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Evidence of chemical communication during the gametogenesis of holothuroids.

INTRODUCTION

Chemical communication is a widely studied topic of ecological importance in the animal kingdom. In recent years, a great deal of knowledge has been gathered from studies in insects and fish on the synthesis, physical structure, and functional properties of pheromones, as well as on how they are perceived and exert their action (Shorey 1976, Leroy 1987, Hardie 1991, Radl and Linsenmair 1991, Cork et al. 1992). Pheromones are also known to play various roles in the maturation, hormonal cycles, and mating events of mammals (Leroy 1987, Agosta 1992).

The number of unresolved questions in the field of chemical communication among marine invertebrates probably justifies the important surge of interest in this subject. A large amount of work in this field deals with predator-prey interactions (Coll and Sammarco 1988, Lawrence 1991, Legault and Himmelman 1993). Nevertheless, pheromones in invertebrates are presumed to serve in congener recognition, search for suitable colonizing area, and communication of potential threats. To date, most of the work has brought significant clues in favor of the existence of chemical mediators during many events related to reproduction in marine invertebrates. Examples include the studies of Boily-Marer (1974), Boily-Marer and L'Homme (1986) and Zeeck et al. (1988) who described the induction of the nuptial dance in polychaetes by pheromones. Pheromones were also proposed to be the major inducers of courtship, moulting, and mating in crustaceans (Atema and Engstrom 1971, Atema et al. 1979, Eisner 1980, Gleeson 1980, 1982, Conner et al. 1981, Atema and Cowan 1986, Cowan 1991).

Reports of chemical communication among echinoderms are very scarce and some remain speculative. Run et al. (1988) indicated that a sex-specific contact chemoreception could partially explain sex recognition in the sea star Archaster typicus. Ormond et al. (1973) showed that an active component released by the gonad of the sea star Acanthaster planci induced reproductive behavior and spawning in nearby mature adults. Furthermore, studies on deep-sea echinoderms imply that chemical cues play a role in gametogenic seasonality (Tyler et al. 1982, Tyler and Gage 1984) and spawning synchronization (Tyler et al. 1982, Young et al. 1992). However, the proximate mechanisms by which deep-sea animals synchronize spawning or gametogenesis to ensure successful fertilization remain unknown (Young et al. 1992). Hamel and Mercier (1995a) found that chemical communication during an aggregative behavior in the sea star Leptasterias polaris seemed to play a role in gametogenic synchronization, mostly during the late stages of gamete maturation before spawning. The paired mating of the sea star Neosmilaster georgianus was also presumed to be regulated by chemical mediators (Slattery and Bosch 1993). McEuen (1988) suggested that synchronization of spawning among sea cucumbers in the field could be enhanced by sex pheromones. Finally, Miller (1989) discussed the periodic presence of a long-lived bio-activated sex-specific substance in natural seawater during the reproductive season of sea stars, and suggested that benthic invertebrates may communicate their sexual identity and readiness to spawn by means of waterborne pheromones.

To our knowledge, the existence and implications of chemical communication among individuals in gametogenesis have not been presented for any invertebrate. Most of the attention has been directed toward the correlation of gametogenesis with environmental conditions. Photoperiod has been shown experimentally to be the main factor responsible for synchronizing gametogenic events of shallow-water echinoderms (Pearse and Eernisse 1982, Pearse and Walker 1986, Pearse et al. 1986a, b, Bay-Schmith and Pearse 1987, McClintock and Watts 1990). Increasing temperature and food supply seem to constitute the other most determinant factors (Ito et al. 1989, Chia and Walker 1991, Pearse and Cameron 1991). It is proposed that the environmental fluctuations perceived by the organisms initiate a reaction leading to somatic changes and modifications in the reproductive metabolism, possibly through gene activation or hormone synthesis (Schoenmakers 1980, Schoenmakers and Dieleman 1981, Shirai and Walker 1988, Hines et al. 1992, Walker et al. 1992, Barker and Xu 1993). It is not as easy to explain the onset and harmonious interindividual development of gametogenesis under total darkness in entire populations, whether exposed to similar or distinct local environmental conditions. It becomes especially important for freely spawning marine invertebrates that may waste a large portion of their gametes if this synchronization is not well orchestrated.

Our investigation revealed that environmental factors, coupled with various endogenous reactions, could not adequately explain the synchronous initiation and harmonious development of gamete synthesis in male and female Cucumaria frondosa. This brought up an interesting question: are chemical mediators involved in the harmonious interindividual development of gametes? We have approached this study of reproductive rhythmicity by examining the progress of gametogenesis in populations of C. frondosa, in the field and in laboratory, under various experimental conditions. This work is the first to explore the role of chemical mediators during gametogenesis of a population of marine invertebrates, while providing basic data and explaining a possible synergism with environmental factors.

METHODS

Our first goal was to investigate which factors were essential to the maintenance of synchronous gametic development among individuals in the same population. We compared the progress of gametogenesis in starved vs. fed sea cucumbers, Cucumaria frondosa, in individuals maintained under naturally varying vs. constant environmental conditions, in individuals from photic vs. aphotic zones, and under isolated and grouped conditions. We finally verified the influence of mature individuals on the gametogenic development of less developed ones.

Laboratory studies were performed at the Station Aquicole de Pointe-au-Pere affiliated with the Universite du Quebec a Rimouski, eastern Canada. The sea cucumbers were dredged locally at Sainte-Anne-des-Monts (48 [degrees] 21 [minutes] N, 68 [degrees] 47 [minutes] W), Lower St. Lawrence Estuary on precise dates, mentioned in the respective experiments. All sea cucumbers were transferred within 6 h of capture to holding tanks supplied with running seawater. Photoperiod was adjusted to simulate seasonally varying conditions of light. As C. frondosa feed mostly on phytoplankton, the running water was left unfiltered, thus providing individuals with a naturally occurring food supply. Except when otherwise mentioned, all experiments in the laboratory were conducted under these conditions and individuals were kept grouped in equal numbers of males and females. The animals were sexed by visual inspection of the sexually dimorphic gonopores. The conditions referred to as constant correspond to a temperature of 6 [degrees] C, a salinity of 28 g/L, a pH of 8, and a daylength of 10 h with a light intensity of 35 [micro]mol photons [center dot] [m.sup.-2] [center dot] [s.sup.-1]. These occur during summer in the field. Individuals maintained under these conditions were either given 4 L of phytoplankton (from a mix of species naturally found in the digestive tract of C. frondosa, in cultures of 2 x [10.sup.6] cells/mL) daily or left without food, depending on the experiment. Experiments in natural and constant conditions were always initiated after 2 or 3 wk of acclimation.

Effect of surgery on gametogenesis

For the purpose of our experiments, periodic evaluation of gametogenic stages in sea cucumbers was necessary. We chose to remove a sample of the gonad by surgery, instead of only extracting a few gametes with a syringe, in order to obtain the most representative portrait of the gonad's level of maturity. This technique allows the determination of (1) the size of the oocytes with their precise location in regard to the germinal epithelium, (2) epitheliae thickness, and (3) tubule diameter. Moreover, the stratification of the maturing gametes and their proportion in the male gonadal tubule can be determined, which is of primary importance in characterizing the level of maturity of the gonad. Experiments by Bouland and Jangoux (1988) in the sea star Asterias rubens demonstrated that this kind of gonad collection by surgery at regular intervals did not affect the health and gonadal development of individuals. To establish the long-term impact of surgery [TABULAR DATA FOR TABLE 1 OMITTED] on gametogenic development and general health of the sea cucumbers, experiments were conducted just after spawning (July 1992) and ended just before the next spawning period in May 1993. We used only individuals having spawned to insure that males and females began the experiment at the same level of gametogenic development. Two groups of 60 individuals (30 males and 30 females) were maintained in two large tanks (500 L). Gonads of the first group were collected every 3 mo by surgery and the second group was kept undisturbed. Similar environmental changes were maintained in the two tanks. At the end of this experiment all individuals were dissected and their gamete and gonad development compared using histological procedures.

Surgery and histological procedures

Histological studies began with a biopsy (0.5 cm long) performed with a scalpel near the midlength of the animal (anal side) to haphazardly collect a tissue sample ([approximately equal to] 1 cm long) close to the apical end of the gonad. To facilitate body-wall regeneration, we placed one stitch to close the aperture in accordance with the technique used by Bouland and Jangoux (1988) for A. rubens. Because gametic development in C. frondosa progresses in two classes of tubules, we always collected 2-3 small ([less than] 2.2 mm in diameter) and 2-3 large tubules ([greater than] 2.2 mm) of the [approximately equal to] 200 found in each individual. Small tubules are associated with the first season of gametic growth and large ones with the second season. For all gonads sampled, we evaluated the number of mature gametes present, their level of maturity, and the thickness of the gonadal epithelium of each tubule collected, using histological techniques (Hamel et al. 1993). Five slides (5-6 [[micro]meter]) per section of the gonadal tubules were examined. The principal stages used to classify gametogenic development for both males and females (recovery, growth, advanced growth, and maturity) are described in Table 1.

Gametogenesis in starved and fed individuals

One hundred fifty males and 150 females, measuring [approximately equal to] 300 mm from mouth to anus, were kept for 20 mo in an aquarium (500 L) with no food under the constant environmental conditions previously described. Control individuals were allowed daily supplies of phytoplankton (4 L from a preparation of 2 x [10.sup.6] cells/mL) under the same environmental conditions. Once every 3 mo, from April 1992 to November 1993, five males and five females were dissected to evaluate their gonadal indices (wet mass of gonad/wet mass of body wall) and mass of the body wall (without the aqua-pharyngeal bulb and the muscle bands). We also estimated the maturity index (MI) of the gonad, calculated separately for both sexes at each sampling period, using the formula:

MI = [1(no. of ind. in postspawning) + 2(no. of ind. in recovery) + ... + 5(no. ind. in maturity)]/no. of ind. in samples.

The postspawning stage is characterized by constricted tubules and the absence of mature gametes and precursor cells. The four other stages are described in detail in Table 1.

Gametogenesis under natural and constant environmental conditions

This experiment was initiated with individuals collected just after spawning in 1992 (mid-June) and immediately transferred to the laboratory. We followed gametogenesis on a monthly basis until July 1993 in individuals (n = 150) kept in large tanks under constant environmental conditions, and individuals kept under naturally fluctuating environmental conditions (n = 150).

Secondly, we collected individuals in early December 1992 (3 wk before winter solstice) and February 1993 (6 wk after it). In each case, 100 sea cucumbers were collected and placed in two tanks (500 L) maintained under constant environmental conditions. The gametogenic stage of all individuals was examined every month from their time of collection until July 1993.

Comparison of gametogenesis and gonadal index between populations found in the photic and in the aphotic zones

We studied the gametogenic development and gonadal index in two populations of sea cucumbers, one found in the photic zone (10 m depth, a depth at which light penetrates all year long) and the other in the aphotic zone (110 m, a depth at which no light penetrates all year long; Babin et al. 1993). Fifteen males and 15 females were collected by dredging every month from April 1992 to November 1993. We selected individuals ranging between 250 and 325 mm in length from mouth to anus as they are the most suitable in establishing gonadal indices.

Gametogenesis in isolated and grouped animals

One hundred fifty C. frondosa were placed individually in 5-L tanks, separately supplied by running seawater, while another group of 150 sea cucumbers was separated in two subgroups of 75 individuals in two large tanks (900 L). The sex ratio was always close to 1:1 (the natural ratio observed in the field; Hamel and Mercier, in press). The gametogenesis of each individual was monitored by periodic histological examination of the gonads (every 2-3 mo, from April 1992 to June 1993). At the end of this monitoring, just prior to spawning in June 1993, whole gonads of all individuals were dissected to establish how many individuals, males and females, were ready to spawn.

Influence of interindividual communication on gametogenesis

In this experiment, we used two connected tanks with a unidirectional water flow. The sea cucumber to be stimulated was placed downstream of the inducing one. To make sure that no contact was possible between the two individuals, a glass wall separated the two tanks. Control experiments were performed with no exposure to upstream individuals. Each experiment was replicated 4 times simultaneously. The individuals used for these experiments were collected in October 1992, well before the first increase of day length, and maintained under constant environmental conditions to verify if day length increase was a necessary factor to induce gametogenesis. Hundreds of sea cucumbers were prevented from maturing by maintaining them under winter conditions (minimal photoperiod and near freezing temperature). Others were kept in-phase and out-of-phase of photoperiod (+ 1, 2, 3, 4, 5, and 6 mo) in running seawater, using the method described by Pearse and Eernisse (1982), Pearse and Walker (1986), Pearse et al. (1986a, b). This insured a regular supply of individuals in all gametogenic stages through the year. The actual stage of an individual was always assessed histologically prior to including it in a given experiment. All experiments lasted at least 8 wk (time enough to observe distinct gonadal change), collecting a gonad sample of large and small tubules in each individual once a week. The gametogenic stage was evaluated by histology using the techniques described earlier. For the females, triplicate measures of the number of mature oocytes spread on a hemacytometer, for each gonadal tubule collected in every individual, were made. In males, we estimated the proportion of mature spermatozoa among all germinal cells present (spermatogonia, spermatocytes, and spermatids) in a 2 cm long section of gonadal tubule, again in triplicate. To insure a constant level of exposure from individuals placed upstream, they were replaced each week with new ones for which the level of gametic development was previously carefully evaluated. Gametogenesis of the tubules in recovery stage was followed in the small tubules and that of tubules in advanced-growth stage was observed in large gonadal tubules.

A second experiment was conducted with individuals collected in February 1993, well after the initiation of day length increase (when gametogenesis is naturally initiated). The experiment was conducted using only males and females in recovery stage maintained under constant environmental conditions. Their stimulation followed exactly the previously described protocol, except only small gonadal tubules were collected. The initial stage of exposed sea cucumbers was chosen to maximize the clarity of their physiological response during the experiments.

RESULTS

Effect of surgery on gametogenesis

After 1 yr of experimentation, histological observations of the gonads in male and female sea cucumbers indicated surgery had no significant influence on gametogenesis. The proportions of individuals ready to spawn, 78 and 81% in experimental and control groups, respectively, were not significantly different (Kruskal-Wallis, P = 0.292). The ratio of mature to immature gametes in the two groups also did not differ significantly (Kruskal-Wallis, P = 0.432). The gonadal tubules did not show any delay in their development due to surgery and were as mature as the undissected tubules in the same individual for a given period. The opening on the external body wall closed after [less than] 1 wk and total recovery occurred in 97% of the individuals manipulated. Individuals resumed their normal posture on the substrate and their feeding and respiratory behaviors within 1 d after the surgery. On average, [less than or equal to] 3 wk were necessary for a sea cucumber to completely heal the external lesion.

Gametogenesis in starved and fed individuals

The mass of the body wall in fed individuals (male or female) remained stable and showed no significant variation throughout the 20 mo of the experiment (Kruskal-Wallis, P = 0.194). In contrast, starved individuals showed a progressive decrease of their body-wall mass [ILLUSTRATION FOR FIGURE 1 OMITTED]. Starved individuals with an initial mass of [approximately equal to] 240 g weighed between 47 and 120 g at the end of the experiment. The decrease in males and in females followed roughly the same pattern [ILLUSTRATION FOR FIGURE 1 OMITTED]. The fastest decrease was during the warmest months in 1992 and in 1993, April to August. The slowest decrease was during the coldest periods, from September to March, in both years. The gonadal index in both experimental conditions followed the same pattern despite the mass depletion in the starved individuals. In both groups for 1992 and 1993, the gonadal index attained a peak in May and dropped rapidly in June, suggesting the occurrence of spawning [ILLUSTRATION FOR FIGURE 1 OMITTED]. The first increase and the level attained by the gonadal index in January-February 1992 were similar in both fed and starved individuals. The only major difference between these two conditions appeared during the 2nd yr of the experiment, from January to June 1993. During that period, the gonadal index in the starved group was significantly higher than that in fed individuals for both males (Kruskal-Wallis, P [less than] 0.01) and females (Kruskal-Wallis, P [less than] 0.01). The maturity index of both sexes in fed and starved individuals followed the same seasonal pattern [ILLUSTRATION FOR FIGURE 1 OMITTED]: it attained a maximum in May of both years and dropped abruptly in June, suggesting that individuals were spawning at that time. This period was followed by a steady state, during which the maturity index remained close to its minimal value. A significant increase of the maturity index occurred in early 1993, attaining a maximum in May when the largest proportion of individuals with mature gonads was noted (Kruskal-Wallis, P [less than] 0.01) [ILLUSTRATION FOR FIGURE 1 OMITTED].

Gametogenesis under natural and constant environmental conditions

Fig. 2 summarizes the gametogenic development of males and females kept either under naturally varying or constant environmental conditions from late June 1992 to July 1993. It considers the activity in small and large gonadal tubules separately. Since a gonadal tubule takes [approximately equal to] 2 yr of growth to reach maturity, early gametogenic stages (postspawning, recovery, and growth stages) predominate in the small gonadal tubules and advanced stages (advanced growth and maturity stages) in the large gonadal tubules.

Male and female sea cucumbers showed close similarity in their gametogenic development when compared in the same tubule size and under the same conditions, either constant or natural [ILLUSTRATION FOR FIGURE 2 OMITTED]. The comparison between the gametogenic stages in the large tubules of sea cucumbers reared in constant vs. natural conditions showed the gradual appearance of an important disparity. While the population under natural conditions passed from 25% of individuals in advanced stages (advanced growth and maturity) in November 1992, to 100% in May 1993, the population under constant conditions never showed [greater than]10% of individuals reaching advanced stages during the same period. Furthermore, the group under natural conditions first presented individuals at maturity in November 1992 (females) and January 1993 (males) while in the group under constant conditions, mature individuals were not detected until March (males) or May (females) 1993 and only in [less than]10% of the population.

An almost identical pattern was observed in the less advanced stages of the small tubules. The majority of gonadal tubules of individuals maintained in constant conditions remained in recovery until the end of the experiment. Less than 20% of the gonadal tubules reached the growth stage. In the sea cucumbers under natural conditions, the gametic growth stage appeared sooner ([approximately equal to]2-3 mo) and involved a growing proportion of individuals, from 20% in November 1992 to [greater than]85% in May 1993. During that same period, the advanced-growth stage was often detected under natural conditions, while that stage was observed only in two sampling dates (April and May 1993) in males (never in females) kept under constant conditions and for [less than] 5% of individuals. Postspawning and recovery stages appeared only in individuals reared under natural conditions, suggesting that spawning occurred in that group but not among individuals kept under constant conditions. This was corroborated by the virtual absence of nutritive phagocytes in the small tubules of individuals maintained under constant conditions. Sea cucumbers kept in natural conditions had small tubules completely filled with nutritive phagocytes in March 1993.

The pattern of gametic development was also greatly influenced by time of collection [ILLUSTRATION FOR FIGURE 3 OMITTED]. Among the individuals collected in February 1993, after the first increase of day length in the field, and maintained subsequently under constant environmental conditions, gametogenesis followed the normal pattern previously described in large and in small tubules under natural conditions [ILLUSTRATION FOR FIGURE 2 OMITTED]. Individuals with tubules in growth stage were progressively replaced by individuals with tubules in advanced-growth stage and eventually by individuals with mature gonads, followed by a normal spawning (June 1993) of the majority of sea cucumbers collected. However, the individuals collected in early December 1992, before the day length increase in the field, did not progress similarly [ILLUSTRATION FOR FIGURE 3 OMITTED]. No more than [approximately equal to] 10% of those individuals attained the advanced-growth and maturity stages in large tubules, and the majority of individuals found during the study remained in gametic recovery and growth stages. No spawning was recorded from histological evidence among those sea cucumbers. These results are similar to those with individuals collected in June 1992 (just after spawning) and maintained under constant environmental conditions [ILLUSTRATION FOR FIGURE 4 OMITTED].

Comparison of gametogenesis and gonadal index between populations in the photic and aphotic zones

The mean gonadal index of males was always higher than that of females [ILLUSTRATION FOR FIGURE 4 OMITTED], but the two sexes showed parallel seasonal cycles throughout the study, in populations collected at 10 and 110 m depths. Although the gonadal index at 10 m remained slightly lower than that observed at 110 m, seasonal pattern was roughly similar at both depths. Spawning was suggested at both depths during the same sampling interval, between May and June in 1992 and in 1993. For each sex, at both depths, spawning was followed by a sharp decline of the gonadal index. Moreover, the first significant increase of the gonadal index occurred during February-March 1993 (Kruskal-Wallis, P [less than] 0.01). After that, the gonadal index at both depths continued to increase and attained a peak in early June 1993, before spawning [ILLUSTRATION FOR FIGURE 4 OMITTED].

Gametogenesis in isolated and grouped sea cucumbers

Figs. 5 and 6 summarize the gametogenic change of males and females kept grouped or isolated under natural environmental conditions from April 1992 to June 1993. Male and female gonadal tubules present similar gametogenic patterns [ILLUSTRATION FOR FIGURE 5 OMITTED]. At the beginning of the experiment (April 1992), all individuals showed a comparable gametic development in both conditions, with the small gonadal tubules in the growth stage (between 80 and 90%) and the large gonadal tubules mainly in maturity stage (between 78 and 85%) and filled with gametes. The release of gametes by [greater than]80% of males and females occurred normally and was particularly visible in the small tubules of the June 1992 samples. The subsequent progressive appearance of nutritive phagocytes in the small tubules, from June to February (female) or April (male), was the main gametic phenomenon observed in those tubules [ILLUSTRATION FOR FIGURE 5 OMITTED]. In the large gonadal tubules, the growth stage remained the most abundant stage present until December 1992. Until then, no substantial departure in the gonad development was recorded between grouped and isolated individuals.

However, from February 1993, the individuals of the two experimental conditions began to show distinct gametic development in both classes of gonadal tubules. After comparing the synchrony between individuals, namely the number of gametic stages observed in all individuals at a definite time, the isolated sea cucumbers had a greater heterogeneity. This asynchrony increased to a peak prior to the next spawning period (April 1993) when the grouped population was mainly represented ([greater than]80%) by individuals with mature gonads and ready to spawn [ILLUSTRATION FOR FIGURES 5 AND 6 OMITTED]. In contrast only 25-28% of the isolated individuals were ready to spawn with 33-45% of the other individuals found in advanced-growth stage and 26-45% in the gametic growth stage. Moreover, histological evidence of gamete release was found in all grouped individuals (coinciding with natural spawning in the field). Only [approximately equal to]10% of the individuals in isolated conditions spawned while a majority remained in recovery or growth and a few in advanced-growth stages [ILLUSTRATION FOR FIGURES 5 AND 6 OMITTED]. The number of individuals ready to spawn in both experimental conditions at the beginning of the study was roughly similar for both sexes (78-96%) and dropped abruptly during spawning in 1992. Grouped individuals showed an increased level of maturity, especially from February and March 1993, whereas isolated individuals did not mature [ILLUSTRATION FOR FIGURE 6 OMITTED]. Moreover, when the grouped individuals were fully mature in May 1993, only [approximately equal to]10% of isolated individuals were mature in both sexes [ILLUSTRATION FOR FIGURE 6 OMITTED]. This very low level of maturity in isolated individuals was observed throughout the study. The only change recorded in isolated individuals was the clear increasing gametogenic asynchrony between individuals [ILLUSTRATION FOR FIGURE 5 OMITTED].

Influence of interindividual communication on gametogenesis

Experiments alternatively exposing males and females, either in recovery or advanced-growth gametic development, to more mature sea cucumbers showed that they were markedly influenced in their maturation processes. The response was sex specific, as an individual of the opposite sex induced no detectable effect [ILLUSTRATION FOR FIGURES 7 AND 8 OMITTED].

The small tubules of female Cucumaria frondosa in recovery stage, collected in October (before the day length increase), did not show any significant gametogenic change during the experiment when exposed to a female also in recovery (Mann-Whitney rank sum test, P [greater than] 0.05; [ILLUSTRATION FOR FIGURE 7 OMITTED]). The same absence of reaction was obtained when recovering females were exposed to immature individuals or to no individual at all (control). However when exposed to a female with gonadal tubules in growth, in advanced-growth or maturity stages, a significant increase of gametogenic activity was recorded in the small tubules (Mann-Whitney rank sum test, P [less than] 0.01). The gonad of the four females with tubules in recovery went on to the growth stage after 8 wk with a slight increase in the number of mature oocytes ([approximately equal to]0.6 oocytes/[cm.sup.2] of tubule section). When exposed to a female with gonadal tubules in advanced-growth stage, the gametogenic development was more rapid, attaining the growth stage in [approximately equal to]7 wk. Moreover, during exposure to individuals with gonadal tubules at maturity, the effect became visible after 4 wk, and the maximum stage attained was the advanced-growth (in the small tubules) during the height week, with [approximately equal to]1 mature oocyte/[cm.sup.2] of tubules recorded. The development of large gonadal tubules onto the advanced-growth stage was observed only after exposure to a female with mature gonadal tubules [ILLUSTRATION FOR FIGURE 7 OMITTED]. In that case, the four females tested passed from the advanced-growth stage to the mature stage in 5 wk. The number of mature oocytes increased abruptly, reaching a peak of [approximately equal to]8 oocytes/[cm.sup.2] of tubules after 5 wk, which represents a value [approximately equal to]800% higher than at the beginning of the experiment. Females exposed to control conditions and all other conditions remained practically unchanged throughout the experiment.

In the case of a male with small tubules in recovery stage, also collected in October, no significant gametogenic development of the gonadal tubules was noted when individuals were exposed to individuals with gonadal tubules in recovery stage, or under control conditions (Mann-Whitney rank sum test, P [greater than] 0.05) [ILLUSTRATION FOR FIGURE 8 OMITTED]. However, after 6 wk of exposure to a male in gametic growth stage, the small tubules, initially in recovery, reached the growth stage. At that time the gonadal tubules contained [approximately equal to]20% spermatozoa. When exposed to a male with gonadal tubules in advanced-growth stage the same effect was first observed 4 wk after the beginning of the experiment. Finally, when individuals were tested with males having mature gonadal tubules, the advanced-growth stage was reached within 4 wk. In that case, the proportion of spermatozoa was [approximately equal to]40% after 5 wk, which is [approximately equal to]50% higher than at the beginning of the experiment. Male sea cucumbers with large tubules initially in advanced-growth stage showed no significant gametogenic activity under all experimental and control conditions (Mann-Whitney rank sum test, P [greater than] 0.05) except when exposed to male individuals with mature gonadal tubules (Mann-Whitney rank sum test, P [less than] 0.01). Under that condition the downstream individual reacted after 6 wk of experimentation, passing from gonadal tubules in advanced-growth to maturity stage. In the same interval, the proportion of mature spermatozoa in the gonadal tubules increased by 38-45%.

Females with gonadal tubules in recovery stage collected in February 1993 (after the first increase of photoperiod) reached the gametic growth stage under all conditions, including control [ILLUSTRATION FOR FIGURE 9 OMITTED]. Nevertheless, individuals with gonadal tubules in recovery stage exposed to individuals with more mature gonadal tubules progressed more rapidly than under control conditions (Mann-Whitney rank sum test, P [less than] 0.01). During exposure to individuals with gonadal tubules in growth stage, gamete synthesis increased significantly (Mann-Whitney rank sum test, P [less than] 0.01), passing onto the growth stage 2 wk before individuals kept under control conditions. This reaction became even more evident with the increasing difference in gametogenic development among individuals. When exposed to individuals with gonadal tubules in advanced-growth stage, the tubule development attained the growth stage 1 wk sooner than when submitted to individuals with tubules in growth stage. Finally, females in gametic recovery exposed to females with gonadal tubules at maturity mostly attained the growth stage and a few reached the advanced-growth stage [ILLUSTRATION FOR FIGURE 9 OMITTED] with [approximately equal to]3-4 mature oocytes/[cm.sup.2] of tubules. The same experiment performed with males in recovery collected after the first increase of day length showed the same patterns: a gametogenic development occurred under all conditions, including controls, but exposure to more mature individuals of the same sex gave the best results [ILLUSTRATION FOR FIGURE 10 OMITTED]. No significant difference in gametogenesis was observed when males or females were exposed to immature individuals (Mann-Whitney rank sum test, P [greater than] 0.05).

DISCUSSION

Field and laboratory investigations demonstrated that gametogenesis in Cucumaria frondosa was influenced by several physical and chemical factors, acting either in synergism, successively or independently of one another. As expected from reports of other marine invertebrates (Pearse and Eernisse 1982, Pearse and Walker 1986, Pearse et al. 1986a, b, Bay-Schmith and Pearse 1987, McClintock and Watts 1990), photoperiod appeared necessary to initiate the gametogenic cycle in C. frondosa, as only individuals collected after the first increase of day length were able to complete their development when subsequently kept under constant conditions. In contrast, the absence of food over a 20-mo period had no significant effect on gametogenic development, even though it resulted in a major decrease in body-wall mass. Interindividual synchrony was obtained only among sea cucumbers allowed some contact with one another, either directly or through the water medium. Furthermore, the influence of more mature individuals of the same sex induced gametic development in less developed sea cucumbers, even when they had not previously been exposed to an increase in day length. All these results help to explain the synchrony in gametogenic cycle of deep-water and shallow-water populations of C. frondosa, even though animals in deep water cannot directly perceive the initial photoperiod cue. Inter-individual communication could play a key role, especially since C. frondosa live in close proximity to one another (5-15 individuals/[m.sup.2]) and often are continuously distributed over a depth range. Waterborne chemicals could be the mode of such communication.

Effect of starvation on gametogenesis

Even when deprived of food over 20 mo, sea cucumbers of both sexes showed an annual reproductive cycle similar to fed individuals. However, we observed a net decrease in body-wall mass of males and females, beginning soon after the experiment was initiated. Gonadal reduction probably was smaller than the corresponding decrease in body-wall mass, so that the gametic development, as revealed by gonadal and maturity indices, was preserved up to the normal spawning event in June 1993. These results suggest that the gametic cycle was maintained despite starvation. Conversely, Bishop and Watts (1994) demonstrated that sea urchins (Lytechinus variegatus) deprived of food showed reduced and eventually no gametic synthesis. Different body-wall types or body wall/total mass proportions may explain these different results.

In fed C. frondosa, the body wall represented [greater than]50% of the total wet mass of the animal. Prim et al. (1976) and Feral and Doumenc (1982) indicated that the body wall has the greatest potential for nutrient storage in holothurians and Ong Che (1990) indicated that the reserves may be used during starvation. Accordingly, Lawrence and Lane (1982) reported that echinoderms have the capacity to reabsorb body-wall tissues and can use the energy stored to support a starvation period, with an eventual decrease in size. Contrary to our findings, Bouland and Jangoux (1988) found that gonads initiating gametogenesis could regress in starving A. rubens. However, they also found that the course of gonad development was similar in both fed and starved individuals when they had already initiated gametogenesis, which is what we found also. Similar results were obtained in sea stars by Lawrence (1973) with Luidia clathrata and by Jangoux and Van Impe (1977) with A. rubens. Andrew (1986) also demonstrated that food availability did not play a major role in the timing of the gametogenic cycle in the sea urchin Evechinus chloroticus. While there is evidence for the influence of food availability on tubule growth or gonad development in many species of echinoderms (Pearse 1965, Crump 1971, Sastry and Blake 1971, Gimazane 1972, Bayne 1975, Walker 1982, Lawrence 1985, Hamel et al. 1993), our results suggest that food abundance did not affect the gametogenic cycle of C. frondosa. A longer starvation period could certainly play a major role in the total amount of gonadal tissues, gametes synthetized, and reserves stored in each gamete.

Effect of constant environmental conditions on gametogenesis

Our results strongly show that photoperiod has a major impact on the initiation of gametogenesis, but a limited one on gametogenic regulation after its initiation, suggesting the possible existence of endogenous rhythms under a photoperiodic control. C. frondosa collected just before the first increase of photoperiod (December 1992) and subsequently kept under constant conditions did not show a similar gametogenic development to those collected in February 1993, just after the first increase of the photoperiod and also maintained under constant conditions. Individuals collected in February demonstrated a gametogenic cycle similar to individuals submitted to natural environmental conditions. These results are consistent with the observations made by Bouland and Jangoux (1988) on A. rubens. Pearse et al. (1986a) reported that the circannual rhythm in P. ochraceus remained unmodified when tested under different day length and temperature conditions. This is also the case in C. frondosa, but only for individuals collected after the winter solstice.

Temperature and salinity seem to have an even lesser effect. Even though those factors were constant throughout the laboratory study, gametogenesis in the February group remained comparable to that of natural populations in the field.

Gametogenesis in photic and aphotic natural habitats

Although we have demonstrated the importance of photoperiod, this factor cannot prevail in populations living in deep water. Nonetheless, sea cucumbers at aphotic depths have a gametic development very comparable to that of individuals collected from the photic zone. What could be the coordinating factors? In the period corresponding to the time of collection in the St. Lawrence Estuary, the temperature below 90 m is relatively stable between 0 [degrees] and 2 [degrees] C (Chasse 1994), primary production is virtually absent (M. Gosselin, personal communication), salinity is constant (Chasse 1994), and upwelling conditions observed during spring and fall are nonexistent (El-Sabh and Silverberg 1990). Fluctuations of temperature, salinity, and food supply only appear in spring, well after the initiation of gametogenesis. The strong synchrony in gametogenic cycles between shallow- and deep-water populations of C. frondosa is intriguing and again suggests that photoperiod is not the only causal factor.

Ferrand et al. (1988) found a restricted seasonal reproductive cycle over the whole bathymetric range of 60-1000 m in the spantagonid Brissopsis lyrifera that showed a close similarity with our results. The findings of Tyler et al. (1982) of a seasonal periodicity in the physical environment and in sediment flux in deep seawater, possibly influencing reproductive seasonality of echinoderms, does not seem applicable to C. frondosa. Phytoplankton downfall during spring and early summer could enhance gametic production, as observed in the shallow-water population of C. frondosa, but it does not occur in early winter when primary productivity is virtually nil. Supplementary observations on C. frondosa indicated that individuals found in shallow-water caves followed a similar gametogenic pattern to those exposed to light at the same depth.

Effect of grouped conditions

Experiments comparing the gametic development of isolated and grouped individuals suggest the importance of an interindividual communication for gametogenic synchrony. Gametogenesis did not progress synchronously among individuals kept separate under natural environmental conditions. This contrasts with individuals maintained in groups that attained gametic maturity simultaneously and were also in synchrony with the natural populations. These results suggest that isolated individuals exposed to the same environmental factors may react differently from one another. This also suggests that some individuals may be more sensitive to environmental fluctuations, whether because they are better located to perceive the stimulus or more sensitive than other individuals in the population. These observations indicate that interindividual communication acts in the synchrony and fine-tuning of gametogenesis within an entire population of C. frondosa.

Our results differ from those describing an endogenous temporal program in the reproductive activity of various marine invertebrates (Olive and Garwood 1983, Pearse et al. 1986a, Fong and Pearse 1992). Those studies demonstrated, for instance, that individuals maintained under constant temperature and day length became sexually mature roughly simultaneously with individuals in the field. However, this type of periodicity was not apparent in C. frondosa.

Influence of interindividual communication on gametogenesis

Contrary to numerous species exhibiting aggregative behavior, in which chemical substances were suspected to play a role in the reproductive behavior (Run et al. 1988, Young et al. 1992, Slattery and Bosch 1993, Hamel and Mercier 1995a), C. frondosa does not show any precise gregarious behavior, aside from fall migrations of individuals reaching the size (60-130 mm) they attain at sexual maturity (Hamel and Mercier, in press). Pheromones may explain in part the epidemic and synchronous spawning observed in many populations of marine invertebrates such as echinoderms (Kanatani and Shirai 1968, Ormond et al. 1973, Komatsu 1983, Run et al. 1988, Young et al. 1992, Starr et al. 1993). However, only a few studies described or proposed the involvement of a chemical mediator during gametogenesis, and none concerned a marine species lacking aggregative behavior.

Message transmission through the water medium is inferred by the experiments allowing water circulation between sea cucumbers of different gametic development. The level of gametic activity progressively reached by the less mature downstream individual considerably increased with increased maturity level of upstream individuals. In other words, the more advanced the upstream individuals, the stronger the stimulation on the less developed downstream individuals. Interestingly, both males and females were not affected or stimulated when tested with the opposite sex. Our results demonstrate that direct contact between individuals was not a prerequisite for transmission of the message. In contrast, no effect on gametogenesis was observed when less developed individuals were placed upstream of a more developed individual. Thus, it appears that a compound develops when individuals reach maturity and is not present in immature individuals. These results may explain how some individuals, although not exposed to variations in photoperiod, initiate gametogenesis roughly in synchrony with individuals exposed to normal light regimes. It could also explain in part the simultaneous initiation of gametogenesis observed during early January 1993 in sea cucumbers from shallow and deep water. The chemical messenger may be carried by the water currents over some distance and stimulate other sea cucumbers located in deeper water. On the North shore of the St. Lawrence Estuary, where the study was performed, sea cucumbers mainly live on a rocky drop-off and the distance between 10 and 110 m is reduced by the steep slope (45 [degrees] - 80 [degrees]). Further, C. frondosa are found in densities of 5-15 individuals/[m.sup.2] (Hamel and Mercier, in press) in areas where the current varies from 0 to 10 m/s (Hamel and Mercier 1995b).

In many species, like Leptasterias polaris (Hamel and Mercier 1995a) or deep-sea echinoderms (Young et al. 1992), the aggregation observed before spawning may be sufficient to fully synchronize the gamete synthesis and permit the synchronous spawning of closely located individuals. However, others like C. frondosa are capable of maintaining reproductive synchrony by dispersing waterborne chemical cues.

ACKNOWLEDGMENTS

We are indebted to M. Dion (Les Entreprises Ondine Inc.) and G. Dugas for their assistance during the collection of sea cucumbers. Drs. J. B. McClintock (University of Alabama at Birmingham), W. Fairchild (Fisheries and Oceans Canada, ESAD), B. Marinier (UQAR) and an anonymous reviewer provided valuable comments on earlier drafts of the manuscript. We thank Dr C. W. Walker (University of New Hampshire) for his encouragement, interest, and comments on this research project. The first author was supported by an FCAR scholarship, and the research was supported by the personal funds of J.-F. Hamel.

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