Contributions to Zoology, 69 (1/2) 57-63 (2000)

SPB Academic Publishing bv, Amsterdam

The -parasite relationship between the rainbow (Oncorhynchus mykiss) and the ectoparasite Argulus foliaceus (Crustacea: ):

cell cortisol and factors which influence epithelial mucous response, may parasite establishment.

D.T. A.L. van der Salm and S.E. Nolan, Wendelaar+Bonga

Department ofAnimal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld 1,

6525 ED Nijmegen, The Netherlands

Key words: ectoparasites, Argulus foliaceus. host-parasite interaction, Oncorhynchus mykiss, skin epider-

mis, cortisol, mucous cells, transmission electron microscopy.

Abstract Introduction

The effects of short-term infectionwith the branchurian The Caligidae and teleost crustacean

the fish hormone cortisol ectoparasite Argulus foliaceus, and stress of ectoparasites are serious parasites fish under (which is reported to stimulate mucus discharge), were studied natural, culture and managed situations (Heckmann, on the mucous cell population ofthe head skin ofrainbow trout 1993). Although much research has been carried (Oncorhynchus mykiss). Argulus infection did not raise plasma of cortisol out on species the marine (Boxshall significantly and hadno effect on the number ofepidermal Caligidae mucous cells in the head skin. twice & little Cortisol was administered Dcfaye, 1993), comparatively attention has to oftrout via the food, groups significantly elevating circulating focused on species of the Argulidae. Nevertheless, plasma cortisol at 24 h post feeding without affecting numbers Argulus species have been implicated in the col- of mucous cells, and increasing the numbers of vesicles in the lapse of a rainbowtrout stock (Mcnczcs et ah, 1990) upper cells ofthe epidermis. Subsequentinfection with the parasite and in (6 are problematic carp farming operations Argulusf/fish) did not affect either plasma cortisol or total

numbers et These ofmucous cells at 48 h post-infection with the parasite, (Singhal ah, 1990). ectoparasites are not

but led to fish in a significantly lower parasite infestation per and can live on a of host host-specific variety spe- the cortisol-administered groups. A 24 h culture system was cies (Lamarre & Cochran, 1992) and have, there- used to expose pieces of trout skin to 50 ng/ml cortisol in vitro to to fore, good potential spread. investigate whether cortisol alone would stimulate reductions

in The skin of the host fish, the mucous addition epithelium target cell numbers. These were unaffected by the of for attachment and feed- cortisol. The in vivo and in vitro results are discussed in relation crustacean ectoparasite

to the of current understanding crustacean host-parasite ing, is a complex epithelium comprised of several interactions. layers of cells which are continuous over the body

surface (Whitear, 1986). The skin epithelium (in-

Contents cluding that of the ) forms the first vital barrier

between the external and the internal environment

Introduction 57 and is protected with a chemically and function-

Materials and methods ' 58 ally complex mucous coat which is discharged by In vivo Argulus infection experiment 58 specialized mucous cells in the epidermis (Shephard, In vitro head skin incubations with cortisol 58 1994). Mucus discharge has been evoked by corti- Sample processing 59 sol administration in vivo (Iger et al., 1995). The Statistical analysis 59 Results 59 correct functioning of these mucous cells is essen-

In vivo Argulus infection experiment 59 tial for maintaining the protective mucus layer on In vitro head skin incubations with cortisol 61 the body surface of the fish. The epidermal struc- Discussion 61 ture changes in fish in response to a wide variety Acknowledgements 62 of stressors, crustacean References 62 including ectoparasites Nolan et al. - cell to in 58 D. T. Epithelial mucous response Argulus

(Johnson & Albright, 1992a, MacKinnon, 1993, Materials and methods

Nolan et al., 1998b).

In A key factor which can affect the health status vivo Argulus infection experiment offish and increase susceptibility to disease is stress of rainbow (Wedemeyer, 1997). The topic of stress in fish is Three treatment groups 70 g trout were each in in 65 1 black tanks complex, but levels of the primary stress hormone set up duplicate plastic

30 fish as described Ruane et al. cortisol are elevated in the of stressed fish containing by

After 2 weeks acclimation, two were and the effects of toxic and non-toxic stressors on (1999). groups fish lead fed food (100 food) on can be severe and to immunosuppression, cortisol-containing mg/kg 2 alternate days 72 and 24 h pre-sampling. The reduced growth and reproduction (for review, see etha- third group received food sprayed with the Wendelaar Bonga, 1997). Links between stress and 24 h nol vehicle The fish were sampled at crustacean ectoparasite infestation have been made only. after the second cortisol-containing meal. Two (Noga et al., 1991, Johnson et al., 1996) and re- then treatments(cortisol fed and normal diet) were cently reviewed (MacKinnon, 1998). Johnson & infected with numbers of sub-adult and adult A. Albright, (1992b) have shown that cortisol admin- foliaceus (6 parasites/fish) and sampled at 48 h istration influenced the host-parasite interaction in infection. coho (Oncorhynchus kisutch) and increased post Ten fish from each treatment (comprised of 5 susceptibility to infection with Lepeophtheirus

fish from each were 24 h salmonis and duplicate tank) sampled by suppressing inflammatory response after feeding the second cortisol-containing diet. epithelial hyperplasia in the skin. Stress-related in 2- Fish were irreversibly anaesthetized changes have also been reported in the skin of phenoxyethanol (1:1000; Sigma) and blood was cortisol-fed rainbow trout, including stimulation of the withdrawn needle from caudal blood ves- of the by mucous cell discharge in the epidermal cells sels and vortexed in eppendorf tubes with head region (Iger et al., 1995). Infection of Atlan- Na,EDTA/apoprotinin (1.5 mg/3000 KIU per ml tic salmon with numbersof the sea L. salmonis for blood; Sigma) on ice cortisol analysis. Samples strongly reduced mucous cell numbers in the skin of skin from the head were fixed in Bouin’s fixa- at 24h post-infection (Nolan et al., 1998b). tive for light microscopy (all groups) and 3% glu- In the present study, mucous cell numbers from taraldehyde buffered in Na-cacodylate (0.09 M, pH the head region ofrainbow trout have been studied 7.3) with post-fixation in 1% osmium tetroxide in infestation in response to crustacean ectoparasite the same buffer for electron microscopy (control and the role of the stress hormone cortisol has been and cortisol fed fish at 24 h post cortisol feeding examined in vivo and in vitro. Immature rainbow only), as described by Nolan et al. (1998b). The trout, including one treatment administered corti- body of each fish was thoroughly examined and sol via the food, were infected with Argulusfoliaceus the numbers of parasites on body surface were (6 parasites/fish), and the total numbers of mucous recorded for each fish. cells in the head skin were quantified at 24 h post

cortisol feeding and 48 h post parasite infection by light microscopy. The effects of the cortisol ad- In vitro head skin incubations with cortisol ministration were assessed by measuring plasma

cortisol and examination of the skin epidermis by Explants ofhead skin measuring approximately 20 electron fish microscopy. Numbers of parasites per x 20 mm were sampled from 8 irreversibly anaes-

were also counted for all treatments. To determine thetized rainbow trout, washed three times in PBS

whether mucous cell numbers were influenced by for 5 min each wash before being bisected. One

of skin from the trout head were cortisol, biopsies in ml piece from each fish was then placed 5 of a also incubated for 24 h in vitro in the of presence complete fish skin culture medium (Mothersill ct

cortisol, and the total number ofmucous cells quan- al., 1995) and the second in 5 ml of the same cul- tified. ture medium with the further addition of 50 ng/ml

cortisol (Hydrocortisone; Sigma). After 24 h in- Contributions to Zoology, 69 (1/2) - 2000 59

Table cells/300 in head skin, and infestation levels I. Plasma cortisol (ng/ml), total number ofepidermal mucous cells (no. mm) the of the rainbow trout (Oncorhynchus mykiss) 24 h after cortisol feeding and 48 h after subsequent infection with Argulus foliaceus (6 parasites/fish).

Treatment Plasma cortisol Epidermal mucous Number Argulus/

cells gram fish

24h post-treatment

1. control (ethanol-sprayed) 2.3±0.7 18± 1.5 0

2. + cortisol 27.6±7.6‘" 19±1.9 0

3. - (ethanol-sprayed) 6.5±2.4 14±4.5 0

48h post-treatment

1. control 6.1 ±1.5 16±2.5 0

2. + cortisol

+ Argulus 9.5±2.9 17±2.3 0.016±0.044‘

3. + Argulus 12.2±3.2 17±2.0 0.050±0.011*

’= '= significantly different from control at PO.OOI; significantly different from each other at P<0.05

the vitro and the count data from cubation at room temperature, skin samples were experiment parasite the differences fixed for 24 h in Bouin’s solution and processed Argulus experiment, between groups

for tested the all light microscopy. were using Mann-Whitney U test. In

cases, statistical significance was accepted at

P<0.05. Sample processing

For quantification of mucous cells, sections from Results each fish were stained with periodic acid-Schiffs

stain to identify total mucous cells, and quantified In vivo Argulus infection experiment as described by Nolanet al. (1998b). For transmis- sion electron microscopy (TEM), fixed skin tissues Plasma cortisol was significantly higher in the were stained in acetate, ethanol-dehydrated uranyl cortisol-fed h groups sampled at 24 post cortisol- and embedded in Spurr’s resin. Sections cut per- feeding compared with controls (Table 1). The

pendicularly to the skin surface were further stained cortisol levels were similar in all plasma groups with lead citrate and examined in a Joel 100 CXI1 sampled at 48 h post-infection with Argulus (Table transmission electron microscope at 40 kV, (Nolan I). The number of parasites attached to the skin of et al., 1998b). Plasma cortisol was measured by fish which had been cortisol-fed was significantly RlA validated for measurements in fish plasma lower than fish which had not been administered (Balm & Pottinger, 1995). cortisol (Table 1). Total numbers of mucous cells

both time Exami- were similar at points (Table 1).

Statistical the skin transmission analysis nation of by electron micros- revealed numbers of vesicles in copy large the

F>ata and filament cells of the arc expressed as means ± S.E.M. for n=10 pavement upper epider- {in vivo Argulus experiment) and n=8 (in vitro skin mis in the cortisol-fed fish at 24 h compared with fish ■ncubate experiment). For the in vivo experiment, control (Fig. 1A & IB).

as there were no differences between replicate tanks,

Pooled In head skin incubations with datawere analyzed by ANOVA at each time vitro cortisol

Point and significance between treatments com-

pared with Total numbers of mucous cells simi- controls was assessed using the epidermal were Bonferroni Multiple Comparisons test. For the in lar in cultured skin explants incubated in full cul- 60 D. T. - in rainbow Nolan et al. Epithelial mucous cell response to Argulus trout

Fig. I. Upper epidermis of the head skin of rainbow trout (Oncorhynchus mykiss). (A) Upperepidermis of control trout skin. The

cell upper layer is comprised ofdifferentiatedpavement cells which have microridges apically (arrows). These cells, and the underlying filament cells (F), contain low amounts of vesicles. Mature mucous cells (M) are close to the surface, where they discharge their contents. (B) Upper epidermis ofcortisol-fed trout skin, 24h post second cortisol feed (see text for details). The pavement (P) and filament (F) cells contain large amounts of vesicles. There is much endoplasmic reticulum visible in each cell (arrows) as more vesicles are synthesized. Both micrographs x5500. Contributions to Zoology, 69 (1/2) - 2000 61

ture with medium and without an additional50 sites which ng/ present on trout groups had previously ml cortisol culture cul- (full medium 30±8.9; full been fed with cortisol suggests that the composi- ture medium 50 cortisol 52±10.8: tion of the plus ng/ml (mean mucous layer at the body surface may

± number of cells 600 SEM) mucous per mm epi- have resulted in a less favourable environment and

= P dermis, n 8, > 0.05. lower establishment rate of the parasite.

Infection of rainbow trout with 6 A. foliaceus

fish did per not significantly elevate plasma corti- Discussion sol levels at 48 h post-infection. Ectoparasites have

co-evolved with their host and species may not evoke

This examines the short-term interaction study a cortisol response in the low numbers used in this between the crustacean A. Increased cortisol levels have ectoparasite, foliaceus experiment. may and the host fish the level of the skin at epithelium. occurred within the 48 h immediately post-infec-

This the role is the first attempt to investigate of a tion and before sampling. Elevated plasma cortisol

host endocrine factor in the to response Argulus levels in Argulus- infested rainbow trout have been infection by combining in vivo and in vitro experi- reported 48 h after a 4 h confinement stress (Ruane mentation. Epithelial responses of fish to infection et ah, 1998) indicating that effect of ectoparasites with caligid crustacean ectoparasites have been may not be seen immediately after infection, but reported in salmonids in & when seawater (Johnson become apparent the response to a second

Albright, and result in stressor is It that 1992a, MacKinnon, 1993) investigated. is also possible a increased mucus discharge and reduced numbers higher clearance rate of cortisol from the blood by of mucous cells in the epidermis of the head skin target tissues (such as the skin) during infection

within 24 h In the andattachment (Nolan et al., 1998b). present of the parasite may be the explana-

tion study using Argulus, total numbers ofmucous cells for the absence of a significant rise in cortisol were in unaffected by the parasite or by cortisol feed- our experiment. mg infection. This be related Cortisol increased cortisol prior to parasite may feeding plasma lev- to the did the different osmotic environments, as the present els but not affect epidermal mucous cell

was carried in freshwater. cells the cell study out Mucous population or mucous response to Argulus

were also quantitatively unaffected after freshwa- infection. Electron microscopy indicated stimula-

ter ofSalmo tion of vesicle in the exposure trutta trutta to polluted water synthesis upper epidermal cell

lr°m the lower river Rhine (Nolan et ah, 1998a). populations. This response has been shown to be The mucous cell populations studied by electron evoked by cortisol administration (Iger et ah, 1995)

microscopy are affected by stressors, including and must confer some advantage on the host dur-

crustacean The the lower ectoparasites (Nolan et ah, 1998b). ing stress, perhaps contributing to para-

dfects of include increased rate mucus discharge, site numbers on cortisol-fed fish. Alterations in

increased of differentiation of new mucous cells and vesicle composition the epidermal cells have also

of immature cells for Salmo salar discharge mucous (indicating been reported following infection mcreased turnover rate in the mucous cell popula- with L. salmonis (Nolan et ah, 1998b).

tlon), as well incubation of the skin as changes in the composition of In vitro head for 24 h,

mucosomes. Similar results havq been reported with and without additionalcortisol, did not stimu-

cortisol reduction in the following administration (Iger et ah, 1995). late a total numbers of mucous

The influence of cells. This culture medium cortisol therefore, may not be supports the growth and

manifest in the absolute differentiation of fish skin in the numbers of mucous cells explants longer P'csent in the epidermis, but in the composition of term (Mothersill ct ah, 1995). Perhaps 50 ng/ml die mucus or the it the stimulation of increased mucus cortisol was too low, as reflected circulating

discharge and in the differentiation of new mucous cells, .cortisol levels fish post-cortisol feeding, but

cell maximum mcreasing turnover within the mucous cell the expected peaks immediately post- Population as a whole, while overall absolute num- feeding following Barton et al. (1987) would be

ers mmain constant. around 100 To address this The lower number of para- ng/ml. issue, a dose - 62 D. T. Nolan et al. Epithelial mucous cell response to Argulus in rainbow trout

24 skin Y, Balm PHM, Jenner IIA, Wendelaar SE. response study to cortisol is required for h Iger Bonga 1995. Cortisol induces stress-related changes in the skin explants incubated under these conditions. Ideally, ofrainbow Gen. trout (■Oncorhynchus mykiss). Comp. Endo- following from the discussion above in relation to crinol. 97: 188-198.

turnover within a mucous cell mea- population, Johnson SC, Albright LJ. 1992a. Comparative susceptibil-

cell in and of the of naive suring proliferation parallel may greatly help ity histopathology response Atlantic,

to resolve the data obtained. chinook and coho salmon to experimental infection with

Lepeophtheirus salmonis (Copepoda: Caligidae). Dis. Aquat. In conclusion, infection of rainbow trout with 6 Org. 14: 179-193. A. foliaceus per fish did not induce elevated corti- Johnson SC, Albright LJ. 1992b. Effects of cortisol im- levels in the 48 h sol blood at post-infection nor the and the of plants on susceptibility histopathology the

reduce the total numbers of cells in the of naive coho'salmon kisutch mucous responses Oncorhynchus to

epidermis of the head skin. Prior cortisol adminis- experimental infectionwith Lepeophtheirussalmonis (Cope- poda: Caligidae). Dis. Aquat. Org. 14: 195-205, tration to the fish elevated blood cortisol levels, Johnson SC, Blaylock KB, Elphick .1, Hyatt KD. 1996. stimulated vesicle in the synthesis upper epider- Disease induced by the sea louse (Lepeophtheirussalmonis) mal cells, and resulted in reduced numbers of the (Copepoda: Caligidae) in wild sockeye salmon (Oncorhyn- 48 h without parasite at post-infection influencing chus nerka) stocks of Alberni Inlet, British Colombia.

Fish. Sci. 53: 2888-2897. the outcome of parasite challenge with respect to Can. J. Aquat. Lamarre E. Cochran PA. 1992. Lack of host species selec- numbers of mucous cells. In vitro 24 h incubation tion by the exotic parasitic crustacean, Argulusjaponicus. of pieces ofhead skin with physiologically relevant J. Freshwater Ecol. 7: 77-80.

cortisol levels resulted in similar num- (50 ng/ml) MacKinnon BM. 1993. Host of Atlantic salmon response

bers of mucous cells with controls. We (Salmo to infection lice compared salar) by sea ((Caligus elongatus).

Can. J. Fish. Sci. 50: 789-792. conclude that this low level of infection with Argulus Aquat.

MacKinnon BM. 1998. Host factors important in sea lice does not increase plasma cortisol levels nor stimu- infections. ICES J. Mar. Sci. 55: 188-192. of late mucus discharge such that numbers mucous Mcnczcs J, Ramos MA, Pereira TG, cla Silva AM. 1990.

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