Gametogenesis in the non-native green mussel, Perna viridis, and the native scorched mussel, Brachidontes exustus, in Tampa Bay, Florida.

ABSTRACT Histologic examination of the normative green mussel, Perna viridis, and the native scorched mussel, Brachidontes exustus, at three locations in Tampa Bay, Florida, indicated that cycles of gametogenesis and spawning were similar between species. Major spawning periods occurred in the spring (April) in conjunction with increasing water temperature and the fall (September to November) as the water temperature was declining. Over the summer, both species appeared to undergo varying levels of redevelopment, partial spawning and resorption of oocytes. Gametogenesis was limited over the winter. Differences between populations in levels of gametogenic activity and the timing of the fall spawn were most likely related to differences in salinity and available food at the three sites. A higher fecundity (caused by the faster growth rate and greater maximum size of P. viridis) may result in displacement of native bivalves in Tampa Bay, including B. exustus.

KEY WORDS: reproduction, Perna viridis, Branchiodontes exustus, Tampa Bay

INTRODUCTION

The tropical euryhaline green mussel, Perna viridis (Linnaeus, 1758), native to the Indo-Pacific, has recently become established in Tampa Bay, on the west central coast of Florida, where it attaches (via byssal threads) to hard substrates in shallow subtidal and intertidal areas (Ingrao et al. 2001, Benson et al. 2001). The introduction likely occurred via ballast water from ships originating from the southern Caribbean where P. viridis had previously become established (Agard et al. 1992, Benson et al. 2001). Since the initial report from Tampa Bay in 1999, populations of P. viridis have become distributed on the west coast of Florida southward to Naples and northward to Tarpon Springs and on the east coast from Daytona Beach to southern Georgia (Power et al. 2004, Fajans & Baker 2005). In its native range, P. viridis is a major fouler of ship hulls, piers, buoys, rafts and mariculture cages (Huang et al. 1985). It is also a valuable species for mariculture because of its rapid growth rate (up to 10 mm per month) and high protein content; market size (60-70 mm) is reached in 7-8 mo (Sivalingam 1977, Lee 1986, Rajagopal et al. 1998). Optimal temperature is 26[degrees]C to 28[degrees]C and optimal salinity is 27-33 ppt (Agard et al. 1992, Sivalingam 1977).

Previous introductions of normative species of marine bivalves, both intentional and accidental, have had negative impacts on native populations (see Barber, 1997). In Venezuela, Perna viridis is displacing previously established populations of P. perna (Segnini de Bravo et al. 1998). The potential ecologic impacts of P. viridis in Tampa Bay include colonization of mangrove prop roots, oyster reefs and seagrass beds, resulting in displacement of native species (Baker & Benson 2002, Baker et al. 2003, Fajans et al. 2005). Green mussels are obviously growing and reproducing successfully in Tampa Bay; densities as high as 4000 per [m.sup.2] have been reported (Fajans & Baker 2003). There is evidence that oyster (Crassostrea virginica) reefs and seagrass (Halodule wrightii) beds in Tampa Bay are already being impacted (Avery & Johansson 2003, Baker et al. 2003).

The scorched mussel, Brachidontes exustus (Linnaeus, 1758), is found from the Carolinas to the Gulf of Mexico and southern Caribbean; recent molecular genetic work has indicated that B. exustus is actually comprised of a complex of four cryptic species (Lee & O Foighil 2004). Most U.S. specimens fall into either Atlantic or Gulf of Mexico lineages; other identified species occur in southern Florida and the Caribbean. Species names have not yet been assigned, and as a result, all references herein shall be made in the context of a B. "exustus" clade.

Brachidontes exustus attaches to the surface of hard substrata and has broad salinity and temperature tolerances, often dominating the molluscan fauna in estuaries (Seed 1980, Brewster-Wingard et al. 2001), on oyster reefs (Hoese 1960, Ortega 1981), and on mangrove prop roots (Marquez & Jimenez 2002). Based on taxonomy and lifestyle, B. exustus is the most similar native to P. viridis in Tampa Bay despite the fact that in Florida, B. exustus seldom reaches 2 cm in shell length.

The ability of one species to out-compete another is largely determined by differences in reproductive strategy. This includes both reproductive effort (fecundity) and the timing and frequency of spawning. The species that produces the most larvae will likely be able to out-compete other species for settlement space and energy. To assess the potential for ecologic impact, we compared the gametogenic cycle of the nonnative P. viridis with that of the native B. exustus at three locations in Tampa Bay, Florida.

MATERIALS AND METHODS

Mussels were obtained from 3 locations in Tampa Bay, Florida (Fig. 1). Safety Harbor (28[degrees]00.22'N; 82[degrees]40.31'W) is in the upper portion of the bay and thus is most influenced by freshwater runoff. The Gandy Bridge (27[degrees]53.40'N; 82[degrees]32.70'W) connects St. Petersburg with Tampa in the midbay region. The Skyway Bridge (27[degrees]35.75'N; 82[degrees]38.10'W) crosses the mouth of Tampa Bay, and thus receives the greatest tidal input. All mussels were taken from vertically oriented artificial substrate (concrete pilings) approximately 1 m below mean high water. Surface water temperature and salinity were recorded periodically throughout the study period at each site using a YSI Model 650 MDS multimeter.

[FIGURE 1 OMITTED]

At approximate monthly intervals between April 2002 and April 2003, individuals of each species were collected from each site and placed immediately into Davidson's Fixative for 24-48 h. Shell length was determined with digital calipers ([+ or -] 0.1 mm). Fixed tissues were dehydrated, cleared, and embedded in Paraplast. Thin sections (6 [micro]m) were placed on glass microscope slides and stained with hematoxylin and eosin (Howard et al. 2004).

Slides were examined with a compound microscope (Nikon LabPhot-2) at x l00. For each individual, sex was determined and gametogenic activity was assigned to one of six stages, similar to those described by Seed and Brown (1975) for Mytilus edulis. These stages are illustrated for Perna viridis in Figures 2 and 3.

[FIGURES 2-3 OMITTED]

Stage 0: Inactive

This stage included mussels with undifferentiated gonads or gonads that contained only a few residual gametes undergoing the last stages of resorption with no development occurring (Fig. 2A).

Stage D1: Early Developing

Mussels in this stage had small follicles that were expanding within gonadal connective tissue. Cells within follicles were mostly primary spermatocytes or oocytes. Sometimes a few residual ova undergoing lysis also occupied shrunken follicles as a new generation of oocytes was being produced (Figs. 2B and 3A).

Stage D2: Late Developing

In this stage, follicles had expanded, displacing most gonadal connective tissue. Follicles contained predominantly spermatocytes and spermatozoa or secondary oocytes (Fig. 2C and 3B).

Stage D3: Ripe

Ripe mussels had gonads comprised almost entirely of follicles filled with primarily spermatozoa and mature ova (Fig. 2D and 3C).

Stage S2: Early postspawning

Mussels in this stage had recently released mature gametes, as indicated by expanded follicles containing space previously occupied by mature gametes. Follicle walls were thin and broken. Resorption of remaining oocytes was common, as was redevelopment (production of primary oocytes and spermatocytes). Thus there was potential for additional spawning activity (Fig. 2E and 3D).

Stage S1: Late postspawning

This stage was characterized by shrunken follicles with a few residual gametes obviously undergoing resorption. Redevelopment was arrested, indicating that spawning activity had ceased (Figs. 2F and 3E).

A gonad index was calculated for each sample by dividing the sum of the numerical stage value (0-3) by the number of individuals (Seed & Brown 1975).

RESULTS

Based on shell lengths, all individuals examined were adults (capable of gametogenesis). Mean shell length for Perna viridis ranged from 28.6-81.3 mm. For Brachidontes exustus, shell length ranged from 5.0-20.0 mm.

Dates of collection and the number of individuals examined histologically are provided in Table 1 (P. viridis) and Table 2 (B. exustus). The frequency of sampling and the number of individuals examined were greater for P. viridis (n = 589) than for B. exustus (n = 252).

Perna viridis

Of the 589 slides examined, 327 were male; 247 were female; and 10 were undifferentiated (sex could not be determined). Thus the sex ratio for P. viridis was found to be significantly different from 1:1 ([chi square] P [less than or equal to] 0.001). In addition, five individuals were hermaphrodites.

At Safety Harbor on April 5, 2002, mussels were mostly late developing, mature, and early postspawning, indicating that gametogenesis was essentially complete and spawning had begun (Fig. 4A). The next month (May 9), mussels were mostly in the late postspawning stage, although some redevelopment was evident. On July 16, most mussels were early postspawning, but remaining oocytes were highly granular and undergoing resorption. Thus it is not clear whether the redevelopment indicated from the May 9 sample continued to completion and resulted in release of gametes (spawning). By August 13, however, gametogenesis had been reinitiated, as most mussels were either in early or late developing stages. Again, it could not be confirmed whether gametogenesis was completed and spawning resulted from this effort as the next month (September 9), mussels were primarily late postspawn or inactive. Active gametogenesis was again indicated on September 29 as all individuals were developing or mature. Subsequent spawning of this generation of gametes was likely; by November 24 most mussels were in the late postspawning stage. Gametogenic activity was minimal over the winter (January 14 and February 5), because most mussels were late postspawning or inactive. Gamete development was underway March 6, 2003, because most mussels were in early or late development stages.

[FIGURE 4 OMITTED]

Gandy Bridge mussels were in the process of spawning on April 5 because most were in late developing, mature or early postspawn stages (Fig. 4B). Gametogenic activity was minimal on May 9 and July 16, with all individuals in either late postspawn or inactive stages. Development was reinitiated on August 13 (mostly early developing) and mostly completed by September 9, when mussels were in late developing, mature or early post spawning stages. Spawning of these gametes was indicated by the fact that all mussels on September 29 were late postspawning. There was evidence of some redevelopment November 24, but for the most part, mussels remained in post spawning or inactive condition over the winter. The April 9 sample contained mussels that were also primarily post spawning, but one individual was in early developing stage.

At the Skyway Bridge site, mussels were undergoing completion of gametogenesis and spawning, because most were in late developing, mature or early postspawning stages (Fig. 4C). This was followed on May 9 by minor redevelopment, but by July 16 all mussels were in the late postspawning stage. This period of little to no gametogenic activity continued through the summer (August 13 and September 9). By September 29, however, some redevelopment was evident, and this continued until November 24, when most mussels were late developing, mature, or early postspawning. Spawning appeared to be complete by December 19 (mostly late postspawning). The population remained inactive over the winter (January 14 and February 5 samples) in either late postspawning or inactive stages. Subsequent redevelopment was indicated on March 6, because most of the mussels were in the early development stage.

Gonad indices, resulting from the staging of individual mussels, indicated that gametogenic activity occurred to a varying extent throughout the year at the three sampling sites (Fig. 5). Values >1.5 (maximal gonad development) occurred for all three sites on April 5, 2002 and again for Safety Harbor mussels on July 16 and September 29; for Gandy Bridge mussels on September 9 and for Skyway Bridge mussels on November 24. Values <1.0 (minimal gonad development) occurred for the Safety Harbor population on September 9; for the Gandy Bridge population on May 9, August 13, and November 24 and for the Skyway Bridge population on May 9, September 9, January 14, 2003 and February 5. Increasing values between consecutive sampling dates were indicative of gonad growth and development, whereas decreasing values suggested that spawning (or resorption) of gametes had occurred. Thus based on gonad indices, all three populations spawned in April 2002 (or earlier) and again in October (Gandy Bridge), November (Safety Harbor) or December (Skyway Bridge). At Safety Harbor, mussels exhibited an additional loss of gonad mass between July 16 and September 9.

[FIGURE 5 OMITTED]

In summary, all three populations of P. viridis underwent at least two cycles of gametogenesis and spawning throughout the year examined in this study. The first of these was in the spring (April) and the second occurred in the fall, with the timing variable among the locations. Fall spawning occurred in early September at Gandy Bridge, in late September to October at Safety Harbor and in late November at Skyway Bridge. Additional periods of gamete development with either spawning or resorption of gametes was possible during the summer months, especially at the Safety Harbor site. Gametogenic activity at all locations was minimal over the winter.

Brachidontes exustus

A total of 252 slides were examined. Of these, 111 were male, 116 were female, and 25 were undifferentiated. The sex ratio for B. exustus was not significantly different from 1:1 ([chi square], P > 0.05). No hermaphrodites were observed.

The Safety Harbor population was undergoing a spawning event on April 5, 2002, because most mussels were in late development, mature and early spawning stages (Fig. 6A). The May 9 and July 16 samples supported possible redevelopment and spawning, because mature and postspawning individuals predominated. Considerable redevelopment had occurred by September 29, because most mussels were mature. Spawning of these gametes was suggested by the fact that the following sample (November 24) contained entirely postspawning and inactive individuals. Gametogenesis was reinitiated on January 14, 2003 and February 5, as indicated by the presence of mostly early developing individuals. Gametogenic development continued on March 6, because most individuals were late developing.

[FIGURE 6 OMITTED]

At the Gandy Bridge site, the first sample collected on May 9, 2002 contained mostly early post spawning individuals, suggested that spawning had already occurred (Fig. 6B). No sample was obtained before this (in April 2002). Redevelopment had obviously occurred by August 13, because all individuals were either late developing or mature. Spawning of these gametes was suggested by the presence of mostly post spawning individuals on September 9 and September 29. By November 24 most mussels were either late post spawning or inactive. Reinitiation of gametes began on January 14, 2003 and continued on February 5, March 6, and April 9, with an increasing proportion of late developing and mature individuals.

Skyway Bridge mussels were spawning on April 5, 2002; all individuals were either mature or early post spawning (Fig. 6C). Redevelopment (both early developing and late developing stages) was underway on August 13. It is possible that gamete development was completed and resulted in spawning, because the following sample (September 9) contained only early post spawning individuals. Gametogenic activity was minimal over the winter; samples on November 24 and December 19 contained entirely late post spawning and inactive individuals. Reinitiation of gametogenesis began with the January 14, 2003 sample and continued with the March 6 sample, which was comprised of either early or late developing mussels.

Gonad indices of 2.0 or greater, indicating periods of maximal gonad development, occurred at Safety Harbor on April 5, 2002 and again on September 29; at Gandy Bridge on August 13 and September 29 (no sample was obtained on April 5, 2002); and at Skyway Bridge on April 5, 2002 and September 9 (Fig. 7). Minimal values ([less than or equal to] 1.0) occurred at Safety Harbor on February 5, at Gandy Bridge on November 24 and January l4, 2003, and at Skyway Bridge on November 24, December 19 and January 14, 2003. Major decreases in gonad index, indicating possible spawning events, were seen for Safety Harbor mussels between April 5, 2002 and May 9 and September 29 and November 24; for Gandy Bridge mussels between August 13 and September 9 and again between September 29 and November 24; and for Skyway Bridge mussels between April 5, 2002 and May 9 in addition to September 9 and November 24.

[FIGURE 7 OMITTED]

Thus the gametogenic staging gonad index data support the existence of dual spawning periods for B. exustus at all three sites in Tampa Bay. The spring spawn occurred in April at all locations, and the fall spawn began earliest in the Gandy Bridge population (early August), followed by the Skyway Bridge population (late August), and the Safety Harbor population (September to October). Continuous development of gametes and partial spawning was possible at Safety Harbor. Gametogenic activity was minimal over the winter at all sites.

Surface water temperature was similar at all 3 locations, averaging 25.1[degrees]C at Safety Harbor, 25.1[degrees]C at Gandy Bridge and 24.5[degrees]C at Skyway Bridge (Fig. 8). Seasonally, temperatures gradually increased from about 25[degrees]C in April 2002-29[degrees]C to 30[degrees]C between July 16 and October 14. Temperatures then declined to a low on January 29 of 13[degrees]C before increasing again in early 2003. Surface salinity varied among the 3 locations, averaging 20.1 ppt at Safety Harbor, 23.4 ppt at Gandy Bridge and 28.6 ppt at Skyway Bridge (Fig. 8). Seasonal patterns were similar among the three locations, however, with salinity being greatest at the beginning of the study, declining to minima in January 2003, and subsequently increasing afterwards.

[FIGURE 8 OMITTED]

DISCUSSION

Gametogenic cycles were generally similar for Perna viridis and Brachidontes exustus in Tampa Bay over the year examined in this study (2002 to 2003), and included two primary spawning periods. The spring spawn was ongoing in April 2002 at the start of the study. There were varying levels of redevelopment with partial spawning and resorption over the summer before the second major spawning period, which occurred between September and November for both species. Gametogenic activity was considerably reduced over the winter. This general pattern of an extended period of gametogenesis with two primary spawning events is typical of populations of marine bivalves from lower latitudes (Giese 1959, Barber & Blake 1990). For example, gametogenesis in the quahog Mercenaria spp. from the east coast of Florida is also bimodal, with spawning peaks occurring in fall and spring (Hesselman et al. 1989). Gametogenesis of B. exustus from Georgia also involves dual spawning periods, with a minor spawn occurring in July and a major spawn occurring in the fall (Sweeney & Walker 1998).

Subtle differences in gametogenesis between the two species of mussels were noted, however. First, Brachidontes exustus had a more clearly defined gametogenic cycle than Perna viridis, as illustrated by the greater range in gonad index values and greater developmental synchrony within samples. For B. exustus, gametogenesis virtually ceased over the winter prior to initiation of another gametogenic cycle early in the following spring; all individuals collected in December were undifferentiated. In contrast, few undifferentiated P. viridis were found, even in the winter months. Instead, most males contained spermatozoa, and females were observed to be creating a new generation of gametes in follicles containing resorbing oocytes, without going through a typical "resting" stage (Fig. 9). Second, few mature (stage D3) individuals were observed for P. viridis, suggesting that in this species, maturation and spawning of gametes occurs very rapidly. All of this indicates that P. viridis is capable of more rapid response to environmental factors that affect gametogenesis than B. exustus. More frequent sampling would be necessary to effectively document these subtle, rapid changes in gametogenic activity.

[FIGURE 9 OMITTED]

Both species of mussels contained gametes (developing and resorbing) throughout the summer, after the spring spawning event. It was not uncommon to observe both processes occurring among individuals within a sample or even within one individual. The extent to which gametes were being spawned or resorbed over the summer, however, was unknown. Either process would contribute to a loss of gonad mass (and reduced gonad index). Continuous production and release of few gametes over an extended period of time is not uncommon, particularly for tropical species like Perna viridis (Walter 1982). It is also not uncommon for bivalves to produce generations of oocytes that are never spawned but instead are resorbed for energy (Le Pennec et al. 1991). It is probable that both processes (spawning and resorption of oocytes) were occurring during this study, perhaps in response to prevailing environmental conditions.

There were differences among sites for both species in terms of extent of gametogenic activity and the timing of spawning. Gametogenesis in both species was more continual over the summer and was initiated earlier in the spring at the Safety Harbor site than at either the Gandy Bridge or Skyway Bridge sites. The fall spawning event of both species occurred earlier at Gandy Bridge than at the other two sites. Among the three sites, the gametogenic cycles of both species was most clearly defined at the Skyway Bridge. Most likely these minor differences in gametogenesis between sites are caused by variations in environmental conditions within Tampa Bay.

The primary exogenous factors regulating gametogenesis in marine bivalves are temperature and food supply (Sastry 1979, Barber & Blake 1990). Temperature was similar among the three locations in this study, with an annual average of 25.1[degrees]C at Safety Harbor, 25.1[degrees]C at Gandy Bridge, and 24.5[degrees]C at Skyway Bridge. Spawning periods for both species of mussels correlated to increasing (spring) and decreasing (fall) water temperatures. Annual minimum water temperature effectively reduced gametogenesis in Perna viridis and Brachidontes exustus over the winter months. Once temperature and food levels increased above threshold levels, gametogenesis was reinitiated in the spring. Differences in the timing of the fall spawning event among locations may be related to other factors, such as salinity or food supply. Salinity did vary among locations; the annual average was 20.1 ppt at Safety Harbor, 23.4 ppt at Gandy Bridge, and 28.6 ppt at Skyway Bridge. Seasonal patterns of salinity, however, were similar among locations and changes were not correlated with spawning events. Therefore it is likely that differences in gametogenic activity between sites over the summer and the timing of the fall spawning period were related to variations in food supply. Water quality monitoring by the Hillsborough County Environmental Commission (Cardinale 2003), showed that overall concentrations of chlorophyll-a in 2002 were greater at Safety Harbor (7.62 [+ or -] 2.93 [micro]/l) than at Gandy Bridge (4.49 [+ or -] 1.50 [micro]/l) or Skyway Bridge (4.61 [+ or -] 1.59 [micro]/l). In addition, maximal chlorophyll-a values (12.4 [micro]/l) occurred at Safety Harbor in June and July, corresponding to the greater gametogenic activity seen at this site during this time. Similarly, Newell et al. (1982) compared gametogenesis in the blue mussel, Mytilus edulis, among seven locations in the north eastern United States and found the greatest temporal difference (3 mo) in reproductive maxima occurred between two populations at the same latitude, but having considerably different amounts of energy availability in the seston.

A pattern of dual spawning peaks (spring and fall) with intermittent spawning over the summer for Perna viridis in Tampa Bay is supported by settlement data collected as part of an ancillary study (Baker et al. unpublished). At Safety Harbor, juvenile mussels (<15 mm shell length) were found at greatest numbers on collection plates from April through November, with a maximum in August 2002. At Gandy Bridge, settlement occurred primarily from May to October 2002, with the greatest number of small (<5 mm shell length) mussels being found in May and July. At the Skyway Bridge, there were two settlement peaks, with numbers of juvenile mussels being greatest from May to August; a smaller peak occurred in January and February 2003.

The gametogenic cycle of Perna viridis in Tampa Bay is similar to that reported from populations in the Indo-Pacific region. In Malaysia, mussels exhibit two distinct spawning periods (March to April and October to November), which are associated with the monsoon seasons (Sivalingam 1977). A similar pattern is seen on the east coast of India, where spawning occurs primarily from May to June and October to November and is more closely correlated to water temperature than phytoplankton standing stock (Rajagopal et al. 1998). Gametogenesis of P. viridis in the Philippines is less well defined, with development of gametes and spawning occurring throughout the year, probably as the result of minimal annual fluctuations of salinity and temperature (Walter 1982). Settlement patterns also support observations on gametogenesis and spawning of P. viridis in its native range. Rajagopal et al. (1997) found that settlement of green mussels on the east coast of India occurred from April to November, with peaks in May to June and October.

Perna viridis has become well established in Tampa Bay. Prevailing environmental conditions are well within the tolerance range of this species, which includes salinities from 0-64 ppt and temperatures from 6[degrees]C to 37.5[degrees]C (Segnini de Bravo et al. 1998). Optimal salinity is above 27 ppt, with 50% survival at 24 ppt; optimal temperature is between 26[degrees]C and 32[degrees]C, with 50% survival at 10[degrees]C (Sivalingam 1977). As demonstrated in this study, reproduction is successful and has resulted in dense populations throughout the bay. Food supplies are adequate for supporting rapid growth and substantial standing stocks of mussels. Even though gametogenic cycles of Brachidontes exustus and P. viridis are similar in Tampa Bay, there are large potential differences in fecundity, simply because of the fact that P. viridis grows much faster and attains a much greater body mass than B. exustus. Thus P. viridis might be able to outcompete B. exustus (and other species in Tampa Bay) for space and energy. The extent to which this occurs remains to be seen; other factors such as substrate availability, primary productivity and predation may eventually limit population size.

ACKNOWLEDGMENTS

The authors thank the following University of Florida students for assisting with field collections and processing: Gabriella Bloehm, Edward DeCasio and Brooke Rimm-Hewitt. Major funding for this research was provided by US Environmental Protection Agency STAR Grant # R82-8898. Additional funds came from a contract with the Suwannee River Water Management District and a contract with the Tampa Bay Estuary Program. Jay Leverone of the Mote Marine Laboratory coordinated histology preparations.

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BRUCE J. BARBER, (1) * JONATHAN S. FAJANS, (2) SHIRLEY M. BAKER (2) AND PATRICK BAKER (2)

(1) Eckerd College, St. Petersburg, FL 33711; (2) Department of Fisheries and Aquatic Sciences, University of Florida, Gainesville, FL 32653

* Corresponding author. E-mail: [e-mail]

TABLE 1.
Numbers of individuals, Perna viridis, collected from Safety Harbor,
Gandy Bridge and Skyway Bridge sites from April 5, 2002 to
April 9, 2003.

Date                 Safety Harbor   Gandy Bridge   Skyway Bridge

April 5, 2002              20             20              20
May 9, 2002                14             13              20
July 16, 2002              20             20              20
August 13, 2002            20             20              20
September 9, 2002          19             20              19
September 29, 2002         20             15              18
November 24, 2002          20             19              20
December 19, 2002                                         19
January 14, 2003           20             21              20
February 5, 2003           20             20              20
March 6, 2003              14                             20
April 9, 2003                             18
Total                     187            186             216

TABLE 2.
Numbers of individuals, Branchidontes exustus, collected from
Safety Harbor, Gandy Bridge and Skyway Bridge sites between
April 1, 2002 and May 1, 2003.

Date                 Safety Harbor   Gandy Bridge   Skyway Bridge

April 5, 2002              8                              2
May 9, 2002               15              11
July 16, 2002             16
August 13, 2002           20              11              3
September 9, 2002          3               6              3
September 29, 2002         3               1
November 24, 2002         12              13              7
December 19, 2002                                        20
January 14, 2003           9              20              6
February 5, 2003          13               9
March 6, 2003             20               8              6
April 9, 2003                              7
Total                    119              86             47