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Observations on mating and reproductive behaviour of Lepeophtheirus salmonis, Krøyer (Copepoda: Caligidae)
Journal
of Experimental
ELSEVIER
Marine Biology
JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY
and Ecology,
201 (1996) 285-298
Observations on mating and reproductive behaviour of Lepeophtheirus salmonis, Kr@yer (Copepoda: Caligidae) G. Ritchie”,*,
A.J. Mordue (Luntz)b, A.W. Pikeb, G.H. Raec
“Nutreco Aquaculture Research Centre, Forusbeen 35, PO Box 353, N-4033 , Forus, Norway ‘Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, UK ‘Scottish Salmon Growers Association Ltd, Drummond House, Scott street, Perth PHI SEJ, UK Received
28 November
1995; revised 9 January
1996; accepted
10 January
1996
Abstract Mating in Lepeophtheirus salmonis was similar to that reported for L. pectoralis in both the procedure and positions attained by both sexes but the timescales were different. Adult males took up precopulatory positions with, predominantly, pre-adult II females. Males held their respective partners around the 4th pedigerous segment using the second antennae. Females approaching the final moult produced temporary frontal filaments that served to anchor the pair as ecdysis proceeded. Males remained attached to the cephalothorax of the female during her final ecdysis before reattaching around the genital complex. After transferring to the ventral surface of the female, copulation occurred between the adult male and recently moulted adult female. The maxillipeds were used to manipulate and raise the genital complex of the female and a pair of spermatophores was transferred by the swimming legs. Following copulation the male returned to the dorsal surface of the female and held the spermatophores on the female for some time, before leaving her. Several specific behavioral patterns were observed during precopula, at the final moult of the female and during copulation. These involved considerable movement of the genital complexes and precise movements of the appendages by the male. Mate searching and testing behaviours were performed by adult males in the presence of pre-adult and adult females. These behaviours provided evidence that mate recognition by short range chemical, or contact stimuli may have occurred in L. salmonis. In addition, males clustered around moulting females which showed the attractiveness of the female at this stage. Postcopula, allometric growth of the female genital complex was observed and quantified from the time of insemination to the production of the first batch of eggs. Keywords:
Behaviour; Caligidae; Lepeophtheirus
salmonis;
Mating; Mate guarding; Pheromones;
Reproduction
*Corresponding
author.
0022-0981/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOO22-098 1(96)000081
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1. Introduction copepods Lepeophtheirus salmonis (Krayer 1837) and Caligus 1832), collectively referred to as sea lice, are a serious problem in commercial Atlantic salmon (Salmo salar L.) farming in Norway, Scotland, Ireland and Canada. Of the two species Lepeophtheirus salmonis is the more problematic pathogen, but high infection levels of both species can result in severe damage and huge economic losses. L. salmonis has a lo-stage lifecycle comprising two free swimming nauplius stages, one free swimming infective copepodid stage, four attached chalimus stages, two pre-adult stages and the adult (Johnson and Albright, 1991a; Schram, 1993). All three post-chalimus stages can move freely over the surface of the fish (Bruno and Stone, 1990; personal observation) and are only immobile during moulting when a temporary frontal filament is produced (personal observation). Until recently there was a dearth of information regarding the mating processes and related behaviour patterns of caligid copepods. Scott (1901) observed several species of Lepeophtheirus and concluded that fertilization of the female was accomplished after the final chalimus stage. It is important to note that Scott (1901) failed to recognise the presence of pre-adult stages. Several authors assumed that copulation proceeded, with the male attached dorsally on the female, folding its genital complex along the ventral surface of the female and discharging a pair of spermatophores. This procedure was described for L. dissimulutus (Lewis, 1963), Culigus spinosus (Izawa, 1969) and several other caligid species (Wilson, 1905). Rae (1979), Wootten et al. (1982) and Hogans and Trudeau (1989) suggested that males copulated with all post-chalimus stages in Lepeophtheirus salmonis and Culigus elongatus, respectively. Kabata ( 198 1) first stated that copulation in the Caligidae began with the apposition of the ventral surfaces with the male gripping the female with its second antennae and maxillipeds. Spermatophores were transferred to the female whilst maintaining this position. The detailed description of the mating procedure in Lepeophtheirus pectoralis by Anstensrud (1990a) supported the observations made by Kabata ( 198 1). There has been considerable speculation as to the existence of post-chalimus filaments. Attachment of pre-adult I and adult females by filaments has been observed by several authors (Lewis, 1963 Hewitt, 1971; Johnson and Albright, 1991a) but most authors assumed that pre-adults and adults were completely mobile (Kabata, 1981; Wootten et al., 1982; Pike, 1989; Hogans and Trudeau, 1989). Anstensrud (1990a) showed that, prior to moulting, pre-adult L. pectoralis produced a temporary frontal filament attaching the animal as ecdysis proceeded. After the final moult, female parasitic copepods of many species show marked and dramatic changes in morphology. Females from the family Pennellidae undergo marked metamorphosis and bear no resemblance to the juvenile stages (Anstensrud, 1990b). In Lepeophtheirus species more subtle changes, including an expansion or swelling of the genital complex, are evident following mating (Scott, 1901; Boxshall, 1974; Anstensrud, 1990b; Johnson and Albright, 1991a), although exact measurements of this expansion have not been made. It was suspected that the mechanisms and behaviour involved during mating by L. pectoralis would also occur in L. salmonis and the present study was set up to The ectoparasitic
elongatus (Nordmann
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investigate this possibility. This study gives a more detailed description of precopulatory and copulatory behaviours displayed by L. salmonis and documents the change in size of the genital complex of adult female L. salmonis from postcopula to the production of the first egg batch.
2. Materials
and methods
Ovigerous female L. salmonis were collected from Atlantic salmon at various farm sites on the west coast of Scotland. The lice were placed in one litre plastic containers filled with oxygenated seawater from the site and then placed in an isotherm box for transportation to the laboratory. Egg strings were hatched, nauplii cultured and resultant copepodids were used to infect 200 g Atlantic salmon smolts. Groups of infected fish were maintained in 100 1 tanks supplied with continuously flowing re-circulating seawater (2 1 min.‘) of 33%0 salinity at lo-12°C and constant aeration. A photoperiod of 18 h was used. All infections were performed under these conditions. When the majority of lice were pre-adults, individual fish were transferred to mirrored glass aquaria (30cm X 30cm X 90cm) to provide views of the entire fish surface and acclimatised for 24 h before observation began. Lice stages were identified according to their size and shape. The number of male and female lice varied between 2 and 10 per fish. The aquaria were supplied with fresh, re-circulated, filtered seawater (2 1 mif ’ ) and constant aeration. Behaviours associated with mating and reproduction were observed continuously, in situ by eye, using lo-15 X lenses. To supplement observations made on laboratory infected fish, mobile lice (pre-adults and adults), collected from the field, were transplanted onto uninfected fish (200 g). These fish were then transferred to the observation tanks and acclimated for 24 h before observations were made. To observe the precise movements and role of the appendages during mating several mating pairs were examined, in beakers with 20 ml of sea water and maintained at IO-12°C using a binocular microscope. All mean measurements of time or size include values for standard error (+SE). For scanning electron microscopy the animals were prepared and examined as described in Ritchie et al. (in press). Inseminated adult females of known age (days postcopulation) were fixed in 10% neutral buffered formalin. The genital complex of each of these females was carefully removed, at the 4th pedigerous segment, and viewed with a JVC 1085 video camera and zoom lens. The change in genital complex area, from insemination to egg production, was measured using PC Image Analysis software. Significant differences in size with time were investigated by analysis of variance followed by Student’s t-tests.
3. Results 3. I. General
observations
Most mobile L. salmonis were located on the dorsal surface of the fish and tended to orientate anteriorly when the fish was resting and when water flowed over the fish.
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Pre-adult II females were positioned mostly antero-dorsally, and prior to precopula, 92% repositioned onto the head (n = 34). From initial observations adult males were more active on the host, whether it was stationary or mobile, than any other stage. Although not quantified, adult males showed an increase in activity in response to the presence of pre-adult females, compared to situations where only males were present on the fish. 3.2. Precopula
and associated
reproductive
behaviour
Mating in L. salmonis began with the formation of a precopulatory pair. In most cases this occurred between an adult male and a pre-adult II female (21 of 26 formed pairs) but adult males were occasionally observed in precopula position with pre-adult I and virgin adult females (5 of 26 formed pairs). If all three stages were established on a fish, in equal numbers, males preferentially selected any free pre-adult II females or recently moulted adult females. If no virgin females or pre-adult II females were available adult males took up precopulatory position with pre-adult I females. In all cases, there were equal numbers of males and females on the fish. Before establishing precopula, adult males, in the presence of females, displayed a type of searching behaviour (n = 12). Males, situated posterior to females, demonstrated these precise movements over large areas of the fish’s surface. From dorsal positions males moved ventrally whilst maintaining an anterior orientation, then moved dorsally again. Males moved anteriorly for a short distance during this movement, and while positioned dorsally. During these movements males often stopped and raised their cephalothoraces for several seconds before continuing. These movements were repeated many times over several minutes. Once in close association with a female, located on the head or dorsal 1 region (Fig. lA), movements became less frequent and were restricted to the area near the female. This behavioral sequence is defined as ‘mate searching’ behaviour. Adult males approached potential mates from the rear and, using the second antennae and maxillipeds, climbed onto the genital complex of the female. In some instances, where males were located anterior to a female, they moved backward over the cephalothorax of the female and positioned themselves over the genital complex. Males in precopula clasped their second antennae firmly around the 4th pedigerous segment of the female (Fig. 2A). In addition, the maxillipeds were often used to grip the ventral side of the female’s abdomen (Fig. 2A). From tank observations, where the fish was relatively stationary, 100% (n = 22) of precopula pairs formed on the dorsal surface of the fish, 73% on the head and 27% in the dorsal 1 position (Fig. 1A). When the fish was mobile and the water flow over the surface of the was fish more natural, 94% of precopulatory pairs formed on the dorsal surface, 23% on the head, 13% in dorsal 1, 58% in dorsal 2 and the remaining pairs in ventral 2 position, particularly behind the anal fin (Fig. 1B). Males were often observed establishing precopula then leaving the mate after less than 1 min. This ‘mate testing’ behaviour occurred more between males and pre-adult I females (86% of encounters) than with pre-adult II females (18.5% of encounters). Once in precopula with a suitable mate, males remained attached until the female’s final moult. The duration of precopula was dependent on the developmental stage of the
C. Ritchie et al. I J. Exp. Mar. Biol. Ecol. 201 (1996)
HEAD
,
DOFISAL1
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0%
HEAD
I I
,
8’
0%
VENTRAL1
,
TAIL
.
TAIL
VENTRAL 2
DORSAL 1
I I
289
0%
#’
VENTRAL1
B
DORSAL 2
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’
DORSAL 2
5.7%
I t
VENTRAL 2
Fig. 1. The different body regions examined on salmon smolts and the percentage distribution of precopula pairs of L. salmonis on fish during, (A) mating observations when the water flow over the fish was irregular (tank observations, n = 22 pairs) and (B) when the water flow was more natural (fish taken from a sea pen, n = 53 pairs).
female at the commencement of precopula and males in precopula with pre-adult II females remained attached for up to 5.6 days (mean, 4.120.2 SE days, n = 14). During this time the pair did not move far from the location where precopula was achieved. Specific aspects of behaviour associated with precopula were observed. Still attached by the second antennae, adult males raised their cephalothorax and genital complex to an angle of approximately 60” from the surface of the fish. Following this movement, males reverted to their original position and held tightly around the genital complex of the female. Males then raised their cephalothorax slightly and curled the lateral edges inward, around the genital complex of the female and remained in this position for up to 5 s. This sequence was repeated every 8-60 s and displayed over several hours (n = 7). 3.3. Female ,@a1 moult As the pre-adult II female approached her final moult a temporary frontal filament was produced that anchored the precopula pair as ecdysis proceeded (Fig. 2B). The filament emerged from the frontal organ (Fig. 2C). The length of the filament was 747.8% 13.8 ,um (n = 8) from the frontal organ to the attachment point, and 101.75+1.54 ,um
Fig. 2. (A-F) SEM analysis of mating pairs of Lepeophrheirus salmonis (A) Precopulatory pair (lateral view) sliced to reveal the second antennae (black arrow) of the male (m) grasped around the 4th pedigerous segment (th) of the female (f). The maxillipeds (white arrow) are observed holding the abdomen (a) of the female. Female genital complex (fg). Bar = 400 pm (B) Frontal filament (white arrow) of an adult female attached to a scale (s). Cephalothorax (c). Bar = 400 pm. (C) Adult female (ventral view) showing the frontal filament (black arrow) emerging from the filament gland (white arrow). Cephalothorax (c). Bar= 100 pm. (D) Temporary frontal filament showing the internal fibres (black arrow). Scale (s). Bar = 40 pm. (E) Filament fibres (white arrow). Bar = IO pm. (F) Postcopulatory pair, ventro-lateral view, sliced to show the maxillipeds (black arrow) of the male (m) holding the newly attached spermatophores (white arrows) onto the genital complex (fg) of the adult female (0. Bar = 400 firn.
(n = 12) in width. The filament comprised many fibres (approx. 0.38 ,um wide) which spiraled approximately 360” along its length (Fig. 2D and Fig. 2E). When the exoskeleton split the male relocated on the emerging cephalothorax of the
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adult female, holding on with the second antennae and maxillipeds, which allowed the old exoskeleton to be discarded. After the exuvium was shed the male resumed attachment with the second antennae around the 4th pedigerous segment of the adult female, as in precopula. The time between the production of the filament and reformation of precopula was 66.323.2 min (n = 7). During the moult of the female, other males aggregated slightly posterior to the pair and were observed raising their cephalothoraces, approximately 30” to the fish, for up to 3 s. Pairs remained attached to the fish via the filament and were observed ‘floating’ off the fish for periods of up to 5 min. The filament was then broken by pulling it in all directions around the attachment point. The female supplemented these movements by raising her cephalothorax and pulling the filament upwards. After 73.7t4.6 min (n = 7), the filament broke and the pair clamped down on the surface of the fish. During these movements the male maintained a tight grip with the second antennae around the 4th pedigerous segment of the adult female (Fig. 3A). Other males remained closely associated with the pair as the female moulted. Prior to copulation, two distinct forms of behaviour were observed. Each paired male repeatedly raised his genital complex approximately 60” from the surface of the fish and then pressed downward. The genital complex was raised aloft for up to 6 s. Associated with the downward movement, the male curled the cephalothorax around the genital complex of the female, rapidly beating the swimming legs in conjunction. This behaviour was repeated continually for several hours. The second, less frequent behaviour, occurred between these gentler movements. The male raised his entire body
nip
E
ai
D
k
Fig. 3. (A-F) Copulation and the mating positions observed between an adult male (m) and a recently moulted adult female (f) of L. salmonis. During copulation the male covered most of the genital complex of the female with his cephalothorax whilst maintaining these positions. For the purpose of clarity the male has been drawn more separated from the female to illustrate the movement of the appendages. (A) Precopula, (B)-(E) copula, (F) postcopula. Arrows represent the movements made by the appendages. a2, second antennae; mp, maxillipeds; ~12, second swimming legs. Bar = 0.8 mm.
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from the female, holding tight around the 4th firmly down on the female. The male remained down. This movement often caused the female During both these behavioral patterns, the male
pedigerous segment, before clamping raised for up to 30 s before clamping to change position slightly on the fish. rapidly vibrated his genital complex.
3.4. Copulation,
behaviour
sperm transfer and associated
After 23.65 1.8 h (n = 9) in precopula, with the newly moulted adult female, copulation proceeded. Fig. 3A-F shows the sequence of events involved in copulation. Only those appendages used during copulation are illustrated. Each paired male crawled sideways over the genital complex of the female, using the second antennae and maxillipeds, until the ventral surfaces opposed (Fig. 3B). Once in copula, the male prodded and manipulated the genital orifices of the female with the maxillipeds, pulling the genital complex tightly toward himself, severely distorting its shape. Occasionally the maxillipeds were moved to the lateral parts of the genital complex, pulling it closer to the male’s ventral surface. Between these events the male raised the genital complex of the female with its maxillipeds and continued to prod the genital orifices. The male then bent his own genital complex to an angle of approximately 60” from the fish and rapidly beat the swimming legs against his and the female’s genital complex (Fig. 3B). Then, using the maxillipeds, the male pulled the genital complex of the female tightly toward himself (Fig. 3C). These actions were repeated more than 25 times until the male transferred two spermatophores simultaneously onto the female’s genital complex. To effect this transfer, the male moved the maxillipeds from the genital orifices to the anterior ventral part of the female’s genital complex, and forced it up to approximately 45” from the surface of the fish (Fig. 3D). The male then raised his own genital complex and expelled a pair of spermatophores through the gonopores. The spermatophores were then transferred by the second swimming legs to the genital complex of the female, in the vicinity of the genital orifices (Fig. 3D). Transfer took approximately 2-3 s. The swimming legs then pushed against the spermatophores on average 15 times. Following this, the maxillipeds were used to position and orientate the spermatophores correctly. The genital complex of the female was raised again and the swimming legs of the male beat rapidly over the surface of the spermatophores for between 30 s and 4 min (Fig. 2E). The transfer of spermatophores was observed only between adult males and recently moulted adult females (n = 9). In all females examined, spermatophore contents emptied into the orifice diagonally opposite. Following this, the male moved sideways over the genital complex and took up a postcopula position, identical to the precopula position (Fig. 3F). Copulation lasted 6.9kO.5 min (n = 9). The maxillipeds held the whitish spermatophores firmly on the female’s genital complex for a few minutes postcopula (Fig. 2F). The cement surrounding the spermatophores darkened and they became less obvious with time. The male then moved backward over the female’s genital complex, abandoning her after 3.220.1 h (n = 9).
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Fig. 4. Change in genital complex area (?-SE) of inseminated adult female 15. salmonis maintained at 12°C from postcopulation (day 1) to production of the first egg batch (day 9). n > 8 for each day (y = - 0.033x3 + 0.533X? - 1.459.X + 3.45 1)
3.5. Postcopulation Following copulation, adult males were observed ‘mate searching’ and ‘mate testing’, presumably in search of new potential mates but it is not clear whether adult males mate once or are polygamous. Mating in L. salmonis was 100% successful (n = 13). Once the spermatophores were attached to the female the genital orifices were sealed, preventing secondary insemination. No evidence was found of misplaced or multiple spermatophores on the female’s genital complex. In the field, no females were found with more than one pair of spermatophores. Neither precopula nor copula positions were observed between adult males and previously inseminated adult females. Mating had a pronounced effect on the size of the adult female genital complex (Fig. 4). The genital complex area increased significantly over the 9 day period, from insemination to egg production, (ANOVA, P < 0.001) and with each passing day (t-test, P < O.Ol), except from day 1 to day 2 and from day 8 to day 9.
4. Discussion The mating processes of L. salmonis are similar to those described by Anstensrud (1990a) for L. pectoralis, although the time periods differed. Mating began with the establishment of a precopulatory pair between an adult male and predominantly a pre-adult II female. In previous studies of caligid copepods, animals observed in precopula as seen here were described as pairs in copula and it was assumed that both pre-adult stages and adult females were capable of copulation (Scott, 1901; Hewitt, 1964; Hwa, 1965; Wootten et al., 1982; Hogans and Trudeau, 1989). It was assumed that males, while attached dorsally, copulated with females by turning their genital complex
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under and toward the ventral surface of the female (Scott, 1901; Wilson, 1905; Lewis, 1963; Izawa, 1969) as in Lernaeoceru branchiulis (Kabata, 1981; Anstensrud, 1989, 199Oc,d). In Lepeophtheirus salmonis however, copulation began with the apposition of the ventral surfaces, as in L. pectoralis (Anstensrud, 1990a). The final moult of the female, from pre-adult II to the adult stage, preceded copulation. A temporary frontal filament was produced by the female prior to ecdysis, analogous to that produced at the first post-chalimus moult (personal observation). The filament structure consisted of one coiled strand compared to two entwined strands seen in L. pectoralis (Anstensrud, 1990a). The thin and semi-transparent nature of the filament, along with the short period of its attachment, may explain why it remained undiscovered in Lepeophtheirus until the work of Anstensrud (1990a). Several authors have observed a filament in a proportion of pre-adult I and adult females of Lepeophtheirus and Culigw (Lewis, 1963; Hewitt, 1971; Johnson and Albright, 1991a) although the majority assumed that only the larval stages possessed an attachment structure (Gurney, 1934; Kabata, 1981 Wootten et al., 1982; Pike, 1989; Hogans and Trudeau, 1989). Rae (1979) was first to comment that, on certain occasions, all post-chalimus stages of Lepeophtheirus salmonis are found attached by frontal filaments. The temporary filament originated and was extruded from the frontal organ, formerly named the median sucker or rugose area (Kabata, 198 1; Oldewage and van As, 1989). Anstensrud (1990a) described this structure as the filament gland since the filament originated from this area. The tugging action of the female to break the filament was similarly seen in 4th chalimus females of Salmincola californiensis (Kabata and Cousens, 1973). A series of complex and delicate behavioral patterns were executed during precopula and copula by adult male Lepeophtheirrds salmonis. Their duration and repetition suggested that they perhaps played a role in courtship. However, the passive nature and inevitable receptiveness of the female following the moult suggested these behavioral patterns were not essential for mate selection. These acts may be required to finalise spermatophore preparation and/or facilitate spermatophore extrusion. During the final ecdysis of females, adult males crawled anteriorly onto the cephalothorax of the emerging adult female. Anstensrud (1990a) found this movement in only 13% of L. pectoralis mating pairs, the rest released their hold on the female and repositioned close to th: moulting female. With males maintaining a hold on their precopula partner during the moult no ‘takeovers’ by other males were observed, in contrast to the 40% change in partner after the moult in L. pectorulis (Anstensrud, 1990a). Pairs of L. pectoralis, after detaching from the filament, remained in precopula for 2 h-6 days (Anstensrud, 199Oa), while in L. sulmonis copulation lasted 19-33 h (mean, 23.6 h) after breakage of the filament. Copulation was very intricate and delicate. The swimming legs and maxillipeds were used in to transfer, orientate and secure attachment of the spermatophores to the genital complex of the female. The spermatophores were covered in a‘cement-like’ substance as described by Anstensrud (1990a,c) for L. pectoralis. The hardening of the ‘cement-like’ substance was probably aided by the beating action of the swimming legs which increased the flow of water over the spermatophore surface. The maxillipeds were utilised effectively in holding the spermatophores firmly on the genital complex of the
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female. This may have prevented the female from expelling the spermatophores which was commonly observed after 2-43 days post-insemination (mean, 26.2 days) in L. pectoralis (Anstensrud, 1990~). Non-virgin female L. salmonis, without spermatophores, were never seen in this study and are only very rarely found in the field (Ritchie et al., in press). This suggests that the seal formed by the spermatophores and cement effectively prevents re-insemination and that female L. salmonis are monogamous. This suggestion is supported by observations that adult males are never found in precopula or copula with inseminated females and that multiple spermatophore placement was never observed. Scott (1901) and Wilson (1905) however found clusters of spermatophores on the ventral surface of several caligid species. The available evidence suggests adult male L. salmonis are polygamous. Postcopulatory males displayed ‘mate searching’ and ‘mate testing’ behaviour in search of a new mate and a range of spermatophores were present in the vasa deferentia of reproductively competent males (Ritchie et al., in press). Male L. pectoralis and Lernaeoceru brunchialis copulated with several females during their lifetime (Anstensrud, 1990~ and d). With approximately 1:l sex ratios in laboratory broods and field populations (pers. obs.), sex specific development rates (Johnson and Albright, 1991b; pers. obs.) and male polygamy, the operational sex ratio of Lepeophtheirus salmonis will be male biased. interaction between males will be Within populations of L. salmonis, competitive confined to locating a precopulatory mate. With a higher proportion of males to receptive females it becomes beneficial for a male to take up precopula with reproductively immature females thus securing a potential mate and guarding her until maturity (Grafen and Ridley, 1983). Mate guarding, which is believed to have evolved under conditions of restricted female availability and time, has been identified in the Copepoda (Grafen and Ridley, 1983), including the Cyclopoida, Harpacticoida, Poecilostomatoida and other members of the Siphonostomatoida (Boxshall, 1990). Where this situation occurs, as in L. salmonis, females that can be mated immediately are rare and as a result the rate at which males find females to mate with is low. Consequently, natural selection will favour males that take up precopula with immature females close to maturity, thereby reducing male searching time. As the proportion of guarding males increases in the population, the density of ‘acceptable’ mates decreases and males must exploit a pool of unguarded females that are further from maturity (Grafen and Ridley, 1983). This theory of sexual selection applies to L. salmonis, where adult males preferentially selected pre-adult II females and recently moulted virgin adult females. Anstensrud (1992) showed that male Lernaeocera branchialis established precopula with females of all stages but preferred chalimus 4 and virgin females. In addition, male Lepeophtheirus pectoralis were associated with, but showed no discrimination between, pre-adult I and II females but preferred newly moulted virgin females. The selection of potential mates implies that males were capable of recognising females and their developmental and reproductive state. Several studies on mating behaviour and sex recognition in the Copepoda (particularly the order Calanoida) showed that males react to chemical signals from conspecifics that increase mate seeking behaviour (Katona, 1973; Griffiths and Frost, 1976 Dunham, 1978; Uchima, 1985; Jacoby and Youngbluth, 1983). Currently however, there is little evidence on the
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presence of sex pheromones in parasitic copepods. Kabata (1958) found male Lemaeoceru brunchialis aggregated around immature females, and Anstensrud (1989, 1992) observed male L. brunchialis clustering around the filament of females and females in coitus. Anstensrud (1990b) suggested that adult male Lepeophtheirus pectorulis could discriminate between pre-adult and adult females by chemical cues. Adult male L. sulmonis increased their level of activity in the presence of pre-adult females, but no quantitative analysis of this change in activity has been made. Associated with increased activity was the display of putative ‘mate searching’ behaviour. These unique and apparently structured movements were performed in the presence of pre-adult and virgin adult females only. In addition, males clustered around precopula pairs formed between males and recently moulted virgin females, and occasionally pre-adult II females. Along with preferences by males for virgin adult females and pre-adult II females, this clustering suggested that any sexual signals released by females to broadcast their receptivity strengthen with development. Adult males were never observed in precopula with inseminated females suggesting that non-virgin females no longer produced any chemical stimuli attractive to males. A similar theory was proposed by Anstensrud (1992) for L. pectorulis. During ‘mate searching’ behaviour and clustering, males were observed raising their cephalothorax from the surface of the fish. This manoeuvre, which may be necessary to ‘sample’ or ‘test’ the water for specific chemical stimuli, was repeated more frequently by males aggregated around recently moulted females. The antero-dorsal position of most pre-adult II females may be a behavioral adaptation that supplements any chemical advertisement of receptivity and makes the process of mate location easier for the male. The larger percentage of precopulatory pairs formed on the head and dorsal 1 regions support this suggestion. Other authors have attributed this aggregation to the absence of scales and the relative thinness of the skin, which may ease feeding (Hastein and Bergsjo, 1976), and the reduced drag associated with this area (Jonsdottir et al., 1992). The ‘mate testing’ behaviour performed by adult males suggested they could not distinguish between post-chalimus stages by chemical signals alone and were attracted, equally, to all female post-chalimus stages. This observation contrasts with Anstensrud (1992) who claimed that chemical cues were initially responsible for male discrimination between pre-adult and adult females. ‘Testing’ allows males to recognise the developmental stage of the female. Recognition of the developmental stage could be achieved by examining the size of the female’s genital complex through mechanoreception or by contact chemoreception. Katona (1973) demonstrated mate selection in three species of planktonic copepod (Eurytemoru afinis, E. herdmani and Pseudodiuptomus coronutus) and suggested that contact chemoreception was probably important. In parasitic copepods, dramatic changes in the morphology, including gigantism of the genital complex and the holdfast often occur after the final moult (Kabata, 198 1; Anstensrud, 1990b). In female Lepeophtheirus sulmonis the main post-moult development, was the increase in size of the genital complex, associated with the development of the reproductive organs and deposition of eggs. A similar increase was observed in female L. pectoralis (Boxshall, 1974; Anstensrud, 1990b). It was believed that these relatively small changes were predetermined rather than induced by mating, sper-
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matophore transfer or seminal fluids inside the female (Anstensrud, 1990b). Johnson and Albright (1991a) reported folds of cuticle at the anterior end of the genital complex of newly moulted adult females and suggested this may serve as a reservoir for cuticle expansion. The apparatus employed in this study was geared for the observation of lice and relied on the passive nature of the fish. This behaviour was not typical of fish found in cage situations so some of the features of mate location and mating, along with time scales, may not be applicable to those found under cage conditions. In addition, observations that suggested sex pheromones may be involved in mating of L. salmonis were obtained from lice in this artificial environment, where water flow over the fish was irregular. Under these circumstances the responses evoked may have been distorted by the slow and artificial current of water. Stimulus concentrations may have been higher or lower than those found under real conditions. Despite these drawbacks, these observations gave a strong indication of a clear selective response by males to post-chalimus females.
Acknowledgments GR would like to thank SERC and the Scottish Salmon Growers Association Ltd. for their financial support during this study. The help and assistance provided by Marine Harvest International in collecting material for this study is gratefully acknowledged.
References Anstensrud, M., 1989. Experimental studies of the reproductive behaviour of the parasitic copepod Lemaeocera branchialis (Pennellidae). J. Mar. Biol. Ass. UK, Vol. 69, pp. 465-476. Anstensrud, M., 1990a. Moulting and mating in Lepeophtheirus pectoralis (Copepoda: Cahgidae). J. Mar. Biol. Ass. UK, Vol. 70, pp. 269-281. Anstensrud, M., 1990b. Effects of mating on female behaviour and allomettic growth in the two parasitic copepods Lemaeocera bronchialis (L., 1767) (Pennellidae) and Lepeophtheirus pectoralis (Muher, 1776) (Caligidae). Crustaceana, Vol. 59, pp. 245-258. Anstensrud, M., 199Oc. Mating strategies of two parasitic copepods Lernaeocera branchiulis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Miiller) (Caligidae) on flounder: Polygamy, sex specific age at maturity and sex ratio. J. Exp. Mar. Bid. Ecol., Vol. 136, pp. 141- 158. Anstensrud, M., 1990d. Male reproductive characteristics of two parasitic copepods, Lemaeocera branchialis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Miiller) (Caligidae). J. Crust. Biol.,Vol. 10, pp. 627-638. Anstensrud, M., 1992. Mate guarding and mate choice in two copepods, Lernaeocera branchialis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Miiller) (Caligidae), parasitic on flounder. J. Crust. Biol., Vol. 12, pp. 3 l-40. Boxshall, G.A., 1974. The developmental stages of Lepeophtheirus pectoralis (Miiller, 1776) (Copepoda: Caligidae). J. Nat. Hist., Vol. 8, pp. 68 l-700. Boxshall, G.A., 1990. Precopulatory mate guarding in copepods. Bijdr. Dierk. Vol. 60, pp. 209-213. Bruno, D.W. and J. Stone, 1990. The role of saithe: Pollachius kens L., as a host for the sea lice, Lepeophtheirus salmonis Kroyer and Cal&us elongatus Nordmann. Aquaculture, Vol. 89, pp. 201-207. Dunham, P.J., 1978. Sex pheromones in Crustacea. Biol. Rev., Vol. 53, pp. 555-583. Grafen, A. and M. Ridley, 1983. A model of mate guarding. J. Theor. Biol., Vol. 102, pp. 549-567.
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Griffiths, A.M. and B.W. Frost, 1976. Chemical communication in the marine planktonic copepods Calanus pacifus and Pseudocalunus sp. Crustaceana, Vol. 30, pp. 1-8. Gurney, R., 1934. The development of certain parasitic Copepoda of the families Caligidae and Clavellidae. Proc. Zool. Sot. Land., Vol. 1934, pp. 177-217. Hastein, T. and T. Bergsjo. 1976. The salmon lice Lepeophtheirus salmonis as the cause of disease in farmed salmonids. Riv. Ital. Piscic. Irtiopatol., Vol. 11, pp. 3-5. Hewitt, G.C. 1964. The postchalimus development of Lepeoptheirus polyprioni Hewitt, 1963 (Copepoda: Caligidae). Trans. R. Sot. N.Z., Vol. 4, pp. 157-159. Hewitt, G.C., 1971. Two species of Cal&us (Copepoda, Caligidae) from Australian waters, with a description of some developmental stages. Pacif. Sci., Vol. 25, pp. 145-164. Hogans, W.E. and D.J. Trudeau, 1989. Preliminary studies on the biology of sea lice, Cal&us elongatus, Cal&us curtus and Lepeophtheirus salmonis (Copepoda: Caligoida) parasitic on cage-cultured salmonids in the Lower Bay of Fundy. Can. Tech. Rep. Fish. Aquat. Sci., Vol. 1715, pp. 14. Hwa, D., 1965. The life cycle of oriental fish louse. Acta Zool. Sin.,Vol. 17, pp. 48-58. Izawa, K., 1969. Life history of Culigus spinosus Yamaguti, 1939, obtained from cultured Yellow tail, Seriola quinqueradiata T. and S. (Crustacea: Caligoida). Rep. Fat. Fish. Prefect. Univ. Mie,Vol. 6, pp. 127-157. Jacoby, C.A. and M.J. Youngbluth, 1983. Mating behaviour in three species of Pseudodiaptomus (Copepoda: Calanoida). Mar. Biol., Vol. 76, pp. 77-86. Johnson, S.C. and L.J. Albright, 1991a. The developmental stages of Lepeop,ptheirussalmonis (Kroyer, 1837) (Copepoda: Caligidae). Can. J. Zool, Vol. 69, pp. 929-950. Johnson, S.C. and L.J. Albright, 1991b. Development, growth, and survival of Lepeoptheirus salmonis (Copepoda: Caligidae) under laboratory conditions. J. Mar. Biol. Ass. UK, Vol. 71, pp. 425-436. Jbnsdbttir, H., J.E. Bron, R. Wootten, and J.F. Tumbull. 1992. The histopathology associated with the pre-adult and adult stages of Lepeophtheirus salmonis on the Atlantic salmon, Salmon salar L. J. Fish Dis., Vol. 15, pp. 521-527. Kabata, Z., 1958. Lemaeocera obtusa n. sp. Its biology and its effects on the haddock. Mar. Res., Vol. 3, pp. l-26. Kabata, Z., 1981. Copepoda (Crustacea) parasitic on fishes: problems and perspectives. Adv. Parasitol., Vol. 19, pp. 1-71. Kabata, Z. and B. Cousens, 1973. Life cycle of Sulmincola californiensis (Dana 1852) (Copepoda: Lemaeopodidae). J. Fish. Res. Board Can., Vol. 30, pp. 881-903. Katona, S.K., 1973. Evidence for sex pheromones in planktonic copepods. Limnol. Oceanogr., Vol. 18, pp. 574-583. Lewis, A.G., 1963. Life history of the Caligid Copepod Lepeophtheirus dissimularus Wilson, 1905 (Crustacea: Caligoida). Pacif. Sci., Vol. 17, pp. 195-242. Oldewage, W.H. and J.G.Van As, 1989. On the sensory (?) structure between the frontal plates of Cal&us O.F. Muller, 1785 (Copepoda, Caligidae). Crustaceana, Vol. 57, pp. 72-78. Pike, A.W., 1989. Sea lice-major pathogens of farmed Atlantic salmon. Parasitol. Today, Vol. 5, pp. 29 l-297. Rae, G.H., 1979. On the trail of the Sea lice. Fish Fmr., Vol. 2, pp. 22-25. Ritchie, G., A.J. Mordue (Luntz), A.W. Pike and G.H. Rae. Morphology and ultrastructure of the reproductive system of Lepeophtheirus salmonis (Kreyer, 1837) (Copepoda: Caligidae). J. Crust. Biol., in press. Schram, T.A., 1993. Supplementary descriptions of the developmental stages of Lepeophtheirus salmonis (Kroyer, 1837) (Copepoda: Caligidae). In, Pathogens of Wild and Farmed Fish: Sea lice, edited by G.A. Boxshall and D. Defaye, Ellis Horwood, New York, NY, pp. 30-50. Scott, A., 1901. On the fish parasites, Lepeophtheirus and Lernaea. Rep. Lams. Sea-Fish. Lab., Vol. 9, pp. 62-95. Uchima, M., 1985. Copulation in the marine copepod Oithona davisae, Ferrari and Orsi. Bull. Plank. Sot. Jpn., Vol. 32, pp. 23-30. Wilson, C.B., 1905. North American parasitic copepods belonging to the family Caligidae. Part I The Caligidae. Proc. US Nat. Mus., Vol. 28, pp. 479-672. Wootten, R., J.W. Smith and E.A. Needham, 1982. Aspects of the biology of the parasitic copepods Lepeophtheirus salmonis and Caligus elongarus on farmed salmonids and their treatment. Proc. R. Sot. Edinb., Vol. 81, pp. 185-197.
of Experimental
ELSEVIER
Marine Biology
JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY
and Ecology,
201 (1996) 285-298
Observations on mating and reproductive behaviour of Lepeophtheirus salmonis, Kr@yer (Copepoda: Caligidae) G. Ritchie”,*,
A.J. Mordue (Luntz)b, A.W. Pikeb, G.H. Raec
“Nutreco Aquaculture Research Centre, Forusbeen 35, PO Box 353, N-4033 , Forus, Norway ‘Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB9 2TN, UK ‘Scottish Salmon Growers Association Ltd, Drummond House, Scott street, Perth PHI SEJ, UK Received
28 November
1995; revised 9 January
1996; accepted
10 January
1996
Abstract Mating in Lepeophtheirus salmonis was similar to that reported for L. pectoralis in both the procedure and positions attained by both sexes but the timescales were different. Adult males took up precopulatory positions with, predominantly, pre-adult II females. Males held their respective partners around the 4th pedigerous segment using the second antennae. Females approaching the final moult produced temporary frontal filaments that served to anchor the pair as ecdysis proceeded. Males remained attached to the cephalothorax of the female during her final ecdysis before reattaching around the genital complex. After transferring to the ventral surface of the female, copulation occurred between the adult male and recently moulted adult female. The maxillipeds were used to manipulate and raise the genital complex of the female and a pair of spermatophores was transferred by the swimming legs. Following copulation the male returned to the dorsal surface of the female and held the spermatophores on the female for some time, before leaving her. Several specific behavioral patterns were observed during precopula, at the final moult of the female and during copulation. These involved considerable movement of the genital complexes and precise movements of the appendages by the male. Mate searching and testing behaviours were performed by adult males in the presence of pre-adult and adult females. These behaviours provided evidence that mate recognition by short range chemical, or contact stimuli may have occurred in L. salmonis. In addition, males clustered around moulting females which showed the attractiveness of the female at this stage. Postcopula, allometric growth of the female genital complex was observed and quantified from the time of insemination to the production of the first batch of eggs. Keywords:
Behaviour; Caligidae; Lepeophtheirus
salmonis;
Mating; Mate guarding; Pheromones;
Reproduction
*Corresponding
author.
0022-0981/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOO22-098 1(96)000081
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1. Introduction copepods Lepeophtheirus salmonis (Krayer 1837) and Caligus 1832), collectively referred to as sea lice, are a serious problem in commercial Atlantic salmon (Salmo salar L.) farming in Norway, Scotland, Ireland and Canada. Of the two species Lepeophtheirus salmonis is the more problematic pathogen, but high infection levels of both species can result in severe damage and huge economic losses. L. salmonis has a lo-stage lifecycle comprising two free swimming nauplius stages, one free swimming infective copepodid stage, four attached chalimus stages, two pre-adult stages and the adult (Johnson and Albright, 1991a; Schram, 1993). All three post-chalimus stages can move freely over the surface of the fish (Bruno and Stone, 1990; personal observation) and are only immobile during moulting when a temporary frontal filament is produced (personal observation). Until recently there was a dearth of information regarding the mating processes and related behaviour patterns of caligid copepods. Scott (1901) observed several species of Lepeophtheirus and concluded that fertilization of the female was accomplished after the final chalimus stage. It is important to note that Scott (1901) failed to recognise the presence of pre-adult stages. Several authors assumed that copulation proceeded, with the male attached dorsally on the female, folding its genital complex along the ventral surface of the female and discharging a pair of spermatophores. This procedure was described for L. dissimulutus (Lewis, 1963), Culigus spinosus (Izawa, 1969) and several other caligid species (Wilson, 1905). Rae (1979), Wootten et al. (1982) and Hogans and Trudeau (1989) suggested that males copulated with all post-chalimus stages in Lepeophtheirus salmonis and Culigus elongatus, respectively. Kabata ( 198 1) first stated that copulation in the Caligidae began with the apposition of the ventral surfaces with the male gripping the female with its second antennae and maxillipeds. Spermatophores were transferred to the female whilst maintaining this position. The detailed description of the mating procedure in Lepeophtheirus pectoralis by Anstensrud (1990a) supported the observations made by Kabata ( 198 1). There has been considerable speculation as to the existence of post-chalimus filaments. Attachment of pre-adult I and adult females by filaments has been observed by several authors (Lewis, 1963 Hewitt, 1971; Johnson and Albright, 1991a) but most authors assumed that pre-adults and adults were completely mobile (Kabata, 1981; Wootten et al., 1982; Pike, 1989; Hogans and Trudeau, 1989). Anstensrud (1990a) showed that, prior to moulting, pre-adult L. pectoralis produced a temporary frontal filament attaching the animal as ecdysis proceeded. After the final moult, female parasitic copepods of many species show marked and dramatic changes in morphology. Females from the family Pennellidae undergo marked metamorphosis and bear no resemblance to the juvenile stages (Anstensrud, 1990b). In Lepeophtheirus species more subtle changes, including an expansion or swelling of the genital complex, are evident following mating (Scott, 1901; Boxshall, 1974; Anstensrud, 1990b; Johnson and Albright, 1991a), although exact measurements of this expansion have not been made. It was suspected that the mechanisms and behaviour involved during mating by L. pectoralis would also occur in L. salmonis and the present study was set up to The ectoparasitic
elongatus (Nordmann
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investigate this possibility. This study gives a more detailed description of precopulatory and copulatory behaviours displayed by L. salmonis and documents the change in size of the genital complex of adult female L. salmonis from postcopula to the production of the first egg batch.
2. Materials
and methods
Ovigerous female L. salmonis were collected from Atlantic salmon at various farm sites on the west coast of Scotland. The lice were placed in one litre plastic containers filled with oxygenated seawater from the site and then placed in an isotherm box for transportation to the laboratory. Egg strings were hatched, nauplii cultured and resultant copepodids were used to infect 200 g Atlantic salmon smolts. Groups of infected fish were maintained in 100 1 tanks supplied with continuously flowing re-circulating seawater (2 1 min.‘) of 33%0 salinity at lo-12°C and constant aeration. A photoperiod of 18 h was used. All infections were performed under these conditions. When the majority of lice were pre-adults, individual fish were transferred to mirrored glass aquaria (30cm X 30cm X 90cm) to provide views of the entire fish surface and acclimatised for 24 h before observation began. Lice stages were identified according to their size and shape. The number of male and female lice varied between 2 and 10 per fish. The aquaria were supplied with fresh, re-circulated, filtered seawater (2 1 mif ’ ) and constant aeration. Behaviours associated with mating and reproduction were observed continuously, in situ by eye, using lo-15 X lenses. To supplement observations made on laboratory infected fish, mobile lice (pre-adults and adults), collected from the field, were transplanted onto uninfected fish (200 g). These fish were then transferred to the observation tanks and acclimated for 24 h before observations were made. To observe the precise movements and role of the appendages during mating several mating pairs were examined, in beakers with 20 ml of sea water and maintained at IO-12°C using a binocular microscope. All mean measurements of time or size include values for standard error (+SE). For scanning electron microscopy the animals were prepared and examined as described in Ritchie et al. (in press). Inseminated adult females of known age (days postcopulation) were fixed in 10% neutral buffered formalin. The genital complex of each of these females was carefully removed, at the 4th pedigerous segment, and viewed with a JVC 1085 video camera and zoom lens. The change in genital complex area, from insemination to egg production, was measured using PC Image Analysis software. Significant differences in size with time were investigated by analysis of variance followed by Student’s t-tests.
3. Results 3. I. General
observations
Most mobile L. salmonis were located on the dorsal surface of the fish and tended to orientate anteriorly when the fish was resting and when water flowed over the fish.
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Pre-adult II females were positioned mostly antero-dorsally, and prior to precopula, 92% repositioned onto the head (n = 34). From initial observations adult males were more active on the host, whether it was stationary or mobile, than any other stage. Although not quantified, adult males showed an increase in activity in response to the presence of pre-adult females, compared to situations where only males were present on the fish. 3.2. Precopula
and associated
reproductive
behaviour
Mating in L. salmonis began with the formation of a precopulatory pair. In most cases this occurred between an adult male and a pre-adult II female (21 of 26 formed pairs) but adult males were occasionally observed in precopula position with pre-adult I and virgin adult females (5 of 26 formed pairs). If all three stages were established on a fish, in equal numbers, males preferentially selected any free pre-adult II females or recently moulted adult females. If no virgin females or pre-adult II females were available adult males took up precopulatory position with pre-adult I females. In all cases, there were equal numbers of males and females on the fish. Before establishing precopula, adult males, in the presence of females, displayed a type of searching behaviour (n = 12). Males, situated posterior to females, demonstrated these precise movements over large areas of the fish’s surface. From dorsal positions males moved ventrally whilst maintaining an anterior orientation, then moved dorsally again. Males moved anteriorly for a short distance during this movement, and while positioned dorsally. During these movements males often stopped and raised their cephalothoraces for several seconds before continuing. These movements were repeated many times over several minutes. Once in close association with a female, located on the head or dorsal 1 region (Fig. lA), movements became less frequent and were restricted to the area near the female. This behavioral sequence is defined as ‘mate searching’ behaviour. Adult males approached potential mates from the rear and, using the second antennae and maxillipeds, climbed onto the genital complex of the female. In some instances, where males were located anterior to a female, they moved backward over the cephalothorax of the female and positioned themselves over the genital complex. Males in precopula clasped their second antennae firmly around the 4th pedigerous segment of the female (Fig. 2A). In addition, the maxillipeds were often used to grip the ventral side of the female’s abdomen (Fig. 2A). From tank observations, where the fish was relatively stationary, 100% (n = 22) of precopula pairs formed on the dorsal surface of the fish, 73% on the head and 27% in the dorsal 1 position (Fig. 1A). When the fish was mobile and the water flow over the surface of the was fish more natural, 94% of precopulatory pairs formed on the dorsal surface, 23% on the head, 13% in dorsal 1, 58% in dorsal 2 and the remaining pairs in ventral 2 position, particularly behind the anal fin (Fig. 1B). Males were often observed establishing precopula then leaving the mate after less than 1 min. This ‘mate testing’ behaviour occurred more between males and pre-adult I females (86% of encounters) than with pre-adult II females (18.5% of encounters). Once in precopula with a suitable mate, males remained attached until the female’s final moult. The duration of precopula was dependent on the developmental stage of the
C. Ritchie et al. I J. Exp. Mar. Biol. Ecol. 201 (1996)
HEAD
,
DOFISAL1
,
0%
HEAD
I I
,
8’
0%
VENTRAL1
,
TAIL
.
TAIL
VENTRAL 2
DORSAL 1
I I
289
0%
#’
VENTRAL1
B
DORSAL 2
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’
DORSAL 2
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I t
VENTRAL 2
Fig. 1. The different body regions examined on salmon smolts and the percentage distribution of precopula pairs of L. salmonis on fish during, (A) mating observations when the water flow over the fish was irregular (tank observations, n = 22 pairs) and (B) when the water flow was more natural (fish taken from a sea pen, n = 53 pairs).
female at the commencement of precopula and males in precopula with pre-adult II females remained attached for up to 5.6 days (mean, 4.120.2 SE days, n = 14). During this time the pair did not move far from the location where precopula was achieved. Specific aspects of behaviour associated with precopula were observed. Still attached by the second antennae, adult males raised their cephalothorax and genital complex to an angle of approximately 60” from the surface of the fish. Following this movement, males reverted to their original position and held tightly around the genital complex of the female. Males then raised their cephalothorax slightly and curled the lateral edges inward, around the genital complex of the female and remained in this position for up to 5 s. This sequence was repeated every 8-60 s and displayed over several hours (n = 7). 3.3. Female ,@a1 moult As the pre-adult II female approached her final moult a temporary frontal filament was produced that anchored the precopula pair as ecdysis proceeded (Fig. 2B). The filament emerged from the frontal organ (Fig. 2C). The length of the filament was 747.8% 13.8 ,um (n = 8) from the frontal organ to the attachment point, and 101.75+1.54 ,um
Fig. 2. (A-F) SEM analysis of mating pairs of Lepeophrheirus salmonis (A) Precopulatory pair (lateral view) sliced to reveal the second antennae (black arrow) of the male (m) grasped around the 4th pedigerous segment (th) of the female (f). The maxillipeds (white arrow) are observed holding the abdomen (a) of the female. Female genital complex (fg). Bar = 400 pm (B) Frontal filament (white arrow) of an adult female attached to a scale (s). Cephalothorax (c). Bar = 400 pm. (C) Adult female (ventral view) showing the frontal filament (black arrow) emerging from the filament gland (white arrow). Cephalothorax (c). Bar= 100 pm. (D) Temporary frontal filament showing the internal fibres (black arrow). Scale (s). Bar = 40 pm. (E) Filament fibres (white arrow). Bar = IO pm. (F) Postcopulatory pair, ventro-lateral view, sliced to show the maxillipeds (black arrow) of the male (m) holding the newly attached spermatophores (white arrows) onto the genital complex (fg) of the adult female (0. Bar = 400 firn.
(n = 12) in width. The filament comprised many fibres (approx. 0.38 ,um wide) which spiraled approximately 360” along its length (Fig. 2D and Fig. 2E). When the exoskeleton split the male relocated on the emerging cephalothorax of the
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adult female, holding on with the second antennae and maxillipeds, which allowed the old exoskeleton to be discarded. After the exuvium was shed the male resumed attachment with the second antennae around the 4th pedigerous segment of the adult female, as in precopula. The time between the production of the filament and reformation of precopula was 66.323.2 min (n = 7). During the moult of the female, other males aggregated slightly posterior to the pair and were observed raising their cephalothoraces, approximately 30” to the fish, for up to 3 s. Pairs remained attached to the fish via the filament and were observed ‘floating’ off the fish for periods of up to 5 min. The filament was then broken by pulling it in all directions around the attachment point. The female supplemented these movements by raising her cephalothorax and pulling the filament upwards. After 73.7t4.6 min (n = 7), the filament broke and the pair clamped down on the surface of the fish. During these movements the male maintained a tight grip with the second antennae around the 4th pedigerous segment of the adult female (Fig. 3A). Other males remained closely associated with the pair as the female moulted. Prior to copulation, two distinct forms of behaviour were observed. Each paired male repeatedly raised his genital complex approximately 60” from the surface of the fish and then pressed downward. The genital complex was raised aloft for up to 6 s. Associated with the downward movement, the male curled the cephalothorax around the genital complex of the female, rapidly beating the swimming legs in conjunction. This behaviour was repeated continually for several hours. The second, less frequent behaviour, occurred between these gentler movements. The male raised his entire body
nip
E
ai
D
k
Fig. 3. (A-F) Copulation and the mating positions observed between an adult male (m) and a recently moulted adult female (f) of L. salmonis. During copulation the male covered most of the genital complex of the female with his cephalothorax whilst maintaining these positions. For the purpose of clarity the male has been drawn more separated from the female to illustrate the movement of the appendages. (A) Precopula, (B)-(E) copula, (F) postcopula. Arrows represent the movements made by the appendages. a2, second antennae; mp, maxillipeds; ~12, second swimming legs. Bar = 0.8 mm.
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from the female, holding tight around the 4th firmly down on the female. The male remained down. This movement often caused the female During both these behavioral patterns, the male
pedigerous segment, before clamping raised for up to 30 s before clamping to change position slightly on the fish. rapidly vibrated his genital complex.
3.4. Copulation,
behaviour
sperm transfer and associated
After 23.65 1.8 h (n = 9) in precopula, with the newly moulted adult female, copulation proceeded. Fig. 3A-F shows the sequence of events involved in copulation. Only those appendages used during copulation are illustrated. Each paired male crawled sideways over the genital complex of the female, using the second antennae and maxillipeds, until the ventral surfaces opposed (Fig. 3B). Once in copula, the male prodded and manipulated the genital orifices of the female with the maxillipeds, pulling the genital complex tightly toward himself, severely distorting its shape. Occasionally the maxillipeds were moved to the lateral parts of the genital complex, pulling it closer to the male’s ventral surface. Between these events the male raised the genital complex of the female with its maxillipeds and continued to prod the genital orifices. The male then bent his own genital complex to an angle of approximately 60” from the fish and rapidly beat the swimming legs against his and the female’s genital complex (Fig. 3B). Then, using the maxillipeds, the male pulled the genital complex of the female tightly toward himself (Fig. 3C). These actions were repeated more than 25 times until the male transferred two spermatophores simultaneously onto the female’s genital complex. To effect this transfer, the male moved the maxillipeds from the genital orifices to the anterior ventral part of the female’s genital complex, and forced it up to approximately 45” from the surface of the fish (Fig. 3D). The male then raised his own genital complex and expelled a pair of spermatophores through the gonopores. The spermatophores were then transferred by the second swimming legs to the genital complex of the female, in the vicinity of the genital orifices (Fig. 3D). Transfer took approximately 2-3 s. The swimming legs then pushed against the spermatophores on average 15 times. Following this, the maxillipeds were used to position and orientate the spermatophores correctly. The genital complex of the female was raised again and the swimming legs of the male beat rapidly over the surface of the spermatophores for between 30 s and 4 min (Fig. 2E). The transfer of spermatophores was observed only between adult males and recently moulted adult females (n = 9). In all females examined, spermatophore contents emptied into the orifice diagonally opposite. Following this, the male moved sideways over the genital complex and took up a postcopula position, identical to the precopula position (Fig. 3F). Copulation lasted 6.9kO.5 min (n = 9). The maxillipeds held the whitish spermatophores firmly on the female’s genital complex for a few minutes postcopula (Fig. 2F). The cement surrounding the spermatophores darkened and they became less obvious with time. The male then moved backward over the female’s genital complex, abandoning her after 3.220.1 h (n = 9).
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Fig. 4. Change in genital complex area (?-SE) of inseminated adult female 15. salmonis maintained at 12°C from postcopulation (day 1) to production of the first egg batch (day 9). n > 8 for each day (y = - 0.033x3 + 0.533X? - 1.459.X + 3.45 1)
3.5. Postcopulation Following copulation, adult males were observed ‘mate searching’ and ‘mate testing’, presumably in search of new potential mates but it is not clear whether adult males mate once or are polygamous. Mating in L. salmonis was 100% successful (n = 13). Once the spermatophores were attached to the female the genital orifices were sealed, preventing secondary insemination. No evidence was found of misplaced or multiple spermatophores on the female’s genital complex. In the field, no females were found with more than one pair of spermatophores. Neither precopula nor copula positions were observed between adult males and previously inseminated adult females. Mating had a pronounced effect on the size of the adult female genital complex (Fig. 4). The genital complex area increased significantly over the 9 day period, from insemination to egg production, (ANOVA, P < 0.001) and with each passing day (t-test, P < O.Ol), except from day 1 to day 2 and from day 8 to day 9.
4. Discussion The mating processes of L. salmonis are similar to those described by Anstensrud (1990a) for L. pectoralis, although the time periods differed. Mating began with the establishment of a precopulatory pair between an adult male and predominantly a pre-adult II female. In previous studies of caligid copepods, animals observed in precopula as seen here were described as pairs in copula and it was assumed that both pre-adult stages and adult females were capable of copulation (Scott, 1901; Hewitt, 1964; Hwa, 1965; Wootten et al., 1982; Hogans and Trudeau, 1989). It was assumed that males, while attached dorsally, copulated with females by turning their genital complex
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under and toward the ventral surface of the female (Scott, 1901; Wilson, 1905; Lewis, 1963; Izawa, 1969) as in Lernaeoceru branchiulis (Kabata, 1981; Anstensrud, 1989, 199Oc,d). In Lepeophtheirus salmonis however, copulation began with the apposition of the ventral surfaces, as in L. pectoralis (Anstensrud, 1990a). The final moult of the female, from pre-adult II to the adult stage, preceded copulation. A temporary frontal filament was produced by the female prior to ecdysis, analogous to that produced at the first post-chalimus moult (personal observation). The filament structure consisted of one coiled strand compared to two entwined strands seen in L. pectoralis (Anstensrud, 1990a). The thin and semi-transparent nature of the filament, along with the short period of its attachment, may explain why it remained undiscovered in Lepeophtheirus until the work of Anstensrud (1990a). Several authors have observed a filament in a proportion of pre-adult I and adult females of Lepeophtheirus and Culigw (Lewis, 1963; Hewitt, 1971; Johnson and Albright, 1991a) although the majority assumed that only the larval stages possessed an attachment structure (Gurney, 1934; Kabata, 1981 Wootten et al., 1982; Pike, 1989; Hogans and Trudeau, 1989). Rae (1979) was first to comment that, on certain occasions, all post-chalimus stages of Lepeophtheirus salmonis are found attached by frontal filaments. The temporary filament originated and was extruded from the frontal organ, formerly named the median sucker or rugose area (Kabata, 198 1; Oldewage and van As, 1989). Anstensrud (1990a) described this structure as the filament gland since the filament originated from this area. The tugging action of the female to break the filament was similarly seen in 4th chalimus females of Salmincola californiensis (Kabata and Cousens, 1973). A series of complex and delicate behavioral patterns were executed during precopula and copula by adult male Lepeophtheirrds salmonis. Their duration and repetition suggested that they perhaps played a role in courtship. However, the passive nature and inevitable receptiveness of the female following the moult suggested these behavioral patterns were not essential for mate selection. These acts may be required to finalise spermatophore preparation and/or facilitate spermatophore extrusion. During the final ecdysis of females, adult males crawled anteriorly onto the cephalothorax of the emerging adult female. Anstensrud (1990a) found this movement in only 13% of L. pectoralis mating pairs, the rest released their hold on the female and repositioned close to th: moulting female. With males maintaining a hold on their precopula partner during the moult no ‘takeovers’ by other males were observed, in contrast to the 40% change in partner after the moult in L. pectorulis (Anstensrud, 1990a). Pairs of L. pectoralis, after detaching from the filament, remained in precopula for 2 h-6 days (Anstensrud, 199Oa), while in L. sulmonis copulation lasted 19-33 h (mean, 23.6 h) after breakage of the filament. Copulation was very intricate and delicate. The swimming legs and maxillipeds were used in to transfer, orientate and secure attachment of the spermatophores to the genital complex of the female. The spermatophores were covered in a‘cement-like’ substance as described by Anstensrud (1990a,c) for L. pectoralis. The hardening of the ‘cement-like’ substance was probably aided by the beating action of the swimming legs which increased the flow of water over the spermatophore surface. The maxillipeds were utilised effectively in holding the spermatophores firmly on the genital complex of the
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female. This may have prevented the female from expelling the spermatophores which was commonly observed after 2-43 days post-insemination (mean, 26.2 days) in L. pectoralis (Anstensrud, 1990~). Non-virgin female L. salmonis, without spermatophores, were never seen in this study and are only very rarely found in the field (Ritchie et al., in press). This suggests that the seal formed by the spermatophores and cement effectively prevents re-insemination and that female L. salmonis are monogamous. This suggestion is supported by observations that adult males are never found in precopula or copula with inseminated females and that multiple spermatophore placement was never observed. Scott (1901) and Wilson (1905) however found clusters of spermatophores on the ventral surface of several caligid species. The available evidence suggests adult male L. salmonis are polygamous. Postcopulatory males displayed ‘mate searching’ and ‘mate testing’ behaviour in search of a new mate and a range of spermatophores were present in the vasa deferentia of reproductively competent males (Ritchie et al., in press). Male L. pectoralis and Lernaeoceru brunchialis copulated with several females during their lifetime (Anstensrud, 1990~ and d). With approximately 1:l sex ratios in laboratory broods and field populations (pers. obs.), sex specific development rates (Johnson and Albright, 1991b; pers. obs.) and male polygamy, the operational sex ratio of Lepeophtheirus salmonis will be male biased. interaction between males will be Within populations of L. salmonis, competitive confined to locating a precopulatory mate. With a higher proportion of males to receptive females it becomes beneficial for a male to take up precopula with reproductively immature females thus securing a potential mate and guarding her until maturity (Grafen and Ridley, 1983). Mate guarding, which is believed to have evolved under conditions of restricted female availability and time, has been identified in the Copepoda (Grafen and Ridley, 1983), including the Cyclopoida, Harpacticoida, Poecilostomatoida and other members of the Siphonostomatoida (Boxshall, 1990). Where this situation occurs, as in L. salmonis, females that can be mated immediately are rare and as a result the rate at which males find females to mate with is low. Consequently, natural selection will favour males that take up precopula with immature females close to maturity, thereby reducing male searching time. As the proportion of guarding males increases in the population, the density of ‘acceptable’ mates decreases and males must exploit a pool of unguarded females that are further from maturity (Grafen and Ridley, 1983). This theory of sexual selection applies to L. salmonis, where adult males preferentially selected pre-adult II females and recently moulted virgin adult females. Anstensrud (1992) showed that male Lernaeocera branchialis established precopula with females of all stages but preferred chalimus 4 and virgin females. In addition, male Lepeophtheirus pectoralis were associated with, but showed no discrimination between, pre-adult I and II females but preferred newly moulted virgin females. The selection of potential mates implies that males were capable of recognising females and their developmental and reproductive state. Several studies on mating behaviour and sex recognition in the Copepoda (particularly the order Calanoida) showed that males react to chemical signals from conspecifics that increase mate seeking behaviour (Katona, 1973; Griffiths and Frost, 1976 Dunham, 1978; Uchima, 1985; Jacoby and Youngbluth, 1983). Currently however, there is little evidence on the
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presence of sex pheromones in parasitic copepods. Kabata (1958) found male Lemaeoceru brunchialis aggregated around immature females, and Anstensrud (1989, 1992) observed male L. brunchialis clustering around the filament of females and females in coitus. Anstensrud (1990b) suggested that adult male Lepeophtheirus pectorulis could discriminate between pre-adult and adult females by chemical cues. Adult male L. sulmonis increased their level of activity in the presence of pre-adult females, but no quantitative analysis of this change in activity has been made. Associated with increased activity was the display of putative ‘mate searching’ behaviour. These unique and apparently structured movements were performed in the presence of pre-adult and virgin adult females only. In addition, males clustered around precopula pairs formed between males and recently moulted virgin females, and occasionally pre-adult II females. Along with preferences by males for virgin adult females and pre-adult II females, this clustering suggested that any sexual signals released by females to broadcast their receptivity strengthen with development. Adult males were never observed in precopula with inseminated females suggesting that non-virgin females no longer produced any chemical stimuli attractive to males. A similar theory was proposed by Anstensrud (1992) for L. pectorulis. During ‘mate searching’ behaviour and clustering, males were observed raising their cephalothorax from the surface of the fish. This manoeuvre, which may be necessary to ‘sample’ or ‘test’ the water for specific chemical stimuli, was repeated more frequently by males aggregated around recently moulted females. The antero-dorsal position of most pre-adult II females may be a behavioral adaptation that supplements any chemical advertisement of receptivity and makes the process of mate location easier for the male. The larger percentage of precopulatory pairs formed on the head and dorsal 1 regions support this suggestion. Other authors have attributed this aggregation to the absence of scales and the relative thinness of the skin, which may ease feeding (Hastein and Bergsjo, 1976), and the reduced drag associated with this area (Jonsdottir et al., 1992). The ‘mate testing’ behaviour performed by adult males suggested they could not distinguish between post-chalimus stages by chemical signals alone and were attracted, equally, to all female post-chalimus stages. This observation contrasts with Anstensrud (1992) who claimed that chemical cues were initially responsible for male discrimination between pre-adult and adult females. ‘Testing’ allows males to recognise the developmental stage of the female. Recognition of the developmental stage could be achieved by examining the size of the female’s genital complex through mechanoreception or by contact chemoreception. Katona (1973) demonstrated mate selection in three species of planktonic copepod (Eurytemoru afinis, E. herdmani and Pseudodiuptomus coronutus) and suggested that contact chemoreception was probably important. In parasitic copepods, dramatic changes in the morphology, including gigantism of the genital complex and the holdfast often occur after the final moult (Kabata, 198 1; Anstensrud, 1990b). In female Lepeophtheirus sulmonis the main post-moult development, was the increase in size of the genital complex, associated with the development of the reproductive organs and deposition of eggs. A similar increase was observed in female L. pectoralis (Boxshall, 1974; Anstensrud, 1990b). It was believed that these relatively small changes were predetermined rather than induced by mating, sper-
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matophore transfer or seminal fluids inside the female (Anstensrud, 1990b). Johnson and Albright (1991a) reported folds of cuticle at the anterior end of the genital complex of newly moulted adult females and suggested this may serve as a reservoir for cuticle expansion. The apparatus employed in this study was geared for the observation of lice and relied on the passive nature of the fish. This behaviour was not typical of fish found in cage situations so some of the features of mate location and mating, along with time scales, may not be applicable to those found under cage conditions. In addition, observations that suggested sex pheromones may be involved in mating of L. salmonis were obtained from lice in this artificial environment, where water flow over the fish was irregular. Under these circumstances the responses evoked may have been distorted by the slow and artificial current of water. Stimulus concentrations may have been higher or lower than those found under real conditions. Despite these drawbacks, these observations gave a strong indication of a clear selective response by males to post-chalimus females.
Acknowledgments GR would like to thank SERC and the Scottish Salmon Growers Association Ltd. for their financial support during this study. The help and assistance provided by Marine Harvest International in collecting material for this study is gratefully acknowledged.
References Anstensrud, M., 1989. Experimental studies of the reproductive behaviour of the parasitic copepod Lemaeocera branchialis (Pennellidae). J. Mar. Biol. Ass. UK, Vol. 69, pp. 465-476. Anstensrud, M., 1990a. Moulting and mating in Lepeophtheirus pectoralis (Copepoda: Cahgidae). J. Mar. Biol. Ass. UK, Vol. 70, pp. 269-281. Anstensrud, M., 1990b. Effects of mating on female behaviour and allomettic growth in the two parasitic copepods Lemaeocera bronchialis (L., 1767) (Pennellidae) and Lepeophtheirus pectoralis (Muher, 1776) (Caligidae). Crustaceana, Vol. 59, pp. 245-258. Anstensrud, M., 199Oc. Mating strategies of two parasitic copepods Lernaeocera branchiulis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Miiller) (Caligidae) on flounder: Polygamy, sex specific age at maturity and sex ratio. J. Exp. Mar. Bid. Ecol., Vol. 136, pp. 141- 158. Anstensrud, M., 1990d. Male reproductive characteristics of two parasitic copepods, Lemaeocera branchialis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Miiller) (Caligidae). J. Crust. Biol.,Vol. 10, pp. 627-638. Anstensrud, M., 1992. Mate guarding and mate choice in two copepods, Lernaeocera branchialis (L.) (Pennellidae) and Lepeophtheirus pectoralis (Miiller) (Caligidae), parasitic on flounder. J. Crust. Biol., Vol. 12, pp. 3 l-40. Boxshall, G.A., 1974. The developmental stages of Lepeophtheirus pectoralis (Miiller, 1776) (Copepoda: Caligidae). J. Nat. Hist., Vol. 8, pp. 68 l-700. Boxshall, G.A., 1990. Precopulatory mate guarding in copepods. Bijdr. Dierk. Vol. 60, pp. 209-213. Bruno, D.W. and J. Stone, 1990. The role of saithe: Pollachius kens L., as a host for the sea lice, Lepeophtheirus salmonis Kroyer and Cal&us elongatus Nordmann. Aquaculture, Vol. 89, pp. 201-207. Dunham, P.J., 1978. Sex pheromones in Crustacea. Biol. Rev., Vol. 53, pp. 555-583. Grafen, A. and M. Ridley, 1983. A model of mate guarding. J. Theor. Biol., Vol. 102, pp. 549-567.
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G. Ritchie et al. I J. Exp. Mar. Biol. Ecol. 201 (1996) 285-298
Griffiths, A.M. and B.W. Frost, 1976. Chemical communication in the marine planktonic copepods Calanus pacifus and Pseudocalunus sp. Crustaceana, Vol. 30, pp. 1-8. Gurney, R., 1934. The development of certain parasitic Copepoda of the families Caligidae and Clavellidae. Proc. Zool. Sot. Land., Vol. 1934, pp. 177-217. Hastein, T. and T. Bergsjo. 1976. The salmon lice Lepeophtheirus salmonis as the cause of disease in farmed salmonids. Riv. Ital. Piscic. Irtiopatol., Vol. 11, pp. 3-5. Hewitt, G.C. 1964. The postchalimus development of Lepeoptheirus polyprioni Hewitt, 1963 (Copepoda: Caligidae). Trans. R. Sot. N.Z., Vol. 4, pp. 157-159. Hewitt, G.C., 1971. Two species of Cal&us (Copepoda, Caligidae) from Australian waters, with a description of some developmental stages. Pacif. Sci., Vol. 25, pp. 145-164. Hogans, W.E. and D.J. Trudeau, 1989. Preliminary studies on the biology of sea lice, Cal&us elongatus, Cal&us curtus and Lepeophtheirus salmonis (Copepoda: Caligoida) parasitic on cage-cultured salmonids in the Lower Bay of Fundy. Can. Tech. Rep. Fish. Aquat. Sci., Vol. 1715, pp. 14. Hwa, D., 1965. The life cycle of oriental fish louse. Acta Zool. Sin.,Vol. 17, pp. 48-58. Izawa, K., 1969. Life history of Culigus spinosus Yamaguti, 1939, obtained from cultured Yellow tail, Seriola quinqueradiata T. and S. (Crustacea: Caligoida). Rep. Fat. Fish. Prefect. Univ. Mie,Vol. 6, pp. 127-157. Jacoby, C.A. and M.J. Youngbluth, 1983. Mating behaviour in three species of Pseudodiaptomus (Copepoda: Calanoida). Mar. Biol., Vol. 76, pp. 77-86. Johnson, S.C. and L.J. Albright, 1991a. The developmental stages of Lepeop,ptheirussalmonis (Kroyer, 1837) (Copepoda: Caligidae). Can. J. Zool, Vol. 69, pp. 929-950. Johnson, S.C. and L.J. Albright, 1991b. Development, growth, and survival of Lepeoptheirus salmonis (Copepoda: Caligidae) under laboratory conditions. J. Mar. Biol. Ass. UK, Vol. 71, pp. 425-436. Jbnsdbttir, H., J.E. Bron, R. Wootten, and J.F. Tumbull. 1992. The histopathology associated with the pre-adult and adult stages of Lepeophtheirus salmonis on the Atlantic salmon, Salmon salar L. J. Fish Dis., Vol. 15, pp. 521-527. Kabata, Z., 1958. Lemaeocera obtusa n. sp. Its biology and its effects on the haddock. Mar. Res., Vol. 3, pp. l-26. Kabata, Z., 1981. Copepoda (Crustacea) parasitic on fishes: problems and perspectives. Adv. Parasitol., Vol. 19, pp. 1-71. Kabata, Z. and B. Cousens, 1973. Life cycle of Sulmincola californiensis (Dana 1852) (Copepoda: Lemaeopodidae). J. Fish. Res. Board Can., Vol. 30, pp. 881-903. Katona, S.K., 1973. Evidence for sex pheromones in planktonic copepods. Limnol. Oceanogr., Vol. 18, pp. 574-583. Lewis, A.G., 1963. Life history of the Caligid Copepod Lepeophtheirus dissimularus Wilson, 1905 (Crustacea: Caligoida). Pacif. Sci., Vol. 17, pp. 195-242. Oldewage, W.H. and J.G.Van As, 1989. On the sensory (?) structure between the frontal plates of Cal&us O.F. Muller, 1785 (Copepoda, Caligidae). Crustaceana, Vol. 57, pp. 72-78. Pike, A.W., 1989. Sea lice-major pathogens of farmed Atlantic salmon. Parasitol. Today, Vol. 5, pp. 29 l-297. Rae, G.H., 1979. On the trail of the Sea lice. Fish Fmr., Vol. 2, pp. 22-25. Ritchie, G., A.J. Mordue (Luntz), A.W. Pike and G.H. Rae. Morphology and ultrastructure of the reproductive system of Lepeophtheirus salmonis (Kreyer, 1837) (Copepoda: Caligidae). J. Crust. Biol., in press. Schram, T.A., 1993. Supplementary descriptions of the developmental stages of Lepeophtheirus salmonis (Kroyer, 1837) (Copepoda: Caligidae). In, Pathogens of Wild and Farmed Fish: Sea lice, edited by G.A. Boxshall and D. Defaye, Ellis Horwood, New York, NY, pp. 30-50. Scott, A., 1901. On the fish parasites, Lepeophtheirus and Lernaea. Rep. Lams. Sea-Fish. Lab., Vol. 9, pp. 62-95. Uchima, M., 1985. Copulation in the marine copepod Oithona davisae, Ferrari and Orsi. Bull. Plank. Sot. Jpn., Vol. 32, pp. 23-30. Wilson, C.B., 1905. North American parasitic copepods belonging to the family Caligidae. Part I The Caligidae. Proc. US Nat. Mus., Vol. 28, pp. 479-672. Wootten, R., J.W. Smith and E.A. Needham, 1982. Aspects of the biology of the parasitic copepods Lepeophtheirus salmonis and Caligus elongarus on farmed salmonids and their treatment. Proc. R. Sot. Edinb., Vol. 81, pp. 185-197.