Ž .

Coho salmon 10 1 10 0.1"0.1

11r96

Ž .

Atlantic salmon 10 0 0 0

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Coho salmon 10 2 20 0.2"0.1

1r97

Ž .

Coho salmon 10 0 0 0

All data

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Rainbow trout 50 254 82 5.1"4.5

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1mm filtered seawater and gently aerated. The seawater at ambient temperature was changed daily.

Four laboratory trials were conducted to determine differences in susceptibility to infection amongst the three salmon species. The number and species of fish used in each trial varied depending on the freshwater hatchery-reared salmonids available at the time. Smoltation of all fish used in these trials was done at the laboratory to ensure no previous exposure to sea lice and the fish were previously kept for at least a week in 500-l tanks with sand-filters and circulating seawater. Infestation took place in shaded and aerated tanks with no water flow and reduced volume of ca. 200 l. The exposure time was from 1 to 3 h after which, seawater flow and rearing volume of 500 l were

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restored. The water salinity was 29–31 ppt and the mean temperatures, 13.68C range:

. Ž . Ž . Ž

11.3–14.7 , 11.18C 11.9–18.5 , 14.28C 11.9–18.5 and 16.38C range: 13.9–19.3,

.

total: 2458C-days for the first, second, third and fourth trial, respectively.

Table 1 summarizes experimental procedures of the four trials. In the first trial, rainbow trout and coho salmon were placed in a tank, and fish were examined for copepods 14 days after copepodid exposure. In trial 2, the fish were placed in two replicate tanks where rainbow trout and atlantic salmon were challenged with copepo-dids and examined for sea lice 14 days post-infection. In trial 3, rainbow trout, atlantic and coho salmon were tested in the two replicate tanks. Each tank received three inoculations of copepodids at 1 to 6 days intervals and fish were examined for copepods at 22 days post-infection. In trial 4, the 3 salmonid species were also tested in two replicate tanks. Each tank was infected with 1540 copepodids and five fish of each species were sampled from each tank on the second day post-infection to determine initial levels of infection. The remaining 74 fish were sampled at 9 days post-infection to determine the number of the copepods and their developmental stages.

The terms prevalence, abundance and density were calculated according to Margolis

Ž . Ž .

et al. 1982 and Bush et al. 1997 definitions. Copepod density is defined as the

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Table 3

Infection of rainbow trout, coho salmon and Atlantic salmon with C. flexispina under laboratory conditions.

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Fish were maintained at 158C–208C and ambient salinity 29–31 ppt

Host species Number of Prevalence Mean

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copepods % abundance

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found "S.D.

Trial one: sampled at 14 d.p.i.

Rainbow trout 58 100 19.2"1.5

Coho salmon 1 8 1.0"0

Trial two: sampled at 14 d.p.i.

Atlantic salmon 283 100 7.2"0.7

Trial three: sampled at 22 d.p.i.

Coho salmon 2 17 0.2"0.3

Trial four: Sampled at 2 d.p.i.

Coho salmon 125 100 11.1"2.2

Sampled at 9 d.p.i.

Coho salmon 142 100 5.0"0.7

number of copepods per 100 g host weight in order to control differences in the size of the various host species. Statistical analyses were carried out on intensity and percentage

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of larval stage data. Kruskal–Wallis and Mann–Whitney U-tests were used to compare the mean number of lice within and amongst the three salmonid species as well as the differences in the developmental stages of C. flexispina between species.

3. Results

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C. flexispina was found to be significantly more prevalent and abundant p-0.001

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on rainbow trout in farmed pens, than on atlantic and coho salmon Table 2, Fig. 2 which show no differences when raised at the same site. The majority of copepods present on rainbow trout were adults, whereas most copepods on coho and atlantic

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Fig. 4. Percentage of chalimus ch , pre-adults and adults pa-a of C. flexispina in netpens in the south of

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salmon were earlier developmental stages Fig. 4A . No ovigerous females were found on coho salmon. Prevalence and density of C. flexispina on rainbow trout was higher than on either atlantic or coho salmon during all cohabitation trials and sampling dates,

Žp-0.01 . With the exception of trial 4, atlantic salmon appears to be the next most.

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susceptible to infection and coho salmon the most resistant Table 3, Fig. 3 . At the end of trial 1 most of the copepods present on rainbow trout were chalimus IV and pre-adults. The one copepod found on coho salmon was an ovigerous female. A coho salmon sampled at 6 days post-infection was infected with six chalimus larvae.

In trial 4, at the 2nd day post-infection, 40.5% copepodids and chalimus I collected in

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all the samples were attached to rainbow trout mean abundances9.9"2.7 , 42.2%

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were on coho salmon mean abundances4.4"1.1 and only 17.2% were on atlantic

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salmon mean abundances11.1"2.2 . At the time, atlantic salmon showed a

signifi-Ž .

cantly lower number of copepodids than the other two salmonid species p-0.001 . At

9 days post-infection, the highest density of copepods collected was on rainbow trout

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followed by coho salmon and then atlantic salmon Table 3, Fig. 3 . Nine days

post-infection a high proportion of the copepods had reached the pre-adult and adult stages with significantly more copepods attaining these stages on rainbow trout than on

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either atlantic or coho salmon p-0.001 . At the time, 26% of copepods collected from

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rainbow trout were pre-adults or adults Fig. 4B . On atlantic and coho salmon 10% and 6%, respectively were pre-adults or adults. No adults were collected from coho salmon.

4. Discussion

Factors, such as host-swimming speed and depth distribution, are thought to be important in determining differences in susceptibility of wild pacific salmon species to

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salmon louse, Lepeophtheirus salmonis, infections Nagasawa, 1987; Nagasawa et al.,

.

1993; Nagasawa and Takami, 1993 . In our laboratory studies, the fish cohabited and were confined in relatively shallow, well-mixed tanks thereby reducing the effect of fish behavior on infection. Therefore, it is likely that other factors may be important in

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determining susceptibility to infection. Johnson and Albright 1992a reported that atlantic salmon was more susceptible to salmon louse, L. salmonis than coho salmon. However rainbow trout was not included in their study. The difference in susceptibility between these two species was explained by differences in the magnitude of tissue response to L. salmonis, with coho salmon showing the greatest response. Suppression of tissue response in coho salmon with cortisol implantation increased their

susceptibil-Ž .

ity to infection Johnson and Albright, 1992b .

Assessment of the initial settlement of C. flexispina copepodids in the fourth trial, could lead to the idea that the larger abundance of this parasite on rainbow trout compared to atlantic salmon could be due to the selective settlement of copepodids.

Ž . Ž

Dawson et al. 1997 also observed differences in the intensity data on sea trout Salmo

.

trutta L. and atlantic salmon experimentally infested with L. salmonis. These authors,

however, thought that the differences were due to a lower survival of chalimus on atlantic salmon rather than to differences in the attachment of copepodids, which they

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salmonids with L. salmonis, observed greater larval settlement on atlantic salmon than on coho salmon one DPI, but after three DPI there were no differences, implying that

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resistant factors began to act. Boxshall 1976 and later De Meeus et al.

¨

1995 ,

demonstrated for some species of Lepeophtheirus that the copepodids hatched from eggs of females living on a particular host species preferred to settle on the same host species. It is possible that such a host preference may have an effect on the number of

C. flexispina recorded on rainbow trout. The importance of the differential settlement of

copepodids of this parasite on the different salmonid hosts needs to be assessed. Another factor that has not been evaluated and could be affecting the sea-lice load on salmonid hosts, is the transfer of mobile stages of the parasite from a more susceptible species such as rainbow trout to a more resistant type like the coho salmon. This was observed in the first trial in which the coho had no copepods except for an ovigerous female that undoubtedly came from the farmed rainbow trout in the same tank. This factor would apparently not be so important under netpen-farming conditions where all three species are reared together because the stages collected on coho were mainly chalimus.

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MacKinnon 1998 in a review of important host factors, indicated that there are numerous interactive parameters that influence fish susceptibility to sea lice infections. Although genetically determined resistance is important, host stress level and inmuno-competency, as well as nutrition should also be taken into account. These last factors could explain the variable results in atlantic and coho salmon in the present study.

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Nevertheless, given the different conditions presented in the present study farm survey

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and experimental infections and previous work with natural infestation of C. flexispina

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on rainbow trout and coho salmon reared in tanks Gonzalez et al., 1997 , rainbow trout

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is always the most susceptible species to C. flexispina. The higher susceptibility of recently smolted rainbow trout to the parasite in the present experimental infections was also demonstrated on rainbow trout kept for several months in seapens and non-induced infections of salmonids reared in salt water tanks.

The C. flexispina life cycle duration does not only depend on temperature but also on the host species. C. flexispina in trial 4 developed twice as fast on rainbow trout than on atlantic and coho salmon. Host species has been previously demonstrated to affect the

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development rate of parasitic copepods. Johnson 1993 reported that the L. salmonis development time was less on atlantic salmon than on chinook. Although the mechanism behind this difference in development remains to be determined, the author suggested that these might be due to differences in nutritional status or defense mechanisms. Further studies are needed to determine if the development rate of C. flexispina differs on immunosuppressed coho salmon affected by Piscirickettsiosis or other diseases. The normally low production of adult stages of the parasite in this more resistant host could be altered under this condition.

The fish immune response to parasites can be affected by a variety of factors such as stress, pollutants, hormone levels, season, diet, secondary infections and temperature. These factors that determine the susceptibility of the host to the infection should be taken into account together with the source and number of infective stages of sea lice when implementing management strategies to reduce sea-lice infection rates on farmed

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Further studies on the mechanisms behind these differences in susceptibility, on the population biology of this species, and on its distribution on wild hosts will further improve our ability to develop new management and control strategies for this economi-cally important parasite.

Acknowledgements

We wish to thank Aissa Soto, Patricia Aros and Edgardo Martel for their technical assistance and Enrique Madrid from Marine Harvest Mc Connell for his assistance in matters pertaining to fish health. We are grateful to S.C. Johnson for his comments on this manuscript and to the aquaculture companies Multiexport and Proyecto Smolt, for allowing us to collect copepod parasites in their sea sites. This research was funded by

FONDECYT Grant 1961151, IFS Ar2076-2 and FDI-CORFO SRS-CALIGUS.

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