Morphological identification of larval king scallops, Pecten maximus (L.) from natural plankton samples.

ABSTRACT For commercial development of a European scallop culture industry based on natural collection of seed, a method of predicting time and location of peak spatfalls is essential. Planktonic identification of scallop larvae using light microscopy, which would allow development of such a prediction technique, has been deemed impossible. Researchers have instead focused on the use of scanning electron microscopy of the hinge structure or development of biotechnological methods to resolve the difficult identification in plankton samples. This study presents details of morphological characteristics, excluding ultrastructural traits of the hinge, that have been used to identify king scallops, Pecten maximus, larvae during their planktonic phase in Mulroy Bay, Ireland over the last 26 years. The most useful features for identification of king scallop larvae in natural plankton samples were the pointed anterior end, the indistinct umbo, the pale color and the length--height relationship. These characteristics for identification of king scallop larvae in natural plankton samples have led to development of a technique for prediction of time and location of scallop spatfall and commercial collection of scallop spat at intensities exceeding 3000 spat per collector bag.

KEY WORDS: Pecten maximus, scallop larvae, morphological identification,

INTRODUCTION

Development and expansion of scallop culture in Japan from the mid 1960s onward with production surpassing 100,000 t by 1975, led to exploratory investigations to examine the feasibility of establishing scallop culture in European waters. Preliminary research trials using spat collector bags in Scotland (McKay 1976, Ventilla 1977a, 1977b, Slater 1978, 1979a, 1980), in England (Pickett 1977, Brand et al. 1980), in Ireland (Minchin 1980, Burnell 1983, Wilson 1987), in France (Buestel et al. 1976) and in Spain (Roman et al. 1985) demonstrated that periods of peak settlement occurred for the king scallop, Pecten maximus, the queen scallop, Aquipecten opercularis and the variegated scallop, Mimachlamys varia. Wide variations in yield of scallop spat from less than 10 spat per collector to in excess of 1000 spat per collector were recorded between sites. Significant temporal variation in spat settlement intensity occurred within any 1 year. Comparison of data collected over several years demonstrated that within a single site, interannual variations of up to 6 weeks could occur in the time of the peak collection.

For commercial development of a European scallop culture industry based on natural spat collection, it became evident that a method of predicting peak spatfall time and location was essential. Comparison of the fledgling European industry with commercial harvests attained in the Far East revealed that in Japan the most effective time for placing collector bags in the sea was assessed using a combination of gonad maturity, plankton analysis and environmental parameters such as water temperature and turbidity (Motada 1977).

Since the mid 1970s determination of spawning patterns of scallop species in European waters has been the subject of considerable research. Unfortunately many of these studies have been of a short-term duration and have focused on gonad histology or biochemistry rather than the relationship between time of spawning and time of spat collection. A review of spawning data between 1980 to 2003 from Mulroy Bay in County Donegal, Ireland demonstrated that spawning times were not a reliable indicator for the time of spat settlement (Slater 2003). In some years, despite large spawnings, larvae either failed to develop in significant abundance or exhibited heavy mortalities during the later stages of larval growth with the subsequent failure to yield a spatfall. For example, in 1993 regular weekly gonad analysis identified five separate spawnings of king scallops in Mulroy Bay during the period April to August inclusive, yet only one of these spawnings produced significant numbers of larvae (Slater 2005a). In summary, while regular gonadal monitoring was useful as a tool for identification of scallop spawning time, it provided no indication that larval development, or more importantly, settlement and metamorphosis into spat would occur.

Several different approaches have been developed to address identification of bivalve larvae in plankton including application of morphological, immunological and DNA-based techniques (Garland et al. 2002).

Prior to the 1960s, identification was based on microscopic observation of larval and postlarval specimens collected from the wild and their sequential arrangement in terms of shape and size, with a view to preparing a record in which the largest specimen could be positively identified. Using this approach descriptions of larvae of several species were prepared although identification always retained an element of doubt because bivalve larvae of guaranteed origin were not available for comparative examination (Stafford 1912, Jorgensen 1946, Sullivan 1948, Rees 1950).

With development of hatchery culture techniques for bivalve larvae (Loosanoff & Davis 1963) it became possible to rear larvae from known parent stock. Photomicrographs and descriptions of larvae of the bay scallop, Aquipecten irradians and a range of other bivalve species produced under hatchery conditions were published (Loosanoff et al. 1966, Chanley & Andrews 1971). In subsequent years photomicrographs and descriptions of hatcheryreared larvae of the king scallop over a range of larval sizes were produced (Gruffydd & Beaumont 1972, Le Pennec 1974, 1978, Sasaki 1979, Slater 1979b). Culliney (1974) and Dix and Sjardin (1975) provided descriptions and photomicrographs of larvae of the giant sea scallop, Placopecten magellanicus (Gmelin) and the commercial scallop from Tasmania, Pecten meridionalis (Tate) respectively. Identification of scallop larvae in the plankton by comparison with photomicrographs of larvae of known origin became theoretically possible. It was predicted that if natural spat collection as a source of scallop seed is to become practiced in the future, larval identification would be essential to provide predictions of spatfall time by tracing larval abundance and development in plankton samples (Dix & Sjardin 1975).

Despite the availability of descriptions and photomicrographs of the larvae of king and other scallop species since the 1970s, limited research has been published on identification of scallop larvae or other bivalve larvae in natural plankton samples. The major constraint to morphological identification of scallop larvae can easily be appreciated by comparison with photomicrographs from a few other bivalve species and is attributed to the possession of few characteristics that can be quantitatively defined, this being particularly true during the period of the straight hinge. Nevertheless techniques have been developed in Japan for identification of larvae of the scallop, Patinopecten yessoensis. These have allowed monitoring of larval abundance, growth and distribution and hence prediction of time and location of peak scallop spatfall in Mutsu Bay (Kanno 1970, Ito 1977). However Ventilla (1980) reported that at this site, a major center for scallop aquaculture production, identification was feasible only because scallop larvae predominated over other bivalve larvae to such an extent that the presence of other larvae did not interfere with counting estimates or identification. Difficulties associated with morphological identification of bivalve larvae are not confined to pectinid species. In research on abundance and distribution of larvae of the mussel, Mytilus galloprovincialis in the Ria de Vigo in NW Spain, CaceresMartinez (1998) considered taxonomic identification of veligers or D-shaped larvae to genus or species level to be practically impossible, whereas mussel pediveligers (0.225-0.470 mm) and postlarvae (>0.470 mm) were easily identified in routine plankton surveys. In a study of the numbers of larvae and primary plantigrades of the mussel, Mytilus edulis, in the western Dutch Wadden Sea, de Vooys (1999) failed to identify pelagic larvae of M. edulis.

Le Pennec (1978), Lutz and Hidu (1979), Lutz et at. (1982), Fuller and Lutz (1989) researchers with considerable experience of bivalve larvae suggested during the 1980s that identification based on morphology, particularly at the early (straight-hinge) developmental stages, without a detailed examination of the structure of the hinge, was impossible. Their research demonstrated that ultrastructural morphological traits of the larval hinge could be used to provide accurate identification. Using hinge structure of larvae of the sea scallop for identification, Tremblay et at. (1988, 1990, 1994) investigated distribution of larvae in the Bay of Fundy, their diurnal vertical migration in the water column and their distribution in the waters of Georges Bank. However use of this technique, involving scanning electron microscopy, for rapid identification of larvae in plankton samples with a view to prediction of time and location of peak scallop spatfall was not considered feasible.

Recent advances in marine biotechnology have led to development of methods for the identification of bivalves based on immunological recognition (Demers et al. 1993, Abalde et al. 2003) and DNA-based recognition (Bell et al. 1998, Hare et al. 2000, Frischer et al. 2000, 2003, Lopez-Pinon et al. 2002, Bendezu et al. 2005). An immunological technique capable of discriminating king scallop larvae from all other bivalve larvae in plankton samples from the Bay of Brest has been developed (Paugham et al. 2000). The technique has been used for monitoring Pecten larval occurrence and distribution at four sites in the Bay of Brest (Paugham et al. 2003). Molecular DNA probes have been developed for identification of the bay scallop and the sea scallop. The bay scallop probe has been shown to be capable of monitoring larval distribution in waters off the coast of Florida (Frischer et al. 2000, 2003). These field investigations using modern biotechnological methods have demonstrated excellent potential for bivalve larval identification and monitoring, though it may be a further 5 to 10 years before such techniques receive widespread acceptance.

This study presents details of the gross morphological characteristics, excluding ultrastructural traits of the hinge, that have been used to identify king scallop larvae during their planktonic phase in Mulroy Bay over the last 26 years. King scallop larval numbers in plankton samples at this site do not predominate over other species, a ratio less than 1:100 being common, quite unlike the predominance of Japanese scallop larvae in Mutsu Bay (Ventilla 1980). Supporting evidence for the positive identification of scallop larvae is presented in terms of the relationship between scallop larvae, their growth rate and the spat settlement in 1981. Using these characteristics for identification of king scallop larvae in natural plankton samples a technique has been developed for prediction of time and location of scallop spatfall and commercial collection of scallop spat at intensities exceeding 3,000 spat per collector bag (Slater 2005b).

MATERIALS AND METHODS

Procurement of King Scallop Larvae and Spat From the Hatchery

King scallop larvae for use as a reference material were produced using standard techniques in an experimental hatchery at Argyll, Scotland (Slater 1979b). Larvae reared at 14[degrees]C to 15[degrees]C were sampled at 2-day intervals and preserved in 5% formaldehyde. External characteristics were examined using an Olympus KH series microscope fitted with an eyepiece graticule. Spat that settled on monofilament netting were detached by immersion in 20% commercial hypochlorite in fresh water for 3 min to break the byssus, sieved on a 45-[micro]m mesh and then examined (Davies 1974).

Following this preliminary study of the external features of hatchery-reared, king scallop larvae, all further requirements for such larvae have been fulfilled by Cartron Point Shellfish hatchery in New Quay, County Clare, Ireland. This is the only shellfish hatchery in Ireland currently producing larvae and spat of the king scallop.

Collection of King Scallop Larvae From Mulroy Bay

Mulroy Bay is located in County Donegal in the northwest of Ireland (Fig. 1). It is a sheltered, marine lough connected to the open sea by a series of long, narrow channels. The Bay is well sheltered from excessive exposure and no oceanic swells penetrate into the two main parts, the Broad Water and the North Water. Seawater enters the Bay through the Narrows that flows into the Broad Water before passing through the Moross Channel into a further and even more enclosed sea lough, the North Water, which is approximately 3 km x 1 km in size and extends to a water depth of 51 m. Tidal range within the Bay and water exchange with the open sea is much diminished within the lough. The Bay provides an ideal location for shellfish spat production because larvae produced by natural populations are retained and settle within the Bay rather than being dispersed to more open waters.

[FIGURE 1 OMITTED]

Plankton samples were obtained from a site at Lurgacloghan on the western side of the North Water (Fig. 1) within the site licensed to Deegagh Point Shellfish Ltd. for scallop aquaculture. Samples were collected using a 63-[micro]m mesh plankton net with a 30 cm diameter opening, hauled at a rate of 5 m/min, from 20 m to the water surface. All planktonic larvae were added to a 1-L wide-mouthed plastic jar containing 100 mL of formaldehyde to preserve the living material immediately. In 1980 and 1981 a single vertical haul provided sufficient numbers of scallop larvae for analysis. From 1985 onwards, after use of TBT (tributyl-tin) antifoulants by salmon farms in the Bay, three vertical hauls were collected to provide sufficient scallop larvae for analysis (Minchin et al. 1987).

In the laboratory, samples were placed in a 1-L beaker and gently stirred. Bivalve larvae, being slightly heavier than most other planktonic organisms accumulate in the center of the beaker as a result of the gentle centrifugal force and can be transferred to 25-mL Sterilin[R] storage containers using a 10-mL pipette. Larvae were examined on a Sedgewick Rafter counting cell using x40 and x100 magnification on an Olympus KH series microscope. Larvae identified as scallops were measured using an eyepiece graticule at x 100 magnification. Identification was possible as a result of expertise gained during hatchery rearing studies performed with king scallops.

Collection of King Scallop Spat From Mulroy Bay

Indicator collectors were used to provide data on time and intensity of settlement of scallop spat. These consisted of a piece of 6-mm Netlon[R] mesh of 0.8 m x 0.3 m dimensions, rolled twice along the long axis into a cylindrical shape and fixed using cable ties. Indicator collectors were attached to ropes at 5 m, 10 m and 15 m beneath the water surface at MLWS and installed in the Bally Hork Bay site in 1981 (Fig. 1, Fig. 2). After 10-11 days immersion, indicator collectors were exchanged for a new set of collectors for spat settlement.

[FIGURE 2 OMITTED]

Bivalve spat settling during the 10-11-day period were removed from the collectors by immersion in 20% sodium hypochlorite in fresh water for 3 min. The solution was sieved through a 90-[micro]m-mesh sieve to collect the newly settled bivalve spat (Davies 1974). Examination of bivalve spat was performed on a Sedgewick Rafter counting cell at x40 and xl00 magnification. Scallop spat were identified and counted from each indicator collector.

RESULTS

Photomicrographs of king scallop larvae, including a complete sequence at 10-[micro]m length intervals between 120-240 [micro]m, are presented in color to assist in larval identification of this species (Fig. 3). The smallest larva examined measured 90.5 [micro]m x 71.2 [micro]m and the largest measured 245.8 [micro]m x 219.1 [micro]m. Digital images have been flipped horizontally to provide the sequence from both sides, because ability to recognize larvae from both perspectives is essential.

[FIGURE 3 OMITTED]

The relationships between shell length and shell height of hatchery-reared and wild scallop larvae were closely correlated ([R.sup.2] = 0.9821 and 0.9871 for hatchery and natural larvae respectively) (Fig. 4). The similarity between the trendlines for both larval batches provided some supporting evidence that larvae collected from the wild were king scallop larvae (Slope [+ or -] 95% CI = 1.025 [+ or -] 0.016 for hatchery larvae and 0.996 [+ or -] 0.014 for wild larvae). The close proximity of the slope of both lines to unity indicated near isometric growth during the larval period.

[FIGURE 4 OMITTED]

Length-height ratios and standard deviations for hatchery and wild scallop larvae of different size groups based on shell length are provided in Table 1. For researchers at the microscope, who following length and height measurement are faced with the decision of whether the length-height data for a larva fits certain specified criteria, the length-height ratio with standard deviation provides a convenient single number to assist in the decision making process. Slight differences in the length-height ratios existed between hatchery-reared larvae and larvae collected in the wild, particularly below 150-[micro]m length, hatchery larvae being slightly longer for a given height than larvae from the wild.

Illustrations based on characteristics used in the morphological identification of larvae of this species including umbo shape (Fig. 5), shell length frequency at different developmental stages (Fig. 6), pointed anterior end and pale color of freshly preserved larvae (Fig. 7); hinge line length (Fig. 8) have been provided for king scallop larvae and compared with other bivalve larvae.

[FIGURES 5-8 OMITTED]

A noticeable eyespot with a mean diameter of 7.59 [micro]m [+ or -] 1.44 [micro]m developed in hatchery-reared and natural samples of king scallop larvae (Fig. 3). Mean shell length and size range of eyespotted king scallop larvae from Mulroy Bay for each year between 1995 to 2003 inclusive demonstrated that the size of eyespotted larvae was similar each year (Table 2). Mean shell length of eyespotted larvae sampled from the wild between 1995 to 2003 was 218.12 [micro]m [+ or -] 7.69 [micro]m within a range extending from 195-246 [micro]m. By comparison mean shell length of eyespotted larvae, derived from Mulroy Bay broodstock, and reared at 18[degrees]C to 20[degrees]C in the hatchery during 2003 was 210.87 [micro]m [+ or -] 7.71 [micro]m within a range extending from 194-230 [micro]m. Mean shell lengths of natural and hatchery eyespotted larvae were significantly different (t = 13.7, df = 1016, P < 0.000). There was no evidence of differences in variability in populations based on an F-test of the two variances (F = 0.994, df = 713, 303, P = 0.471). Size distributions of eyespotted king scallop larvae sampled from the wild and hatchery are provided in Figure 9.

[FIGURE 9 OMITTED]

Settlement size was determined by measuring the prodissoconch shell length in recently settled spat from indicator collectors. Mean shell length at settlement was 220.9 [micro]m in 1980, 220.4 [micro]m in 1981 and 225.7 [+ or -] 8.33 [micro]m in 2000 within a range of 207-246 [micro]m. Length frequency distributions of king scallop larvae at settlement for each of these years are provided in Figure 10. The frequency of occurrence of the different length classes in 1980 and 1981 was similar ([chi square] = 6.137, df = 5, P = 0.293), however the frequencies of the length classes differed when the year 2000 data was included ([chi square] = 48.6, df = 10, P < 0.0001).

[FIGURE 10 OMITTED]

Data supporting planktonic identification of scallop larvae in natural samples using their morphological characteristics alone are provided in Figure 11. In 1981 two scallop spawnings were recorded using gonadal monitoring between June 10-20 and the June 26 to July 2. Two batches of larvae, tentatively identified as king scallops, were recorded corresponding to these spawnings. Growth of these two larval batches between 140-150 [micro]m to settlement size and their eventual settlement on indicator collectors was monitored. Mean number of scallop spat per indicator collector over a period extending through settlement periods demonstrated two peaks in settlement intensity. The skewed nature of the size distribution of scallop spat on the indicator collector immersed from June 30 to July 10, indicated that the main part of this settlement occurred in the latter part of the 10-day period. Normal distribution of the size frequency of spat on the indicator collector immersed from July 10 to July 20 indicated that the main part of this settlement occurred in the middle of the 10-day period. The relationship between time of spawning, time over which scallop larvae were recorded in the plankton and time of settlement estimated from size frequency of dissoconch shell height has been summarized in Table 3.

[FIGURE 11 OMITTED]

DISCUSSION

Photomicrographs of king scallop larvae, showing the key distinguishing characteristics over the full range of larval sizes are an extremely useful aid for successful identification of these larvae in natural plankton samples. For the inexperienced observer, preparation of photomicrographs serves a dual purpose, increasing familiarity with characteristic larval shapes at different sizes while at the same time providing a source of reference for comparative purposes during identification of unknown larvae sourced from the wild. Photomicrographs of larvae of this species at some developmental stages are available in Gruffydd and Beaumont (1972), Le Pennec (1974, 1978), Sasaki (1979) and Slater (1979b). The sequence of digital color photographs of larvae provided here, by comparison with the black and white images of earlier studies, illustrated the additional value of color photomicrographs. In accordance with the proposal of Lutz et al. (1982) micrographs of all the ontogenetic stages of larval development have been included rather than representative micrographs from the larval period.

Comparative examination of photomicrographs of P. maximus larvae with those of other pectinid larvae, for example the American bay scallop, (L.) (Loosanoff et al. 1966, Chanley & Andrews 1971), the calico scallop (L.) (Costello et al. 1973), the Tasmanian scallop, (Tate) (Dix & Sjardin 1975), the giant scallop, (Gmelin) (Culliney 1974), E. bifrons (L.) (Dix 1976) and A. opercularis (L.) (Sasaki 1979) illustrated the similarity that existed between larvae of these different scallop species. Rather than providing a generalized discussion of characteristics that contribute to these similarities, the features used have been considered individually and comments included as to the value of each for the morphological identification of scallop larvae.

Length-height Relationship

Many descriptions of hatchery-reared pectinid larvae provided during the 1960s to the 1970s included measurements of shell length, shell height and the equation of the best line fit through the data (Chanley & Andrews 1971, Culliney 1974, Dix & Sjardin 1975, Dix 1976, Sasaki 1979, Slater 1979b). Taking account of the minimum and maximum sizes of king scallop larvae measured in this program the regression line results indicated that shell length was between 23-28 [micro]m greater than shell height. This difference between length and height was smaller than the 25-35 [micro]m difference quoted in earlier work with this species (Sasaki 1979).

Given the natural variation that exists within any batch of larvae and the considerable overlap that has been reported between different bivalve species (Chanley & Andrews 1971), the length-height relationship cannot be considered in practice as a key diagnostic feature for scallop larval identification. Its principal value is as a tool to eliminate non-king scallop larvae from further consideration. It is recommended that if following measurement of the length, the calculated larval height differs by more than 4-5 [micro]m from the actual measured height; it is more than probable that the larva is not P. maximus. With increased experience and familiarity with larval appearance, the actual measurement of the larval length and height becomes unnecessary and an unconscious comparison of the two dimensions rapidly informs the observer whether the larva possesses the required length--height characteristics.

Umbo Type and Stages of Development

The umbo in bivalve larvae refers to a small prominence that develops along the hinge line and gives rise to the term umbonate larvae, that is those larvae that having developed an umbo have lost their earlier straight hinge or D-shaped appearance. Chanley and Andrews (1971) subdivided different types of umbo into the following 5 categories and provided line drawings to illustrate each: (1) round or indistinct; (2) broadly rounded; (3) angular; (4) knobby and (5) skewed.

Color images of umbonate larvae exhibiting each type of umbo are illustrated in Figure 5. Intermediate and transitional shapes were described as occurring frequently. While this terminology has not been widely adopted, researchers preferring to describe the pectinid umbo in their own terms, the overall descriptions and photomicrographs from the literature implied that the pectinid umbo belonged to the round or indistinct category. For example the bay scallop was described as having a poorly defined, low umbo, first appearing at 125 [micro]m but remaining inconspicuous throughout development (Chanley & Andrews 1971), the calico scallop as having a rounded and poorly defined umbo above 140 [micro]m, which remained inconspicuous throughout larval development (Costello et al. 1973), the Tasmanian scallop developed an umbo between 150-160 [micro]m, but this was not prominent throughout development (Dix & Sjardin 1975) and the Tasmanian queen scallop developed an insignificant umbo with only a slight bump appearing along the hinge (Dix 1976). These examples indicated that the pectinid umbo belonged to the indistinct category and that the larval size when the umbo developed varied between species.

Sasaki (1979) recognized four stages in larval development; straight hinge or D-shaped larvae with a straight hinge line, two stages of umbonate larvae; an early umbonate stage in which a slight protrusion could be observed at the center of the hinge line, which as a result could no longer be regarded as straight; a late umbonate stage in which the straight hinge line bad completely disappeared and the umbo extended over the full length of the hinge line and an eyespotted stage in which a noticeable eyespot was present (Fig. 3). According to Sasaki, the king scallop exhibited the early umbonate stage between 140-160 [micro]m and the late umbonate stage between 180-210 [micro]m. By comparison the queen scallop exhibited the early umbonate stage between 110-139 [micro]m and the late umbonate stage between 150-169 [micro]m.

In this program, king scallop larvae of a range of sizes were closely examined and subdivided into the four stages defined by Sasaki (1979). The percentage frequency of each of the four groups at 10-[micro]m size intervals over the course of larval development is presented in Figure 6. The majority of king scallop larvae < 135-[micro]m length were straight hinge larvae, early umbonate larvae dominated between 135-170 [micro]m, late umbonate larvae dominated between 170-215 [micro]m, with larvae above this size generally being eyespotted. These size ranges for each developmental stage are comparable to those of Sasaki (1979) and in broad agreement with the comments of Gruffydd and Beaumont (1972) that the straight hinge was lost as a result of umbo formation at 180 [micro]m. Le Pennec (1974) similarly reported that umbo development in king scallop larvae commenced at 150 [micro]m and was well developed at 170 [micro]m.

In practice, the indistinct, rounded umbo developing in larvae above 135 [micro]m in length has proved to be a useful indicator that the larvae should be examined for the presence of further king scallop characteristics.

Pointed Anterior End

Like the bay scallop, larvae of king scallops are asymmetrical at all stages of development (Loosanoff & Davis 1963). Asymmetry is more pronounced in larger, older larvae (Loosanoff et al. 1966) to the extent that Chanley and Andrews (1971) described the shape of A. irradians as being almost triangular. Comparing the umbonate larvae of P. maximus with those of M. edulis, Sasaki (1979) described the scallop as being wedge-shaped compared with the more egg-shaped mussel. This asymmetrical shape feature, resulting from the more pointed anterior end and more rounded posterior end, is clearly evident in all but the smallest pectinid larvae where it proved more difficult to observe. The pointed anterior end and general wedge-shape of the scallop compared with two other more symmetrical larvae of similar size is illustrated in Figure 7.

In practice this proved to be a key feature in distinguishing king scallop larvae from other bivalve larvae in a natural plankton sample.

Hinge Line Length

A selection of straight hinge larvae exhibiting a range of hinge line lengths and including the king scallop, have been provided in Figure 8 to illustrate some of the variations observed. Chanley and Andrews (1971) described hinge line length as an important characteristic of bivalve larvae that remained constant during larval development and ranged in length between different bivalve species from 35 [micro]m to over 100 [micro]m. As a diagnostic feature, the significance of the hinge line length was diminished by the considerable overlap that was reported to exist between different species, particularly where the hinge line length ranged between 50-80 [micro]m.

The hinge line length of king scallop larvae measured in this study ranged between 65-75 [micro]m, slightly larger than the 60-70 [micro]m recorded by Sasaki (1979). Variations in hinge line length occur between different pectinid species, for example in queen scallops it ranged between 50-59 [micro]m (Sasaki 1979) and in sea scallops it ranged between 75-82 [micro]m (Culliney 1974).

In practice, identification of straight hinge larvae below 135 [micro]m length remains, even to the most experienced observer, a formidable task and even after years of experience major difficulty still exists with larvae in this size range.

Color

Both hatchery-reared and wild king scallop larvae were pale in color and almost translucent by comparison with the more usual yellow-brown coloration of most other larvae (Fig. 7). Similar comments have been recorded for other pectinid larvae, for example larvae of the bay scallop were described as considerably lighter than most other larval forms, and although color darkened as the larvae increased in size, bay scallop larvae remained transparent compared with other species (Loosanoff & Davis 1963, Chanley & Andrews 1971, Loosanoff et al. 1966). The pale color of larvae of the calico scallop was suggested as a characteristic feature that may assist identification of this species (Costello et al. 1973). Larvae of the king scallop were described as much paler than those of the mussel, M. edulis, when observed under identical light conditions (Sasaki 1979).

In practice the difference in color between king scallop larvae and other bivalve larvae, particularly at the smaller straight hinge stage, is an important characteristic in natural plankton samples containing mixed populations of bivalve larvae. This feature is easily observed in freshly preserved specimens and with experience can be used in combination with other characteristics to aid identification of pectinid veligers. With longer-term preservation of samples over several weeks, color difference diminishes significantly reducing the value of this feature for diagnostic work.

Similar fading of the general color of clam larvae, Venerupis pullustra (Montagu) and other anatomical features such as the eyespots has been recorded in the literature (Quayle 1950).

Eyespot Development

The eyespot refers to a small black spot, located slightly off-center, which has been observed in some bivalve larvae as settlement size is approached. The presence or absence of an eyespot and the larval size at which it develops has been recorded as varying within different pectinid species. In the bay scallop the eyespot was inconspicuous (Loosanoff & Davis 1963) and was particularly difficult to observe in larger larvae (Loosanoff et al. 1966). According to Chanley and Andrews (1971) the eyespot developed in bay scallops at shell lengths between 150-180 [micro]m, though Sastry (1965) made no mention of eyespots in this species. Significant eyespots were similarly never observed in the Tasmanian scallop although the Tasmanian queen scallop developed conspicuous eyespots that were common in larvae of 165-175 [micro]m (Dix & Sjardin 1975, Dix 1976). In the European queen scallop, the eyespot was reported at shell lengths over 180 [micro]m (Sasaki 1979) and in the sea scallop at shell lengths above 230 [micro]m (Culliney 1974).

Conspicuous eyespots were recorded in hatchery-reared king scallop larvae at lengths greater than 210 [micro]m when reared at 15[degrees]C (Comely 1972), at lengths greater than 210 [micro]m when reared between 17[degrees]C to 18[degrees]C (Sasaki 1979) and in larvae exceeding 225 [micro]m when reared at 16[degrees]C (Gruffydd & Beaumont 1972). In this study, eyespots were observed in king scallop larvae at a smaller size than reported elsewhere in the literature, 194 [micro]m in the hatchery and 195 [micro]m under natural conditions. Mean shell length of eyespotted larvae from the hatchery was less than that of eyespotted larvae in the wild. This may be attributed to higher water temperatures used in the hatchery (18[degrees]C to 20[degrees]C) compared with ambient temperatures in Mulroy Bay (mean 16.4[degrees]C [+ or -] 0.89[degrees]C, n = 6) although there was no significant correlation between ambient temperature and mean shell length of eyespotted larvae for six of the years reported in Table 2 ([R.sup.2] = 0.16, [F.sub.1,4] = 0.76, P = 0.43, n = 6). An alternative explanation for this difference may be that it resulted from the time of sampling of larvae, derived from a single hatchery spawning that resulted in a larval batch of a more uniform size, which did not demonstrate the size range recorded in the wild (Fig. 9).

The eyespot and size at which it is recorded are characteristics that can be useful in larval identification. For example based on data from natural samples between 1995 to 2003 inclusive, approximately 10% of king scallop larvae will have an eyespot by 210 [micro]m shell length yet 90% of larvae will have developed one by 230 [micro]m. However, for the purpose of predicting the time of the scallop spatfall, the close proximity of the time of eyespot development to the time of larval settlement means that this characteristic is of little practical value in an aquaculture context.

Settlement Size in Nature

After settlement and metamorphosis, heavier dissoconch or spat shell, with engraved markings, was deposited around the edge of the lighter prodissoconch or larval shell. The prodissoconch-dissoconch boundary provides a morphological feature useful in distinguishing true juveniles from metamorphosing postlarvae (Lutz & Hidu 1979). Measurement of the length of the prodissoconch shell in recently settled spat can thus be used to provide the larval shell length at settlement.

Settlement size has been reported to exhibit variations between different pectinid species. The bay scallop metamorphosed between 175-200 [micro]m (Loosanoff et al. 1966, Chanley & Andrews 1971), though Sastry (1965) recorded a mean prodissoconch length of 190 [micro]m within a larger range extending from 174-213 [micro]m. The calico scallop settled at a larger shell length between 235-270 [micro]m (Costello et al. 1973). The Tasmanian scallop metamorphosed between 220-240 [micro]m, whereas the Tasmanian queen scallop settled when larvae exceeded 200 [micro]m, though individuals were recorded as small as 170 [micro]m (Dix & Sjardin 1975, Dix 1976).

Under natural conditions king scallop larvae settled at mean shell lengths of 220.5 [micro]m, 219.6 [micro]m and 225.7 [micro]m in the years 1980, 1981 and 2000 respectively. The range of settlement sizes in these years extended between 200-246 [micro]m (Fig. 10). Based on a limited number of small spat collected in waters off the Isle of Man, the prodissoconch shell length of the king scallop was recorded at 240 [micro]m, whereas that of the queen scallop was recorded at 190 [micro]m (Eggleston 1962). In the hatchery king scallop larvae metamorphosed at approximately 250 [micro]m at 16[degrees]C (Gruffydd & Beaumont 1972). Sasaki (1979) working with king scallops in Loch Creran measured the prodissoconch shell length of recently settled king scallop spat from two sources. In nature settlement occurred at a mean shell length of 247 [micro]m within a size range from 220-270 [micro]m (n = 100) considerably larger than the values recorded in this study. By contrast scallop larvae derived from broodstock of the same origin as the natural samples but reared under artificial conditions in a hatchery settled at a mean shell length of 223 [micro]m within a size range from 190-250 [micro]m (n = 60). The difference in settlement size between natural and hatchery conditions of 20-30 [micro]m, given broodstock of the same origin, may be the result of differences in water temperature, the hatchery culture being performed between 17[degrees]C to 18[degrees]C compared with the natural temperatures of approximately 14[degrees]C (Sasaki 1979). Differences in water temperature, during the scallop larval period, between Mulroy Bay and Loch Creran, temperatures at the former being approximately 15[degrees]C to 16[degrees]C, differences in other environmental conditions and possible genetic differences between Scottish scallops and Mulroy Bay scallops may contribute to variations in the settlement size of king scallop larvae.

Comparison of the mean size of eyespotted larvae of 218.12 [micro]m (Table 2) and mean size of larvae at settlement over the three years in which this was measured of 221.19 [micro]m (Fig. 10) and assuming a growth rate of 4-5 [micro]m/day demonstrated that the eyespot is a good indicator of the imminence of settlement in king scallop larvae under natural conditions.

Supporting Evidence for Planktonic Identification Based on Morphological Characteristics

Results of this research demonstrated the relationship between the scallop spawning time, larval growth in the plankton and spat settlement in 1981 (Table 3 and Fig. 11) and provided supporting evidence that scallop larvae can be distinguished from other bivalve larvae based only on their morphology and that their identification can be used to predict scallop settlement.

Based on 26 years of experience observing and identifying scallop larvae in mixtures of bivalve larvae from natural plankton samples, the principal features, arranged in order of their practical value, for identification of both umbonate and straight hinge scallop larvae have been provided in Table 4. Using these features for identification of scallop larvae in natural plankton samples a technique for prediction of time and location of scallop spatfall has been developed. This technique has been applied for commercial collections of scallop spat in Mulroy Bay since 1981 with spat collection intensities exceeding 3,000 spat per collector bag (Slater 2005b).

ACKNOWLEDGMENTS

The authors thank Dr. Ryo Sasaki of the Miyagi Prefectural Fisheries Experimental Station and Professor. Y. Uno, Tokyo University of Fisheries for their encouragement to persevere with plankton analysis during this time. Initial work on this project was financially supported by the White Fish Authority Marine Farming Unit at Ardtoe, Argyll, Scotland. Preliminary investigations in Mulroy Bay during 1980 and 1981 were financed by Beirtreach Teoranta, a wholly owned subsidiary of Udaras na Gaeltachta, the development agency for the Irish-speaking regions of Ireland. Further experience and commercial application of the technique since 1981 has been undertaken by Deegagh Point Shellfish Ltd, a private company, supported at different times by financial grant assistance from Bord Iascaigh Mhara, the Irish Sea Fisheries Board and Udaras na Gaeltachta. Letterkenny Institute of Technology provided facilities for photomicroscopy of scallop larvae. The Marine Institute in awarding post-doctoral fellowship number PDOC/01/004 to University College Cork provided the impetus for the author to register for a Ph.D. and to prepare this manuscript as part of the program of work under the supervision of Dr. Gavin Burnell, Department of Zoology and Animal Ecology.

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JOHN SLATER 

Letterkenny Institute of Technology, Department of Science, Port Road, Letterkenny, County Donegal, Ireland.

E-mail: [e-mail]

TABLE 1.
Length/height ratio of different size groups of hatchery-reared
and wild king scallop larvae, Pecten maximus (n = 255 each).

Hatchery Larvae

 Size     n          Mean Length                Mean Height
 Range           ([micro]m) [+ or -]        ([micro]m) [+ or -]
                 St. Dev. ([micro]m)        St. Dev. ([micro]m)
80-100
100-120   42     111.15 [+ or -] 3.56        84.43 [+ or -] 4.18
120-140   17     131.92 [+ or -] 6.03       106.46 [+ or -] 5.83
140-160   58     148.25 [+ or -] 6.34       120.45 [+ or -] 7.25
160-180   36     169.93 [+ or -] 6.32       145.11 [+ or -] 9.56
180-200   26     189.14 [+ or -] 6.57       162.39 [+ or -] 10.15
200-220   60     210.71 [+ or -] 4.89       187.02 [+ or -] 8.14
220-240   15     224.94 [+ or -] 3.40       198.88 [+ or -] 6.22
240-260    1     245.78 [+ or -] 0.00       219.06 [+ or -] 0.00

 Size     n           L/Ht. Ratio
 Range             [+ or -] St. Dev.

80-100
100-120   42      1.32 [+ or -] 0.04
120-140   17      1.24 [+ or -] 0.03
140-160   58      1.23 [+ or -] 0.05
160-180   36      1.17 [+ or -] 0.05
180-200   26      1.17 [+ or -] 0.04
200-220   60      1.13 [+ or -] 0.04
220-240   15      1.13 [+ or -] 0.03
240-260    1      1.12 [+ or -] 0.00

Wild larvae

 Size     n          Mean Length                Mean Height
 Range           ([micro]m) [+ or -]        ([micro]m) [+ or -]
                 St. Dev. ([micro]m)        St. Dev. ([micro]m)

80-100     8      92.21 [+ or -] 1.78        73.79 [+ or -] 1.58
100-120    8     112.35 [+ or -] 3.86        88.27 [+ or -] 4.82
120-140   33     130.48 [+ or -] 5.96       106.72 [+ or -] 6.67
140-160   59     150.39 [+ or -] 5.20       125.92 [+ or -] 4.70
160-180   47     169.47 [+ or -] 6.63       143.43 [+ or -] 8.08
180-200   44     190.06 [+ or -] 6.23       164.34 [+ or -] 7.45
200-220   40     209.80 [+ or -] 5.80       184.28 [+ or -] 6.90
220-240   16     226.53 [+ or -] 5.21       205.20 [+ or -] 6.82
240-260

 Size     n           L/Ht. Ratio
 Range             [+ or -] St. Dev.

80-100     8      1.25 [+ or -] 0.02
100-120    8      1.27 [+ or -] 0.03
120-140   33      1.22 [+ or -] 0.03
140-160   59      1.19 [+ or -] 0.03
160-180   47      1.18 [+ or -] 0.03
180-200   44      1.16 [+ or -] 0.03
200-220   40      1.14 [+ or -] 0.02
220-240   16      1.10 [+ or -] 0.02
240-260

TABLE 2.
Mean shell length and size range of eyespotted larvae of the
king scallop, Pecten maximus from Mulroy Bay between 1995-2003.

              Mean length [+ or -]    Size range
Year    n     St. Dev. ([micro]m)     ([micro]m)

1995    92    215.22 [+ or -] 6.18     198-230
1996    22    212.95 [+ or -] 4.15     206-220
1997   336    218.95 [+ or -] 7.45     200-240
1998    37    215.32 [+ or -] 5.00     202-224
1999    29    212.24 [+ or -] 6.10     203-229
2000     5    222.40 [+ or -] 10.04    206-230
2001    75    218.00 [+ or -] 7.02     206-243
2002   112    221.85 [+ or -] 8.67     202-246
2003     6    209.33 [+ or -] 8.33     195-220

TABLE 3.
Relationship between spawning period, larval period and settlement
time for the king scallop Pecten maximus in 1981.

                                    1st Spawning      2nd Spawning

Spawning period from gonadal
  monitoring                       June 10-20 June   June 26-2 July
Larval period from plankton
  analysis                         June 20-10 July   July 7-30 July
Period of immersion of indicator
  collectors                       June 30-10 July   July 10-20 July
Settlement time from size
  frequency of dissoconch
  shell height                     July 7-10 July    July 14-16 July

TABLE 4.
Principal features, arranged in order of their practical value, for
identification of straight hinge and umbonate king scallop larvae.

      Straight Hinge Larvae           Umbonate Larvae
          <135 [micro]m               135-215 [micro]m

1   Pointed anterior end         Indistinct rounded umbo
2   Pale color                   Pointed anterior end
3   Hinge line length            Pale color
4   Length-Height relationship   Length-Height relationship
5                                Eyespot