Experimental Parasitology 125 (2010) 51–54

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Experimental Parasitology

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The reproductive output of sea lice Caligus rogercresseyi under controlled conditions

Sandra Bravo *

Universidad Austral de Chile, Casilla 1327, Puerto Montt, Chile

a r t i c l e i n f o

Article history:Received 1 April 2009Received in revised form 7 October 2009Accepted 1 December 2009Available online 11 December 2009

Keywords:Sea liceCaligus rogercresseyiLepeophtheirus salmonisLife time fecundityEgg sizeFecundity rate

0014-4894/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.exppara.2009.12.001

* Fax: +56 65 255583.E-mail address: sbravo@uach.cl

a b s t r a c t

Gravid females of Caligus rogercresseyi were collected from Atlantic salmon (Salmo salar) from a farmlocated at Chiloe Island (42�400S73�150W), Chile, to obtain information about the reproductive outputof this parasite in vitro. The egg strings removed from the females were incubated under controlled con-ditions to obtain virgin adult females. One female which had mated only once produced eleven genera-tions of eggs strings in a period of 74 days. The first egg strings of the females obtained in vitro wereproduced at 389 degree days (�D) after egg incubation, while the next generations of eggs strings wereproduced with a periodicity between 4 and 6 days dependent on the water temperature. The averagelength of the egg string was 3.1 mm and the mean number of eggs per string was 31. The values recordedin captivity for the egg string length and the number of eggs per string, were lower than the valuesrecorded in gravid females from the field. One female survived for 79 days and males, maintained sepa-rately from the females, survived for 60 days.

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1. Introduction

Caligus rogercresseyi has been the most important parasite forthe salmon industry in Chile since it was recorded for the first timein 1997 infecting Atlantic salmon (Salmo salar) (Boxshall and Bra-vo, 2000; Bravo, 2003; Johnson et al., 2004). C. rogercresseyi is anon-specific parasite of several wild marine fish, which are fre-quently found in the vicinity of salmon cages attracted by thewaste feed (Carvajal et al., 1998; Bravo, 2003). It has also been re-ported as a parasite of the introduced anadromous brown trout(Salmo trutta) in southern Argentina since 1998 (Bravo et al., 2006).

Despite the impact of C. rogercressyi on the salmon farming inChile, there is still limited information regarding its biology. Thelife cycle of C. rogercresseyi comprises of eight developmentalstages, and there are no preadult stages. The life cycle dependson water temperature, at 10 �C it is about 45 days and it is com-pleted in 26 days at 15 �C (González and Carvajal, 2003). The pre-dominant temperature in Region X of Chile is around 12 �C, andhere the life cycle of the parasite is completed in around 32 daysfrom egg extrusion to mature adult (personal observation).

Adult females of C. rogercresseyi collected from the field attainedan average length of around 5.0 mm, including caudal rami, with-out important differences in size between females and males (Box-shall and Bravo, 2000; Bravo et al., 2009). Although C. rogercresseyiproduces larger females in spring–summer, longer egg strings anda greater number of eggs per string are produced in winter at a

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temperature around 10 �C compared with summer temperaturesaround 15 �C (Bravo et al., 2009). This concurs with reports forLepeophtheirus salmonis, where longer egg strings are produced atlow temperature and there are more eggs per string on averagethan at higher temperatures (Ritchie et al., 1993; Heuch et al.,2000; Boxaspen, 2006).

The number of eggs per string reported for female C. rogercres-seyi is around 45 (Bravo et al., 2009), lower than the number ofeggs per string reported for L. salmonis, which is reported in therange 152–296 eggs per string (Heuch et al., 2000). AlthoughC. rogercresseyi is negatively influenced by low salinity (Bravoet al., 2008), longer egg strings and greater numbers of eggs perstring are produced at sites with salinity around 20‰ comparedwith sites where the salinity is higher (Bravo et al., 2009).

The aim of this study was to produce information about thereproductive output of C. rogercresseyi to provide a better under-standing of the infestation pressure of sea lice in salmon farmingareas and to improve the strategies used for an effective control.

2. Materials and methods

Samples of gravid female C. rogercresseyi were collected in Feb-ruary 2008 from Atlantic salmon (S. salar), in a farm located in theChiloe Island area (42�400S73�150W). Parasites were carefullyremoved from 10 fish anaesthetized with benzoacaine (10% inethanol, 1 ml/L), and transported live in containers with cooledseawater supplied with aeration to the laboratory of the Aquacul-ture Institute (Universidad Austral de Chile) in Puerto Montt(41�46́S72�56́W). Egg strings of these naturally produced lice were

52 Sandra Bravo / Experimental Parasitology 125 (2010) 51–54

carefully removed from 50 females. The total body length recordedfor the gravid females was 4.82 ± 0.28 mm, the length of eggstrings was 3.49 ± 1.11 and the number of eggs per string41 ± 14.7. The mean length of eggs was calculated as describedby Heuch et al. (2000), where sections of 10 eggs per string weremeasured and divided by 10. The number of eggs per string wascalculated by dividing the total egg string length by the length ofa single egg. Measurements were carried out with an ocular micro-metre at 10� magnification for the whole parasite and egg string,and 45� in the case of the eggs.

Egg strings were incubated at 12 �C in containers with filteredsea water in a chamber with a photoperiod of 12 h light and 12 hof darkness until the copepodid stage had developed to around650 lm total length. Two Atlantic salmon per tank, weighing about165 g, were maintained in tanks of 0.5 m3 volume, supplied withseawater at ambient temperature and salinity. The salmon wereinfested with 140 copepodids per fish to produce the adult stage.To avoid the loss of copepodids through the effluent water, thewater level in each tank was lowered, the water inflow stoppedand the water oxygenated before the 140 copepodids per fish wereadded. This situation was keeping for 8 h to avoid the loss of cope-podids through the effluent. Then the water flow was resumed andthe copepodids that had not infected the fish, were trapped in a250 lm mesh installed in the outflow of each tank, counted and re-moved. The temperature (�C) and salinity were recorded daily. Toobtain information about the development stage of the life cycleof the parasite attached to the host, five parasites were collecteddaily from one of the fish kept isolated in one tank. Parasites weremeasured, the stage of development recorded, and then they werestored in 70% ethanol.

Once the parasites reached the mature adult stage, at approxi-mately 389 �D, the developmental stages of males and femaleswere recorded daily until the first pair of egg strings was extruded.Twenty gravid females, with average size 4.5 ± 0.27 mm, were re-moved from the anaesthetized fish and kept alive in an insulatedcontainer with aeration. The males were removed from the fishto avoid new mating and these were transferred to another tankto infest another fish. Once the egg strings were carefully removed

Table 1Developmental stages of the first laboratory generation of Caligus rogercresseyi obtained fr

Date Cumulative days �D Cum

February 05 0 0 0February 14 10 103 103February 18 14 62 165February 23 19 88 253February 25 21 31 284February 27 23 30 315February 29 25 30 345March 03 28 44 389

Table 2Survival of Caligus rogercresseyi females through the brood generations under laboratory c

Brood number Mean temperature between broods (±SD)

12 12.8 ± 0.793 12.8 ± 0.564 13.9 ± 0.665 14.1 ± 0.836 13.7 ± 1.157 12.1 ± 1.308 11.3 ± 0.449 11.5 ± 0.45

10 10.9 ± 0.1611 10.7 ± 0.35

from the female using a fine forceps and dissection needle, thefemales were returned to the same fish. The egg string length,number of eggs per string and the egg size were measured accord-ing to the procedure described above. This procedure was repeatedfor each generation of egg strings until the last brood generationwas produced.

To evaluate the natural survival of both sexes when infestingthe fish, 20 adult females were monitored on one fish and 20 maleson another fish kept isolated in a tank. The same monitoring wasdone with adult parasites kept without host in seawater. The dif-ferences between groups were tested by analysis of variance andthe Multiple Range Test Fisher (LSD).

3. Results

The life cycle of the parasites obtained in captivity was com-pleted in 389 degree days (�D). The immature adult was reachedat 345 �D with little differentiation between sexes (Table 1). Thegravid females produced an average of 35 eggs per string in thefirst brood, with a mean egg string length of 3.42 mm (Table 3).Mature eggs showed a brown colour and approximately 40%reached the copepodid stage at 103 �D, under ambient controlledlaboratory conditions (12 �C), while 30% of the survival copepodidsreached the adult stage. Ambient temperature in the period of thestudy ranged from 10 �C to 18.5 �C, with an average of 13.9 �C,while the salinity ranged between 28‰ and 31‰.

The second egg string pair was extruded at 64 �D after theappearance of the first strings in all females, with an average pro-duction of 29 ± 6.71 eggs per string (Table 2). The third pair of eggsstring was extruded at 51 �D after the second pair and twelve grav-id females survived to this stage (60%). Nine gravid females (45%)survived to the extrusion of the fourth egg string pair and one grav-id female (5%) survived the extrusion of the 7th pair of egg strings.This was alive until the 11th egg string production. The egg stringswere extruded with a periodicity of 4 days between broods fromthe 3rd to 7th brood inclusive (Table 2). The 11th pair of egg strings(the last brood), was extruded at 74 days (954 �D), this period in-cluded the complete development of the parasite from hatching

om gravid females collected from the field.

ulative (�D) Development stage Length (lm)

Egg string incubationCopepodid (infestation) 658Chalimus I 830Chalimus II 1271Chalimus III 2146Chalimus IV 4201Immature adult 4380Mature adult 4880

onditions.

Cumulative (�D) Cumulative days No. of females

389 28 20453 33 20504 37 12560 41 9616 45 8671 49 4720 53 1777 58 1846 64 1901 69 1954 74 1

0

20

40

60

80

100

1 6 9 13 17 21 25 30 32 33Days

Surv

ival

(%)

Females *FemalesMales

Fig. 1. Survival of 20 undisturbed Caligus rogercresseyi on Atlantic salmon (Salmosalar) in tanks. Females (�), correspond to disturbed females from which the eggstrings were removed.

0

20

40

60

80

100

1 2 3 4 5 6 7 8Days

Surv

ival

(%)

FemalesMales

Fig. 2. Survival of 20 detached adult Caligus rogercresseyi in seawater from the host.

Table 3Characteristics of the eggs string generations obtained in Caligus rogercresseyi under laboratory conditions.

Brood number No. of females Mean ± SD egg string length (mm) Range (mm) Mean ± SD eggs/string Range Egg size (width � length) (mm)

1 20 3.42 ± 0.44 2.56–4.25 35 ± 6.12 24–48 0.36 � 0.082 20 2.72 ± 0.56 2.00–3.56 29 ± 6.71 20–37 0.32 � 0.083 12 2.76 ± 0.55 2.00–3.81 28 ± 6.18 22–43 0.31 � 0.084 9 3.16 ± 0.40 2.56–3.94 32 ± 7.34 22–49 0.33 � 0.085 8 3.36 ± 0.25 3.13–3.81 36 ± 2.14 32–38 0.36 � 0.086 4 3.38 ± 0.27 3.13–3.88 32 ± 4.89 27–41 0.34 � 0.087 1 3.03 2.88–3.19 31 31–31 0.36 � 0.088 1 2.69 2.63–2.75 29 28–30 0.33 � 0.089 1 2.13 1.94–2.31 16 13–19 0.33 � 0.10

10 1 2.47 2.31–2.63 22 18–25 0.32 � 0.1011 1 2.08 2.06–2.10 16 14–17 0.31 � 0.11

Average 3.10 31 0.33 � 0.09

Sandra Bravo / Experimental Parasitology 125 (2010) 51–54 53

to maturity (Table 2). The last surviving female remained alive for79 days, four additional days after the extrusion of the 11th pair ofegg strings.

The mean length of the egg strings for the 11 broods obtained un-der laboratory conditions was 3.1 mm and the average number ofeggs per string was 31 (Table 3). There was a reduction in the lengthof the egg strings and in the number of eggs per string after the 8thgeneration in the only surviving female. The average egg size for the11 broods was 0.33 mm (wide) � 0.09 mm (length), with an in-crease of the egg length after the 8th generation (Table 3).

Adult males which were kept isolated from females on salmonin different tanks survived for 61 days on the host, and showed ahigher mortality compared with the females kept under the sameconditions (without handling), which at the same date showed asurvival of 35% (Fig. 1). In contrast, the survival in females whichwere handled during the period of study was lower (5%), comparedto undisturbed females, and this was significantly different(p < 0.005). By other way, males and females, isolated from thehost and keeping free in seawater, survived for 7 days, withoutrecording significant differences between sexes (p < 0.005) (Fig. 2).

4. Discussion

Caligus rogercresseyi has a similar size to Caligus elongatus,where females are 5–6 mm long and males are 4–5 mm long(Kabata, 1992). It also has a similar life cycle, which consists ofeight stages separated by moults, and without preadult stages(Piasecki and MacKinnon, 1995; Piasecki, 1996). The egg strings

in both species also have approximately 80 eggs (Pike et al.,1993). However, even if C. rogercresseyi is more similar to C. elong-atus than L. salmonis with respect to size, biology and reproductiveoutput, more scientific information is available for L. salmonis, andthis permitted comparison with the results obtained in this study.

Although the number of eggs per string produced by C. roger-cresseyi is lower than that reported for L. salmonis (Heuch et al.,2000; Boxaspen, 2006), females of both species produce 11 gener-ations of egg strings with just one copulation. It has been reportedthat females of L. salmonis can mate again and polyandry is com-mon (Todd et al., 2005; Costello, 2006), and this may also be thecase for C. rogercresseyi. Females of C. rogercresseyi survived for79 days and males for 60 days in comparison with the 191 days re-ported for female adult L. salmonis (Heuch et al., 2000).

Under laboratory conditions, undisturbed males had a shorteradult life on the host than undisturbed adult females. However,both sexes survived free in seawater without the host for 7 days.However, the mortality in males was higher than in females inthe first few days (Fig. 2). This explains the major influence of fe-males in the infestation pressure on farmed salmonids. C. rogercres-seyi showed a lower production of eggs in vitro, in comparison withthe number of eggs per string recorded in the field (Bravo et al.,2009). This could be associated with the stress imparted to the fe-males during the laboratory study. In fact, the gravid females werehandled and anaesthetized each time the egg strings were re-moved, a situation which also may explain the higher mortality re-corded through the brood generations, and it contrasts with themortality pattern of the undisturbed females (Fig. 1).

An average of 50 eggs per string has been reported for C. roger-cresseyi collected from the field, with a maximum of 130 eggs perstring in areas with salinity below 25‰ (Bravo et al., 2009),

54 Sandra Bravo / Experimental Parasitology 125 (2010) 51–54

conditions less favourable for this species. However, in areas wherethe salinity is higher and more constant through the year, as is thecase of the site where the parasites were collected, the egg produc-tion can be as low as 9 per string in the summer (Bravo et al.,2009). In this study, the parasites were collected in summer, atan average temperature of 15.5 �C. This is in agreement with thereport by Ritchie et al. (1993) where it is suggested that in L. salmo-nis the alteration in the reproductive strategy is the consequence offluctuations in environmental parameters.

The number of eggs in the different sea lice species varies withtime of year, louse length, louse age, host species and differentpopulations (Costello, 2006). This study showed that C. rogercres-seyi has an egg production about five times less than in L. salmonis.Ritchie et al. (1993), reported rates of 142–246 eggs per string inthe summer period while, in contrast, Heuch et al. (2000) reported55–704 eggs per string. However, an important consideration isthat the female L. salmonis is about three times longer than C.rogercresseyi. Heuch et al. (2000) observed that the first pair ofegg strings in L. salmonis was significantly shorter than the subse-quent brood generations. However, in C. rogercresseyi there was areduction in egg string length from the 8th brood, but since onlyone female survived to this stage, it cannot be concluded that thisis a general pattern for the species. The first generations showed asimilar reproductive output. No important difference in the aver-age egg size was recorded in the egg obtained in vitro (0.33 �0.09 mm) in comparison with the egg sizes of C. rogercresseyicollected from the field (0.34 � 0.08 mm) (Bravo et al., 2009). Inboth situations all eggs showed a similar development stage inthe string.

This study may contribute to improvements in control strate-gies by providing an understanding of the reproductive output ofC. rogercresseyi. The high number of broods produced by one fe-male will most likely influence the re-infestation rate in the sal-mon stocks, mainly when suitable strategies of managementagreements are not well implemented. By other way, if resistantlice are present, female fecundity will also be important for anunderstanding of reinfection dynamics.

Acknowledgments

This research was carried out in the framework of the projectFondef D04I12555 supported by Conicyt. I want to thank ClaudiaLagos for her help with the statistical analyses, to Francisca Erranz,

Pier Bassaletti and Veronica Pozo for their assistance in the field.Special thanks to Dr. Peter Heuch and Dr. James Treasurer for theirvaluable advice to improve this paper.

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