Performance Progress Report - NA14NMF4270033

Title: Demonstrating Shellfish Aquaculture Technology in Pilot and Commercial Scale Projects: Creating New Opportunities for Maine’s Coastal Communities

PI: Brian Beal

Organization: Downeast Institute for Applied Marine Research & Education (Beals, Maine)

Award Period: July 1, 2014 through June 30, 2016

Report Period: July 1, 2015 through December 31, 2015

The project proposed to: 1) demonstrate marine aquaculture technologies in pilot and commercial scale projects designed to create jobs in coastal communities, produce healthful, local seafood, revitalize working waterfronts and support traditional fishing communities; and, 2) provide training for fishermen and others in coastal communities in aquaculture production methods.

The proposed project goals were to: To increase soft-shell clam harvests locally in the face of increasing threats due to invasive green crab predation, warming seawater temperatures, and ocean acidification, and to create a model shellfish management program for coastal Maine communities facing unprecedented declines in clam landings.

The proposed project objectives were to:

1) Determine spatial and temporal variability of green crabs, Carcinus maenas, in the Harraseeket River and intertidal areas adjacent to the river using specially designed and tested traps in an attempt to remove crabs from the ecosystem that, otherwise, would remain to prey on soft-shell clams of all age-classes;

2) Test the efficacy of “green crab fencing” of intertidal areas using methods similar to those used in the middle of the last century to deter green crabs from preying on soft-shell clam juveniles and adults;

3) Examine whether sediment buffering, under the lowest sediment pore pH conditions, will result in enhanced numbers of soft-shell clam settlers and recruits compared to control areas where no buffering occurs.

4) Examine the interactive effects of stocking density of cultured soft-shell clam juveniles (10-15 mm shell length, SL) in netted plots and crab trapping on clam survival and growth.

5) Determine whether the use of predator deterrent netting, in combination with various densities of adult soft-shell clams, will result in an enhancement of wild clam recruits; and,

6) Train clammers and other interested fishermen in the use of aquacultural techniques to improve local clam harvests.

The following highlights each objective separately along with preliminary results.

I. Determine spatial and temporal variability of green crabs, Carcinus maenas, in the Harraseeket River, Freeport, Maine and intertidal areas adjacent to the river using specially designed and tested traps (May to October 2014; May to October 2015).

Information about population dynamics of green crabs in the Harraseeket River during 2014 were presented and discussed in the First Progress Report. Below is a discussion of results from 2015 in the same general areas.

The Harraseeket River was divided into two LOCATIONS (an “Upper” and “Lower” area) based on previous observations of green crab densities. Within each location, five trapping sites were chosen – three intertidal and two subtidal (Fig. 1). Within each site, five traps (Fig. 2) approximately 100 m apart were fished every four days from 10 May to 29 October using one of two kinds of bait: crushed, adult soft-shell clams (live or frozen; Fig. 3), or salted, dead herring (similar to what local fishermen use to capture lobsters from their traps). To ensure that hauls were independent, excess bait was not discarded overboard in the vicinity of the traps, but was taken ashore where it was then disposed. Crabs from all five traps at each site were combined, and the mass of the combined sample recorded to the nearest (0.1 kg, or 0.1 lb). When a sample from the five traps at a given site had a mass > 0.91 kg (2 lbs.), a random sample (0.91 kg) was taken and the carapace width of each crab in the sample recorded to the nearest 0.01 mm using a digital caliper. In addition, when possible, the sex of each crab was determined. When the mass of a sample of five traps was < 0.91 kg, all crabs in the entire sample were measured and sexed as described previously.

The sampling design allowed us to estimate spatial and temporal variability in green crab abundance, size-frequency distributions, and sex ratios between locations and between intertidal and subtidal sites in the Harraseeket River. In addition, the use of different baits provided some insight on catch rates between locations and sites.

Figure 1. Schematic of LOCATION within the Harraseeket River where green crab traps were deployed. Five traps were fished at each site within each location every four days from 10 May to 29 October. Traps were baited with crushed live/frozen soft-shell clams or salted/dead herring that is typically used by local fishermen to capture lobsters. Clams and Herring alternated as the bait type throughout the sampling period. In both the Upper and Lower Harraseeket: Blocks 1, 2, & 5 = Intertidal; Blocks 3 & 4 = Subtidal. Traps were fished during periods of high tide.

Figure 2. Green crab trap (18-inch diameter x 36-inch long with a 4-inch diameter entrance on each end. The yellow object in the middle of the trap is a collapsed bait bag – see Fig. 3.

Figure 3. Cracked, whole soft-shell clam adults were used as bait on every other day when traps were hauled (10 May to 29 October 2015). Green crab abundance varied significantly between locations (P = 0.0196), and the pattern did not vary across sampling dates (P = 0.7348; Table 1). Overall, crabs were approximately 18% more abundant in the Lower vs. Upper Harraseeket ( x  95%CI  0.20 ± 0.04 kg/trap vs. 0.17 ± 0.03 kg/trap; n = 220; Fig. 4). Abundance varied significantly across sampling dates (P < 0.0001), and explained ca. 52% of the total variation in mean weight per trap. Mean crab weight per trap did not rise above 0.1 kg (ca. 0.2 lbs) until early August, after which, mean weight increased nearly exponentially through late September (Fig. 4). Highest mean weight per trap occurred in the Lower Harraseeket on 5 October (0.77 ± 0.47 kg/trap, or 1.7 ± 1.04 lbs/trap; n = 5) and on 17 October in the Upper Harraseeket (0.76 ± 0.64 kg/trap, or 1.67 ± 1.41 lbs/trap; n = 5). Traps baited with herring (.19 ± 0.04 kg/trap, n = 210) produced approximately 10% higher catch rates than those baited with adult clams (0.17 ± 0.04 kg/trap, n = 230); however, the effect of bait on catch rates varied significantly across sampling dates (P < 0.0001, Table 1; Fig. 5).

Table 1. Mean weight of green crab (Carcinus maenas) per trap in the Harraseeket River (10 May to 29 October 2015). Location refers to the Upper and Lower Harraseeket. Circular wire traps (91 cm long x 46 cm diameter) were hauled every four days at five sites in both locations. Within each site, a series of five traps (ca. 50 m apart) were fished. Three sites within each location were intertidal while two sites within each location were subtidal. Bait (crushed adults of soft-shell clams, Mya arenaria or dead and salted herring, Clupea harrengus) was changed on each haul throughout the sampling period. Date (a = 23), Bait (b = 2), Location (c = 2), and Site (d = 5) within Location were all considered fixed factors. Because two successive dates in May (10 & 14) and two other successive dates (30 May & 3 June) used clams as bait, data are not balanced; therefore, Type III Sums of Squares are reported.

Source of Variation df SS MS F Pr > F

Location 1 0.469 0.469 5.56 0.0196 Bait 1 1.729 1.729 20.49 <.0001 Location x Bait 1 0.000 0.000 0.00 0.9784 Date 22 83.322 83.322 44.87 <.0001 Location x Date 22 1.466 0.067 0.79 0.7348 Bait x Date 19 5.161 0.272 3.22 <.0001 Location x Bait x Date 19 2.016 0.106 1.26 0.2203 Site(Location) 8 16.049 2.006 23.77 <.0001 Site (Location = Lower) 4 7.592 1.898 22.49 <.0001 Site (Location = Upper) 4 8.457 2.114 25.04 <.0001 Date x Site(Location) 176 41.002 0.233 2.76 <.0001 Bait x Site(Location) 8 0.989 0.124 1.47 0.1742

Error 152 12.831 0.084 Total 429 159.767

1.8

1.6 Lower Harraseeket 1.4 Upper Harraseeket

1.2

1.0

0.8

0.6

Weight (lbs) per Trap Weight 0.4

0.2

0.0

10 May 15 June 25 July 3 Sept 13 Oct 29 Oct

Figure 4. Mean weight of green crabs on each sampling date (10 May to 29 October 2015) in the Lower and Upper Harraseeket River, Freeport, Maine. Each point represents the contents of 25 traps (Fig. 2) Over all sampling dates, the average weight per trap was approximately 18% greater for traps fished in the Lower vs. Upper Harraseeket River.

Crab abundance varied significantly from site-to-site within each location (P < 0.0001; Table 4; Fig. 6). In the lower Harraseeket, crab abundance was highest at two of the intertidal sites (Spar Cove and Staples Cove) and one of the two subtidal sites (adjacent to the Freeport Yacht Club). In the upper Harraseeket, green crab abundance at only one site (subtidal off Collins Cove) averaged less than 0.1 kg/trap (0.2 lbs/trap). No significant difference in mean green crab weight was observed between the other sites.

Mean sex ratios varied significantly across dates and locations (P = 0.016; Fig. 7), but were, for most dates, male-biased (i.e., above 50% male). Beginning in mid-September, there was a trend towards a 50% ratio in both locations.

Mean green crab carapace width (CW) varied significantly with most sources of variation (Table 2). Prior to mid-July when catch/trap typically was low (Fig. 4), there was high variation in mean CW (Fig. 8). After that period, however, variation in CW decreased between sampling dates at both locations, and trends with time could be seen (Fig. 8). That is, CW gradually increased from mid-June to early September. For the final 14 sampling dates beginning 3

1.8

1.6 Clam 1.4 Herring

1.2

1.0

0.8

0.6

Weight (lbs) per Trap Weight 0.4

0.2

0.0 10 May 15 June 25 July 3 Sept 13 Oct 29 Oct

Figure 5. Effect of different baits on green crab catch across all sampling dates in the Harraseeket River, Freeport. The pattern observed for crabs caught using adult soft-shell clams as bait was significantly different than that observed for crabs caught using herring (P < 0.0001; Table 1).

September, mean CW (± 95% CI) for crabs in the Lower Harraseeket was 45.7 ± 0.8 mm and 51.2 ± 0.7 mm for crabs in the Upper Harraseeket. This gradual increase in mean CW through time was also observed in crab size-frequency distributions (Fig. 9).

Mean number of green crabs per pound (Fig. 10) showed similar variability to that observed for mean crab CW. Early in the spring and summer when crabs were small, number per pound varied between 50-200 animals. As the size of crabs in the population increased through at least one molt, number per pound leveled off around 3 September. At that time, mean number per pound from traps hauled in the Lower Harraseeket River was 17.1 ± 1.92 crabs/lb (n = 15) whereas the mean number from traps hauled in the Upper Harraseeket River was 12.6 ± 0.9 crabs/lb (n = 15). These two means are significantly different (T = 4.59, df = 28, P < 0.001) suggesting that the average size of crabs was somewhat larger in the Upper vs. Lower portion of the Harraseeket River after 3 September (see Fig. 8).

1.0 Lower Harraseeket Upper Harraseeket

0.8

0.6

0.4

Weight (lbs) per Trap per (lbs) Weight 0.2

0.0

Intertidal -Spar Subtidal -Yacht IntertidalIntertidal -Across -StaplesSubtidal -Ledges Intertidal -CollinsSubtidal -Collins Intertidal -Pettengill Subtidal -Waterman's Intertidal -Sandy Beach

Site

Figure 6. Mean weight (+ 95% CI) of green crabs per trap at each site within both locations (Upper & Lower Harraseeket). Each bar represents 44 observations.

0.016). sampling date Figure 7

Mean Mean Percent of Males Percent of Males . 100 100 20 40 60 80 80 20 40 60 Analysis of variance showed thatthere was signi a sex ratio 0 0 10May 10May

of green crabs 15June 15June at

both locations (Upper& LowerHarraseeket) 25July 25July Lower Lower Harraseeket 3 Sept3 3 Sept3 ficant Datex Location interaction (P = Upper Harraseeket 13Oct 13Oct 29Oct 29Oct

on each on

Table 2. Analysis of variance on mean carapace width of green crabs from traps (Fig. 2) hauled every four days from 10 May to 29 October 2015 in the Harraseeket River, Freeport, Maine. Five traps were assigned to each of five sites (three intertidal and two subtidal) at two locations (Upper vs. Lower River). Two baits were used--herring (salted), and soft-shell clam adults (crushed). Each bait type was used on every other sampling date. Crabs were taken from a 0.91 kg (2 lb) random sample of the combined catch from the five traps at each site on each sampling date. When the total mass from a particular site did not equal 0.91 kg, all crabs were measured.

Source of Variation df SS MS F Pr > F

Location 1 124.099 124.099 9.65 0.0026 Bait 1 111.374 111.374 8.66 0.0042 Location x Bait 1 11.359 11.359 0.88 0.3500 Date 22 11620.791 528.217 41.08 <.0001 Location x Date 22 3398.491 154.476 12.01 <.0001 Bait x Date 18 1207.779 67.098 5.22 <.0001 Location x Bait x Date 14 221.917 15.851 1.23 0.2688 Site(Location) 8 930.683 116.335 9.05 <.0001 Date x Site(Location) 143 7556.290 52.841 4.11 <.0001 Bait*Site(Location) 8 119.103 14.887 1.16 0.3348

Error 81 1041.521 12.858 Corrected Total 322 35650.415

70 Lower Upper 60

Mean Mean Carapace Width (mm)

0 10 May 15 June 25 July 3 Sept 13 Oct 29 Oct

Figure 8. Mean carapace width of green crabs on each sampling date from two locations in the Harraseeket River, Freeport, Maine. Each point represents the mean CW from 25 traps.

UPPER HARRASEEKET LOWER HARRASEEKET

50 50 30 May 30 May 40 N = 5 40 N = 22

30 30

20 20

10 10

0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

50 50 27 June 27 June 40 N = 22 40 N = 14

30 30

20 20

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0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

50 50 29 July 29 July 40 N = 67 40 N = 66

30 30

20 20

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0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

50 50

40 30 August N = 124 40 30 August N = 165

Percent Frequency

30 30

20 20

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0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

50 50

40 27 September N = 99 40 27 September N = 135

30 30

20 20

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0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

50 50

40 29 October N = 141 40 29 October N = 173

30 30

20 20

10 10

0 0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90

Carapace Width (mm)

Figure 9. Size-frequency distribution of green crabs on selected dates from two locations in the Harraseeket River, Freeport, ME. N = number of crabs in the sample. represents information from 25 traps. (Uppervs. LowerHarraseeket River, Freeport, Maine) from 10 May to 29 October 2015.Each point Figure 10. Mean Number of Crabs Per Pound 100 150 200 50 10May 0

Mean number of green crabs per pound from traps hauled 15June

25July 3 Sept3 every four days intwo locations 13Oct Upper Lower 29Oct

II. Test the efficacy of “green crab fencing” of intertidal areas using methods similar to those used in the middle of the last century to deter green crabs from preying on soft-shell clam juveniles and adults;

This portion of the project was presented in the First Progress Report. The data suggested that the use of fencing is not any more effective than using plastic netting alone. Fences are expensive to build and difficult to maintain compared to plastic, flexible netting. Survival and growth rates were similar between fenced and unfenced controls. Netted plots within each fence resulted in higher overall survival than control plots within the fenced areas.

III. Examine whether sediment buffering, under the lowest sediment pore pH conditions, will result in enhanced numbers of soft-shell clam settlers and recruits compared to control areas where no buffering occurs.

This field trial was designed to test the relative importance of coastal acidification vs. predation on 0-year class soft-shell clams. Increasing acidification lowers the availability of carbonate ions in water and sediments, and can impair the ability of calcium carbonate-bearing organisms, such as the soft-shell clam Mya arenaria, to build and maintain their shells. Small bivalves, such as newly-settled clams, may dissolve completely. This process, termed “death by dissolution,” is considered a leading cause of mortality in young clams (Green 2004, 2009).

Results from 2014 (6-8 May 2014) were presented in the second performance report. The experimental design used in 2014 was repeated in 2015, but instead of conducting the trial at one intertidal location, two locations were chosen based on previous pH determinations. The locations in Freeport, Maine were the mid-tide at Little River (Lat. 43o 49’ 36.8862”N; Long. -70o 04’ 36.224”W) and Winslow Park (Lat. 43o 48’ 1.4724”N; Long. -70o 07’ 9.6558”W).

To determine the interactive effects of crushed soft-shell clam shells (designed to buffer sediments) and predator exclusion (using plastic, flexible netting – 4.2 mm aperture, as described above), the experiment was initiated on 29 and 30 April 2015 at Little River and Winslow Park, respectively. Thirty 2m x 2m plots were established in a 6 x 5 matrix with 5 m spacing between rows and columns. Five replicates of six treatments were used: 1) Control, no netting; 2) Control, netting; 3) 13 lbs of crushed Mya arenaria shells, no netting; 4) 13 lbs of crushed Mya shells, netting; 5) 26 lbs of crushed Mya shells, no netting; 6) 26 lbs of crushed Mya shells, netting. Treatments were randomly assigned to positions within the matrix. This was a completely randomized design. Netting was a polypropylene (flexible) material with an aperture of 4.2 mm x 4.2 mm (see: http://www.industrialnetting.com/ov7100.html).

Crushed shells of Mya were obtained from a recent commercial shell heap located on Great Wass Island in the town of Beals, ME that had been aging for ca. 1 year (A. Carver, A.C. Inc., Beals, ME, pers. comm.). Shells were crushed manually into pieces that varied in size from 6-25 mm (greatest length). Shells were spread by hand within each of the plots designated as shell treatment plots (N = 20; Fig. 11). Pieces of flexible netting (2.3 m x 2.3 m) were used to cover

Figure 11. Stewards of the Sea president, Chad Coffin, spreading crushed soft-shell clams in a 2m x 2m plot. one-half of the plots (Fig. 12). Each net was secured in place by walking along the periphery and forcing the edge (15-20 cm) into the sediments (as described above). Each net had two Styrofoam floats affixed to the side nearest the sediment (as described above; Fig. 13).

Winslow Park – Large Scale Sediment-buffering Experiment (thirty 2m x 2m plots)

On 5 August 2015, two pH readings were taken from the center of at least two replicates (chosen at random) of each treatment at Winslow Park (Fig. 14). Readings were made by Michael Doan (Friends of Casco Bay) who used a Fisher Scientific Accumet AP 115 pH meter and 13-620-AP50A combination electrode. The electrode was tilted at an acute angle to the mudflat surface and its tip placed approximately 6 mm into the sediments. Measurements were recorded when the meter read “STABLE.” The electrode was rinsed with distilled water between each measurement. Prior to taking samples from netted plots, each net was pealed back exposing two-thirds of the sediment. Nets were immediately re-secured around each plot after sampling. ANOVA on the mean pH reading (Table 3) indicated a significant an effect due both to netting and to the presence of shell. That is, pH was significantly lower in netted (7.26 ± 0.18, n = 5) vs. unnetted (7.48 ± 0.29; n = 10) plots. Also, pH was significantly lower in plots without shell (7.20 ± 0.45, n = 5) vs. plots with shell (13-lbs = 7.48 ± 0.39, n = 6; 26-lbs = 7.56 ± 0.49, n = 4).

On 30 October 2015, two pH readings were taken from the center of each of the 30 plots at Winslow Park. In addition, two benthic cores (A = 0.01824 m2) were taken from each plot. The sixty samples were washed separately through a 1 mm mesh sieve and all individuals of Mya arenaria and Mercenaria mercenaria were enumerated and the SL of each taken to the nearest 0.01 mm using digital calipers.

Figure 12. Crushed shell (13 lbs) and protective netting in 2m x 2m plot.

Mean pH readings taken from Winslow Park on 30 October demonstrated a clear trend towards significantly lower (more acidic) surface sediments in plots that were protected with netting (Fig. 15). Crushed shell had no significant effect on sediment pH (Table 4), suggesting that they had no effect on sediment buffering.

There was no significant effect of adding shells to the 2m x 2m plots at Winslow Park on soft- shell clam recruitment (Table 5; Fig. 16). Mean clam abundance in plots with and without shell was 3.9 ± 8.1 ind/core (19.8 ind/ft2; n = 10) and 3.4 ± 5.9 ind/core (17.3 ind/ft2; n = 20), respectively. Conversely, plots that were covered with the polypropylene predator deterrent netting contained approximately 70x more clam recruits than plots without netting, regardless whether shell was added to plots. For example, mean number of soft-shell clam recruits in netted plots was 7.0 ± 9.2 ind/core (35.6 ind/ft2; n = 15) vs. 0.1 ± 0.1 ind/core (0.5 ind/ft2; n = 15). This result is similar to that observed in 2014 in the sediment-buffering experiment conducted at Staples Cove.

Figure 13. Netted plot at Winslow Park (1 May 2015). Lanes Island is visible in the background.

The shell length of clam recruits (Fig. 16) varied from 1.42 mm to 7.47 mm with a mean ± 95% CI of 2.84 ± 0.15 mm (n = 213). For netted plots, mean SL did not vary significantly between shell treatments (P = 0.5783), the size-frequency distributions did (3 x 3 G-test of independence, P = 0.0076). A higher percentage of clam recruits < 2 mm occurred in plots without shell compared to plots with crushed shell.

Although sediment pH did not appear to be influenced by the addition of crushed shells to the substrate, it is possible that the physical environment created by the crushed shells may act as a spatial refuge from small predators. That is, if 0-year class clam recruits had been found at higher densities in plots with crushed shells vs. plots without crushed shells, there could have been at least two very different reasons: 1) sediment buffering, or 2) enhanced habitat. The large-scale experiment (2m x 2m plots) could not, however, distinguish between these two competing hypotheses.

Winslow Park – Small-Scale Sediment-buffering Experiment

On 30 April 2015, a small-scale experiment was initiated at Winslow Park, Freeport, Maine, adjacent to the large-scale, sediment buffering experiment in an attempt to extend and interpret results from the larger-scale experiment. Eight treatments were employed using a factorial design with a = 4 substrate amendments and b = 2 levels of predator exclusion netting. The four levels of substrate amendments were: 1) control, only ambient sediments; 2) crushed shell; 3) marble chips (see: http://www.lowes.com/pd_2527-29629- 901835_0__?productId=50315629); and, 4) small granite cobbles. The two levels of predator exclusion were: 1) control – no netting; and, 2) netting (flexible netting with 4.2 mm aperture similar to that used in the larger-scale experiment). Experimental units were plastic horticultural pots (15 cm diameter x 15 cm deep – see Beal, 2006). Units were dug into the substrate so that a 3-4 mm lip extended above the sediment surface. Units were filled with ambient sediments and for those treatments with substrate amendments, the shell, chips, or cobbles were added to top of the substrate in the units (Figure 17). A piece of the netting (45 cm x 45 cm) was secured around one-half of the units and was held in place with a rubber band. Ten replicates of each of the eight treatments were employed, and treatments were assigned randomly to positions within an 8 x 10 matrix. All units were removed from the mudflat on 30 October 2015 and the contents of each sieved through a 1 mm mesh. All live Mya and Mercenaria were enumerated and the SL of each measured to the nearest 0.01 mm using digital calipers.

9.5 n = 2

9.0

n = 3

8.5 n = 2

8.0 n = 5 n = 3

pH + 95% CI pH 7.5

7.0

6.5 No Shell 13-lbs Shell 26-lbs Shell 13-lbs Shell 26-lbs Shell No Net No Net No Net Net Net

Figure 14. Mean pH (+95% CI) from five of six treatments taken on 5 August 2015 at Winslow Park, Freeport, Maine. Number above each error bar refers to the number of plots (out of five) sampled. Table 3. ANOVA on mean pH readings from five of six treatments at Winslow Park on 5 August 2015.

Source of Variation df SS MS F Pr > F

Shell 2 1.6004 0.8002 18.43 0.0049 Shell vs. No Shell 1 1.5703 1.5703 36.18 <.0001 13 vs. 26 1 0.0300 0.0300 0.69 0.4432 Net 1 1.2322 1.2322 28.37 0.0031 Shell x Net 1 0.0013 0.0013 0.03 0.8678 Sample(Shell x Net) 5 0.2171 0.0434 0.47 0.7947

Error 20 1.8515 0.0926 Corrected Total 29 4.0412

No Netting Netting

8.0

7.5

7.0

Mean pH Mean

6.5

6.0 0 13-lbs 26-lbs 0 13-lbs 26-lbs

Shell

Figure 15. Mean pH readings taken at Winslow Park, Freeport, Maine on 30 October 2015. Significantly lower pH readings occurred in netted vs. unnetted plots (Table 4). No significant differences were observed due to the presence of crushed soft-shell clam shells. (n = 5) Table 4. Analysis of variance on mean pH readings from sediment-buffering plots at Winslow Park on 30 October 2015. Five replicates of each of six treatments (a fully factorial combination of three levels of shell [0, 13, or 26 pounds] and two levels of netting [4.2 mm aperture – present vs. absent] were assigned randomly to 4m2 plots. Two replicate pH readings were recorded from each plot.

Source of Variation df SS MS F Pr > F

Shell 2 0.0445 0.0223 0.39 0.6807 Net 1 2.5958 2.5958 45.57 <.0001 Shell x Net 2 0.0573 0.0287 0.50 0.6108 Plot(Shell x Net) 24 1.3670 0.0569 1.89 0.0495

Error 30 0.9038 0.0301 Corrected Total 59 4.9685

Table 5. Analysis of variance on the ranked number of soft-shell clam recruits (0-year class individuals) in large plots at Winslow Park (established on 30 April and sampled on 30 October 2015 – 183 days). Two core samples (0.01824 m2) were taken from each of the thirty 2m x 2 m plots. Treatments were the fully factorial combination of three levels of crushed shell (0, 13-lbs, 26-lbs per plot) and two levels of predator deterrent netting (present vs. absent). Each treatment was randomly assigned to five plots within a 6 x 5 matrix.

Source of Variation df SS MS F Pr > F

Shell 2 37.975 18.988 0.08 0.9275 Net 1 1440.600 1440.600 5.73 0.0248 Shell x Net 2 37.975 18.988 0.08 0.9275 Plot(Shell x Net) 24 6032.200 251.342 2.67 0.0059

Error 30 2827.750 94.258 Corrected Total 59 10376.500

40 No Shell (N = 77)

0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

40 13-lbs Shell (N = 15)

Percent Frequency 10

0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

26-lbs Shell (N = 118) 40

0 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 Shell Length (mm)

Figure 16. Size-frequency distribution of soft-shell clam recruits in netted plots at Winslow Park on 30 October 2015. A single clam was found in one of the five unnetted plots containing no shell (3.38 mm), containing 13-lbs of shell (2.40 mm), and containing 26-lbs of shell (2.80 mm). A 3 x 3 G-test of independence indicated that the distributions were significantly different between the netted treatments (P = 0.0076).

a b

) )

Figure 17. Examples of experimental units used in the small-scale sediment buffering experiment at Winslow Park (30 April – 30 October 2015). a) Marble chips; b) crushed shell. Both treatments are without predator exclusion netting. Units are 15 cm in diameter x 15 cm deep and were filled with ambient sediments prior to the addition of the chips or shells.

Number of 0-year class soft-shell clams did not vary significantly over the four substrates (ambient sediments, crushed shell, marble chips, or small granite cobble) (P = 0.6727), but significant differences were detected between units with vs. without the flexible netting (P = 0.0017; Table 6; Fig. 18). Approximately 4x as many recruits were found in netted vs. unnetted experimental units (5.35 ± 2.4 ind/unit vs. 1.35 ± 0.67 ind/unit (n = 40), respectively.

Table 6. Analysis of variance on the mean number of 0-year class individuals of the soft-shell clam, Mya arenaria, from the small-scale sediment-buffering experiment at Winslow Park, Freeport, Maine (30 April – 30 October 2015). Four levels of substrate (ambient sediments; crushed clam shell; marble chips; small granite cobble) and two levels of predator protection (flexible netting – aperture 4.2 mm; no netting) were combined factorially to produce 8 treatments. (n = 10)

Source of Variation df SS MS F Pr > F

Substrate 3 46.700 15.567 0.52 0.6727 Netting 1 320.000 320.000 10.60 0.0017 Substrate*Netting 3 100.700 33.567 1.11 0.3498

Error 72 2172.800 30.178 Corrected Total 79 2640.200 clams in granitechipmarble in clamsthe cobble and treatments were 25 SL P36.90, = (G = 0.002). Approximately 70% clams of withchipsin marbleunits were mm < 10 wascompareusedindependence to were 0.0001). (Fig. units unnetted 19) G compared 5were usinga x2 treatmentssubstrate Size(P0.4542). = frequencyofcl distributions (P0.0039). significant = significant No SLmeandifference wasin observed between four the those unitsinthan ±unprotected (7.0 2.63 mm, 24). Thisndifference= wasstatistically shelllength Mean ofrecruitsin units=was 2.35netted mm, (12.5 33) 75% ±n more than larger approximately greater 4x numbersfrom netted unnettedvs. experimental units. (n= 10) not.Netting,or however, No significantdifferences occurred scale sediment Figure 18. , -

28%controlinclamsthe ofshell and were treatments whereas whereas w thisrate

< Mean Number per Unit (+ 95% CI)

10mm in units with protected predator Ambient sediments 10 12 14 16

Ninety inclamsNinety percent unitsof unnetted were 0 2 4 6 8 Mean number of 0 -

buffering experiment at Winslow Park, Freeport, Maine (30 April to 30 October 2015). Crushed clam shell No No Netting as between and substrates. 38% thefor 51% other addition, In

resulted in a significantenhancement clamof recruits (Table6) with Marble chips - year class individuals of the soft Small granite cobble between substrate types regardless whether treatments werenetted sizefrequency distributions

Ambient sediments deterrentInnetting. addition,

Crushed clam shell - test ofindependencetest (G= P 63.74, < < - > shell clam,

Netting 10 whereas10 mm, 42% clamsonly of

20 mmwhile SL, only20 10 >

20 mm SL. 20mm Marble chips between Small granite cobble am recruitsam in netted vs. My a arenaria

the fourthe substrates ax G 4 5 , fromsmall the

- 15% of15% between between - test oftest

- 50

40 No Netting (N = 54) Netting (N = 215) 30

PercentFrequency 10

0 0 5 10 15 20 25 30 35

Control (N = 54) 30 Shell (N = 59) Marble (N = 62) Rock (N = 93)

PercentFrequency 10

0 0 5 10 15 20 25 30 35

Shell Length (mm)

Figure 19. Size-frequency distribution of 0-year class soft-shell clams from the small-scale sediment- buffering experiment at Winslow Park, Freeport, Maine (30 April to 30 October 2015). Top panel: a 5 x 2 G-test of independence yielded a test statistic of 63.74, P < 0.0001. Bottom panel: a 5 x 4 G-test of independence yielded a test statistic of 36.90, P = 0.0002. Little River – Large Scale Sediment-buffering Experiment (thirty 2m x 2m plots)

Prior to establishing the 6 x 5 matrix of 2m x 2m plots at Little River, Freeport, Maine on 29 April 2015, 10 benthic cores (A = 0.01824 m2) were taken and the contents of each washed through a 1 mm sieve. Every core contained at least one and one core contained 29 clams. Clams ranged in SL from 3.1 mm (animals that apparently settled late in the fall of 2014) to 37.7 mm, and this resulted in a bi-model size-frequency distribution (Fig. 20).

N = 71

Percent Frequency Percent 5

0 0 5 10 15 20 25 30 35 40

Shell Length (mm)

Figure 20. Size-frequency distribution of wild soft-shell clams in ten benthic cores taken near the mid- intertidal at Little River, Freeport, Maine on 29 April 2015. Density = 389.2 ± 340.1 individuals/m2 (36.2 ± 31.6 individuals/ft2, n = 10).

Crushed clam shells established in large plots at a rate of 13- or 26-lbs did not result in a significant enhancement of 0-year class recruits compared to plots without shells (P = 0.6835; Table 7); however, plots covered with flexible netting yielded approximately 20x more recruits than plots without netting (5.27 ± 2.59 ind/unit vs. 0.23 ± 0.18 ind/unit, n = 15).

Table 6. Analysis of variance on the ranked clam recruit data from the large (2m x 2m) plots at Little River, Freeport, Maine. Plots (N = 30) were established on 29 April 2015 and sampled on 30 October 2015. Experimental design and sampling effort were similar to that described for the same field experiment at Winslow Park (Table 5).

Source of Variation df SS MS F Pr > F

Shell 2 138.775 69.388 0.39 0.6835 Net 1 7866.150 7866.150 43.83 <.0001 Shell x Net 2 75.625 37.813 0.21 0.8115 Plot(Shell x Net) 24 4306.950 179.456 1.54 0.1305

Error 30 3495.000 116.500 Corrected Total 59 15882.500

No Netting Netting

Mean # of Juvenileofcore # Meanper Soft-Shell Clams 0 13-lbs 26-lbs 0 13-lbs 26-lbs

Shell

Figure 21. Mean number of 0-year class recruits of Mya arenaria in the large plots at Little River (30 October 2015). No significant difference was detected due to the presence of crushed shells. Densities in netted plots were approximately 20x greater than those in unnetted plots (P < 0.0001; Table 6). Mean SL of recruits (5.8 ± 0.69 mm, n = 165; Fig. 22) did not vary significantly with either shell or netting treatments (P > 0.35). Most (89%) of the clams were < 10 mm SL. A 3 x 5 (shell treatments x 5 size classes) G-test of independence indicated that there was no significant difference in the frequency distribution of clam recruits in netted plots (control, 13-lbs, and 26-lbs) (G = 9.47, P = 0.3045).

Percent Frequency Percent

0 0 5 10 15 20 25 30

Shell Length (mm)

Figure 22. Size-frequency distribution of 0-year class soft-shell clam recruits at Little River from large- scale plots. (N = 165)

Little River – Small-Scale Sediment-buffering Experiment

No significant differences were observed in mean number of 0-year class clam recruits for any of the sources of variation (Substrate: P = 0.7747; Netting: P = 0.6683; Substrate x Netting: P = 0.4967). Mean density per experimental unit was 1.40 ± 0.57 individuals (n = 80; Fig. 23). No significant difference in mean SL was observed among any of the sources of variation (Substrate: P = 0.1138; Netting: 0.5152; Substrate x Netting: P = 0.7960). Mean SL pooled across all treatments was 9.05 ± 0.99 mm (n = 112). Size-frequency distribution of these individuals demonstrated that approximately 80% were less than 12 mm (Fig. 24).

sampled on 30 October 2015 atLittle River,Freeport, Maine. (n = 10) Figure 23.

Ambient sediments Mean Number per Unit (+ 95% CI)

0 2 4 6 8

Mean number of 0 Crushed clam shell No No Netting

Marble chips Small granite cobble - year class soft

Ambient sediments - shell clam recruits insmall

Crushed clam shell Netting

Marble chips Small granite cobble

- scale experimental units

Percent Frequency Percent

0 0 5 10 15 20 25 30

Shell Length (mm)

Figure 24. Size-frequency distribution of 0-year class soft-shell clam recruits from the small-scale sediment-buffering experiment at Little River (29 April to 30 October 2015. (N = 112)

Little River – Large Scale Experiment -- 1-yr age class clams (30 October 2015)

Because densities of wild clams on 29 April 2015 at Little River were relatively high (389.2 ± 340.1 individuals/m2, n = 10; Fig. 20), it was possible to follow the fate of that cohort in the sediment- buffering 2m x 2m plots during the 30 October 2015 sampling (see above). All 1-year class clams in the core samples had a distinct overwinter mark (disturbance line) in their shell that was somewhat less distinct than that associated with cultured clams (see Beal et al., 1999; Fig. 25). Clams with this disturbance line were sampled at the same time that 0-year class soft-shell clam recruits were because the core sampler was pushed into the sediments approximately 15 cm. All live 1-yr class individuals in each of the sixty samples were enumerated, and the SL of the overwinter mark and the final SL of each live clam was measured to the nearest 0.01 mm using digital calipers. Mean number and SL of the 1- year class clams were examined in terms of the six sediment buffering treatments (see above). Specifically, there was an interest in determining whether densities of these clams would be higher in netted vs. unnetted plots.

Figure 25. Wild individual of a soft-shell clam from cores taken on 30 October 2015 at Little River within the large-scale sediment buffering experiment. A distinct overwinter mark is visible in the shell of this clam. The length of the overwinter disturbance line and the final shell length of each live wild clam that was sampled in the two cores per plot were measured.

Table 7. Analysis of variance on the mean number of wild (1-year class) adult clams in benthic core samples from the 2m x 2m plots at Little River, Freeport, Maine on 30 October 2015. “Shell” refers to one of three substrate amendments: None (the ambient control); 13-lbs of crushed shell added to each plot; or, 26-lbs of crushed shell added to each plot. “Net” refers to one of two levels of predator deterrent netting: None (the ambient control); or, a piece of flexible netting (4.2 mm aperture) that covered the entire plot. The two fixed factors were combined to make six factorial treatments on 29 April 2015. Two benthic core samples (A = 0.01824 m2) were taken from each plot.

Source of Variation df SS MS F Pr > F

Shell 2 214.433 107.2167 2.11 0.1433 Net 1 322.017 322.0167 6.33 0.0189 Shell x Net 2 19.633 9.8167 0.19 0.8257 Plot(Shell x Net) 24 1220.000 50.833 1.12 0.3809

Error 30 1362.500 45.417 Corrected Total 59 3138.583 Wild clam densities were significantly greater (by 2.3 x) in plots that received the predator netting than in plots that did not receive the netting (P = 0.0189; Table 7), but the addition of crushed shell did not affect numbers significantly (P = 0.1433; Table 7). Mean number of 1-year class individuals in plots that were netted was 8.2 ± 3.16 individuals/core, or 451.4 ± 173.36 individuals/m2 (n = 15), vs. 3.6 ± 2.43 individuals/core, or 197.4 ± 133.48 individuals/m2 (n = 15).

No Netting Netting 20

Mean # of 1-yr old Soft-Shell Clams per core per Soft-Shellold Clams 1-yr of # Mean 0 13-lbs 26-lbs 0 13-lbs 26-lbs

Shell

Figure 26. Mean number of 1-year class soft-shell clams in the 2m x 2m plots at Little River, Freeport, Maine on 30 October 2015. ANOVA (Table 7) indicated that over twice as many clams occurred in plots protected with flexible netting (regardless of the level of shell added to the plots) compared with clam numbers in unprotected (no netting) plots. (n = 5).

Mean final SL and absolute growth (Final SL – Initial SL) did not vary significantly across the six treatments (Table 8). Overall mean SL increased from 26.2 ± 2.3 mm (n = 71) on 29 April to 42.3 ± 1.6 mm (n = 26, which represents the number of plots that live clams were sampled from, not the number of clams measured [= 355]) on 30 October. Mean absolute growth (new shell from the overwinter disturbance line to the ventral margin) was 16.3 ± 0.76 mm (n = 26). Approximately 15% of the 1-year class clams attained legal size (50.8 mm, or 2-inches in SL) by 30 October (Fig. 27). Table 8. Mean (± 95% CI) of final shell length and absolute growth of 1-year class soft-shell clams at Little River (29 April to 30 October 2015). n refers to the number of plots (of 5) that contained clams. ANOVA on these means indicated no significant differences for any of the main or interaction sources of variation (P > 0.20).

Shell Treatment Netting n Final SL (mm) Absolute Growth (mm)

Ambient (Control) No 3 44.1 ± 19.3 16.7 ± 6.3 Ambient (Control) Yes 4 43.5 ± 5.5 17.3 ± 3.5

13-lbs No 5 44.1 ± 5.2 17.0 ± 1.8 13-lbs Yes 5 41.0 ± 3.8 15.6 ± 0.4

26-lbs No 5 39.3 ± 2.7 15.6 ± 3.7 26-lbs Yes 4 42.7 ± 4.3 16.1 ± 2.0

N = 71

Percent Frequency Percent 5

0 0 10 20 30 40 50 60 Shell Length (mm)

N = 355

Percent Frequency Percent 5

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 27. Initial (29 April 2015) and final (30 October 2015) size-frequency distribution of wild soft-shell clams that occurred in 2m x 2m plots at Little River, Freeport, Maine. Clams were individuals that settled to the flat during the summer and fall of 2014 that occurred naturally in the sediments when the sediment-buffering experiment (see above) was initiated. Approximately 15% of animals attained legal size (50.8 mm, or 2-inches) by 30 October 2015.

IV. Examine the interactive effects of stocking density of cultured soft-shell clam juveniles (10-15 mm shell length, SL) in netted plots on clam survival and growth.

In 2014, a comparative experiment was deployed at two lower intertidal sites (Collins Cove and Wolf Neck) in the Upper Harraseeket River (19-21 April 2014) to examine effects of density and predator exclusion on the growth and survival of cultured juveniles of Mya arenaria. At each site, a total of forty 22-ft x 14-ft (28.6 m2) plots were established with a planting area of 20-ft x 12-ft (22.3 m2). Cultured clam seed (8-12 mm SL) was planted at a density of 15 or 30 individuals ft-2 (161-323 ind. m-2) within each plot. Nets were deployed in blocks of four nets (2 replicates of each planting density per block; Fig. 28) resulting in 10 blocks at each site.

Figure 28. A block of four nets at Collins Cove, South Freeport, Maine on 12 July 2014.

Two large (A = 0.01824 m2) benthic cores were taken from each netted plot at both sites from 8-10 November 2014. Each sample was washed separately through a 1.0 mm mesh and all live soft-shell clams (wild and cultured) were enumerated. At Collins Cove (western side of the Harraseeket River), the mean number of live cultured clams was extremely low overall (22.6 ± 12.2 ind. m-2) and depended upon stocking density. Compared to initial planting densities (161 or 323 ind. m-2), estimated survival rates in the lower density plots was 1.67% (mean number per core = 2.7 ± 3.9 ind. m-2, n = 40) and 13.2% in the higher density plots (mean number per core = 42.5 ± 23.0 ind. m-2, n = 40). In addition to live clams, dead clams also were discovered in the benthic cores. The distribution of animals that were found dead, with undamaged valves compared with the distribution of animals that were seeded indicated that ca. 55% had added shell prior to death. Clams that increase in shell length and then perish without signs of shell damage are likely to have been preyed upon by the nemertean, Cerebratulus lacteus. In addition, a number of clams were recovered dead with damaged valves typical of crustacean (e.g., Carcinus maenas, Cancer irroratus). Wild clam recruits were observed in only 17 of the 80 cores (ca. 21%) taken at Collins Cove in November 2014. Mean density of wild recruits was 48.7 ± 12.7 ind. m-2 (n = 80), and no sources of variation were significant for mean recruit density. The density of wild recruits in November was not significantly different than initial wild recruit density taken in April (33.3 ind. m-2).

At Wolfe Neck (eastern side of the Harraseeket River), cultured clam survival was not significantly different than 100% for both stocking densities. Mean percent survival in plots stocked initially at 161 m-2 was 96.7 ± 37.5% (n = 40) compared to 107.8 ± 20.8% (n = 40) in plots initially stocked at twice that density. That is, mean number of cultured clams in the low density plots was 156.3 ± 60.6 ind. m-2 and 348.1 ± 67.2 ind. m-2 in the high density plots (n = 40 in both instances). Only initial stocking density had a significant effect on mean density of clams at the end of the study. While some dead clams with undamaged valves and chipped/ crushed valves were found in the benthic cores, the fact that overall survival was so high outweighs the observed mortality at this site. Growth was relatively fast (Fig. 29), with 26% of cultured clams attaining a size > 50.8 mm (legal size, or 2-inches in SL). Wild juvenile soft-shell clams (0-year class individuals) were extremely abundant in the benthic cores. For example, four of the 80 cores contained > 1,000 animals (Figs. 30-32). Overall, densities of wild recruits per core ranged from 0 to 1,102. The mean number of wild recruits per square meter was 14,830.0 ± 3,597.3 (n = 80). If this estimate is extrapolated to the size of the seeded plots (22.3 m2), then the mean number of wild recruits per plot would be 330,709.98 ± 80220.77. That is, for every 3.02 plots, approximately 1 million wild seed clams occurred. The size-frequency of these clams indicated that approximately 75% were < 12 mm SL.

Work that occurred in 2015 was designed to examine if differences in cultured clam survival and wild recruitment between the two sides of the Harraseeket River are consistent between years, or whether the results obtained in 2014 are unique to that year and those sites.

Figure 29. Cultured clams from a core sample from the Wolf Neck site on 10 November 2014. A distinct “hatchery mark” occurs near the umbo of each clam that distinguishes it from a wild clam (see Beal et al. 1999). Approximately 25% of cultured clams planted in April 2014 attained legal size (> 50.8 mm, or 2-inched SL).

Figure 30. Benthic core (15 cm diameter x 15 cm deep) on the surface of a plot at Wolf Neck flat (10 November 2014) that had been seeded with cultured clams in April 2014, then covered with protective, flexible netting. Most of the small holes are wild, 0-year class individuals of soft-shell clams, Mya arenaria. Some of the core samples contained > 1,000 soft-shell clam recruits.

Figure 31. Soft-shell clams taken from a single core sample in a netted plot at Wolf Neck on 10 November 2014. Most of the clams are wild and settled into the plot during the experimental period that began on 18 April 2014. One cultured clam (ca. 50 mm SL) can be seen near the bottom of the photo.

Figure 32. An example of the contents of a single benthic core from a netted plot at Wolf Neck flat (10 November 2014) washed through a 1.0 mm sieve. Approximately 12 cultured clams can be seen in the lower right hand corner of the sieve, the remaining clams are wild and were not in the netted plot at the beginning of the experiment.

To determine if the stark differences in cultured clam survival and wild recruitment observed between Collins Cove and Wolfe Neck were anything but a random chance phenomenon, we established ten study sites on both sides of the Harraseeket River. Each site was approximately 200 m apart. At each site, we placed four nets (22.3m2, as in 2014) with all receiving soft-shell clam seed grown in the upweller from 2014 (Fig. 33) and overwintered in Freeport using techniques outlined in Beal et al. (1995) at 10.5 individuals per square foot (113 ind. m-2).

Figure 33. Clams from the upweller on 25 September 2014. Most clams had attained shell lengths > 20 mm; however, fouling organisms (tunicates – both colonial and solitary; mussels; and hydroids) attached to the shells making cleaning difficult and time-consuming. Some of these clams were overwintered in Freeport using techniques outlined in Beal et al. (1995), and were planted in April 2015.

Beginning in mid-April 2015, nets were deployed at each of the 20 stations along the Harra- seeket River (Figs. 34-37). A benthic core (A = 0.01824 m2) was taken from the middle of each netted area at each station (N = 80) prior to seeding with cultured clams to establish natural densities and size-frequency distributions of wild clams at the beginning of the field trial.

Between 4-10 November 2015, two benthic cores (A = 0.01824 m2) were taken from each netted plot. The contents of each were washed through a 1 mm sieve and all live and dead cultured clams and all wild clams enumerated and measured to the nearest 0.01 mm using digital calipers. Cultured clams were assigned one of three fate categories: 1) alive; 2) dead, with undamaged valves (typical of nemertean – Cerebratulus lacteus – predation ; and, 3) dead with crushed or chipped valves (typical of crustacean predation).

Figure 34 Deploying predator exclusion netting near Collins Cove, South Freeport Maine in April 2015.

Figure 35. Chad Coffin, President of the Maine Clammer’s Association spreading out predator netting on a plot in South Freeport, Maine in April 2015.

Figure 36. The periphery of each net is pushed into the soft sediments by walking on it, then filling in the furrow with the surrounding sediments. This secures the net in place.

Figure 37. A secured net that is protecting seed clams (12-20 mm SL). The Styrofoam floats are affixed to the underside of the net and act to lift the netting 8-10 inches (20-25 cm) off the mudflat surface during tidal inundation so that the netting does not interfere with clam feeding.

The percent of live cultured clams was estimated for each core sample by taking the total number of clams in the sample (live and dead) and then dividing the number of living clams by the total number of live and dead clams. When no live or dead clams were encountered in a sample, the percent alive was set to zero. Analysis of variance was performed on the mean arcsine-transformed percent survival data. The full linear model contained side of river, site nested within side of the river, and net nested within site and side of the river. Because all three sources of variance were statistically significant, the linear model was decomposed to examine both sides of the river separately.

Seeded field plots – East Side of the Harraseeket River (fate of cultured juvenile clams)

Core samples taken at each of the ten sites along the east side of the Harraseeket River (Fig. 38) demonstrated mean rates of live clams between 0 and 55% ( x = 25.1 ± 10.4%, n = 40; Table 9 & 10); however, no clear spatial (i.e., site-to-site) pattern was apparent. Although few nemerteans (Cerebratulus lacteus) and green crabs (Carcinus maenas) were found in the core samples (4 of 80 for each species), effects of predation on the fate of clams at each site was important. The combined mean for dead undamaged and dead crushed categories (Table 9) was 59.9 ± 11.4% (n = 40).

VIII

VII

III

Figure 38. Study sites located on the east side of the Harraseeket River. Four 14-ft x 22-ft plots were installed at each site and seeded with cultured clams at a density of ca. 10.5/ft2 in mid- April 2015. Two benthic cores were taken from each netted plot during early November 2015.

Table 9. Mean (± 95% confidence interval) percent alive, dead undamaged, and dead crushed cultured soft-shell clams at ten sites along the east side of the Harraseeket River. Clams were seeded at a density of 10.5/ft2 in four netted plots at each site in mid-April 2015. Two core samples were taken within each plot in early November 2015. Means per site are pooled across the two core samples (n = 4). Percentages across the three fate categories may not sum to 100% for some sites because some samples contained no live or dead clams.

Site Mean % Mean % Mean % Alive Dead Undamaged Dead Crushed

I 21.8 (46.7) 12.6 (23.2) 28.1 (25.0) II 0.0 ( - ) 25.0 (32.5) 50.0 (56.3) III 53.5 (67.7) 17.1 (35.7) 29.4 (75.2) IV 43.2 (55.8) 12.3 (25.2) 19.5 (32.7) V 26.1 (66.4) 40.8 (42.1) 33.1 (38.6) VI 0.0 ( - ) 37.5 (22.9) 62.5 (22.9) VII 23.8 (43.8) 31.5 (34.7) 32.2 (44.1) VIII 0.0 ( - ) 50.0 (72.6) 25.0 (32.5) IX 27.8 (28.4) 51.7 (61.7) 8.0 (18.8) X 55.2 (64.4) 3.1 ( 9.9) 29.2 (39.8)

Table 10. Analysis of variance on the arcsine-transformed mean percent alive for cultured soft-shell clams planted in protected plots along the east side of the Harraseeket River (April-November 2015). Clams were seeded into four 22-ft x 14-ft plots at each of ten sites (Fig. 38) at a density of 10.5/ft2. Two benthic cores were taken from each netted plot in early November 2015.

Source of Variation df SS MS F Pr > F

Site 9 24276.563 2697.396 2.14 0.0568 Net(Site) 30 37732.645 1257.755 3.22 0.0003

Error 40 15640.842 391.021 Corrected Total 79 77650.050

The size-frequency distribution of clams recovered dead with undamaged valves (Fig. 39) showed that at least 65% were larger than the largest size clam seeded into the plots in April. This suggests that many of the clams had grown prior to dying. While few C. lacteus were sampled in the cores, many of these nemertean worms were seen in the sediments during the sampling, and information from other field trials in 2015 (see below) indicates that these have become a major predator of clams. Studies have shown that these animals consume their soft- shell clam prey without leaving any damage to the valves (Bourque et al., 2001). Mean SL of clams with undamaged valves was 30.1 ± 1.8 mm (n = 124).

50 N = 158

Percent Frequency Percent

0 0 10 20 30 40 50 60 Shell Length (mm)

25 N = 124

Percent Frequency Percent

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 39. Size-frequency distribution of clams planted in protected plots in April 2015 on the east side of the Harraseeket River (top-blue) and the distribution of dead undamaged clams. Approximately 65% of dead animals were > 25 mm SL.

Chipped or crushed clams are typical of damage due to crustaceans such as the green crab, Carcinus maenas, or rock crabs, Cancer irroratus. The frequency distribution of clams recovered with chipped or crushed valves (Fig. 40) demonstrates that crabs preyed on smaller clams than did nemertean worms. Mean SL of crushed clams was 23.6 ± 1.4 mm (n = 58).

50 N = 158

Percent Frequency Percent

0 0 10 20 30 40 50 60 Shell Length (mm)

N = 58

Percent Frequency Percent 10

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 40. Size-frequency distribution of clams planted in protected plots in April 2015 on the east side of the Harraseeket River (top-blue) and the distribution of dead clams with crushed or chipped valves. Approximately 25% of dead animals were > 25 mm SL.

Less than 2% of the live clams reached the legal size of 50.8 mm SL (2-inches; Fig. 41). Mean SL of live clams was 35.9 ± 0.9 mm (n = 142).

50 N = 158

Percent Frequency Percent

0 0 10 20 30 40 50 60 Shell Length (mm)

N = 142

Percent Frequency Percent 10

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 41. Size frequency distribution of soft-shell clams in April 2015 (top-blue) and in early November 2015 from sites sampled on the eastern side of the Harraseeket River. Seeded field plots – West Side of the Harraseeket River (fate of cultured juvenile clams)

Mean percent of live clams was extremely low (70% of the sites [Fig. 42] yielded no live clams; Table 11). Overall, only 5.7 ± 5.4% (n = 40) of clams were recovered alive. Although mean percent of live clams was as high as 33.3% (site X – Pettengill), there was no significant spatial variation in survival from site-to-site on the western side of the Harraseeket River. Nearly 75% of clams were either dead with undamaged valves or with chipped or crushed valves (74.3 ± 10.4%, n = 40; Table 11).

VIII

VII

III II

Figure 42. Study sites located on the west side of the Harraseeket River. Four 14-ft x 22-ft plots were installed at each site and seeded with cultured clams at a density of ca. 10.5/ft2 in mid- April 2015. Two benthic cores were taken from each netted plot during early November 2015. Table 11. Mean (± 95% confidence interval) percent alive, dead undamaged, and dead crushed cultured soft-shell clams at ten sites along the west side of the Harraseeket River. Clams were seeded at a density of 10.5/ft2 in four netted plots at each site in mid-April 2015. Two core samples were taken within each plot in early November 2015. Means per site are pooled across the two core samples (n = 4). Percentages across the three fate categories may not sum to 100% for some sites because some samples contained no live or dead clams.

Site Mean % Mean % Mean % Alive Dead Undamaged Dead Crushed

I 0.0 ( - ) 74.3 (30.8) 13.2 (21.8) II 0.0 ( - ) 44.8 (50.6) 55.2 (50.6) III 0.0 ( - ) 83.3 (44.6) 16.7 (44.6) IV 0.0 ( - ) 56.3 (50.1) 6.3 (19.9) V 0.0 ( - ) 56.9 (46.5) 5.6 (10.5) VI 10.8 (25.0) 74.2 (33.7) 2.5 ( 7.9) VII 0.0 ( - ) 75.6 (34.7) 11.9 (21.9) VIII 0.0 ( - ) 52.1 (59.7) 10.4 (19.9) IX 12.5 (30.1) 40.2 (27.5) 47.3 (43.8) X 33.3 (64.9) 16.7 (37.5) 0.0 ( - )

Table 12. Analysis of variance on the arcsine-transformed mean percent alive for cultured soft-shell clams planted in protected plots along the west side of the Harraseeket River (April-November 2015). Clams were seeded into four 22-ft x 14-ft plots at each of ten sites (Fig. 38) at a density of 10.5/ft2. Two benthic cores were taken from each netted plot in early November 2015.

Source of Variation df SS MS F Pr > F

Site 9 3538.731 393.192 2.12 0.0593 Net(Site) 30 5560.410 185.347 0.71 0.8334

Error 40 10437.437 260.936 Corrected Total 79 19536.578

Over 55% of clams recovered in the benthic cores from netted plots were dead with undamaged valves (57.4 ± 9.8%, n = 40). The size-frequency distribution of clams from this category (Fig. 43) indicates that most clams added new shell prior to dying, and it is presumed that milky ribbon worms, C. lacteus, were responsible for most of the clam deaths. Mean SL of dead clams with undamaged valves was 26.7 ± 1.1 mm (n = 157). Five core samples (of 80; ca. 6%) contained milky ribbon worms, and, while this density appears to be low, other field trials conducted at sites I and VII (see below) suggest that this predator was a major source of clam mortality in these field plots during the summer and fall of 2015.

50 N = 158

Percent Frequency Percent

0 0 10 20 30 40 50 60 Shell Length (mm)

25 N = 157

Percent Frequency Percent

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 43. Size-frequency distribution of clams planted in protected plots in April 2015 on the west side of the Harraseeket River (top-blue) and the distribution of dead undamaged clams. Approximately 50% of dead animals were > 25 mm SL.

Few clams were recovered that had been chipped or crushed by crustaceans, and only four green crabs were sampled in the cores (2 from site I: 15.26 mm and 21.69 mm CW; 1 each from site III and V – 13.71 mm and 4.02 mm, respectively). The distribution of sizes of crushed/chipped clams (Fig. 44) indicated that many of these animals were consumed earlier than those that were found dead with undamaged valves because the sizes were generally smaller and the mean SL of clams in this category was 21.2 ± 2.5 mm (n = 31).

50 N = 158

Percent Frequency Percent

0 0 10 20 30 40 50 60 Shell Length (mm)

N = 31

Percent Frequency Percent 10

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 44. Size-frequency distribution of clams planted in protected plots in April 2015 on the west side of the Harraseeket River (top-blue) and the distribution of dead clams with crushed or chipped valves. Approximately 25% of dead animals were > 25 mm SL. Only 14 clams were recovered live from the benthic cores (Fig. 45). Mean SL was 31.7 ± 3.7 mm.

50 N = 158

Percent Frequency Percent

0 0 10 20 30 40 50 60 Shell Length (mm)

N = 14

Percent Frequency Percent 10

0 0 10 20 30 40 50 60

Shell Length (mm)

Figure 45. Size frequency distribution of soft-shell clams in April 2015 (top-blue) and in early November 2015 from sites sampled on the western side of the Harraseeket River. Seeded field plots – East & West Side of the Harraseeket River (0-year class wild clam recruits)

In November 2014, there was a stark contrast in numbers of 0-year class wild clam recruits between the site on the eastern side of the Harraseeket River (Collins Cove), and the intertidal flat immediately across the river from that site on the western side of the Harraseeket River. The difference in mean number of recruits was 33.4/m2 (3.1/ft2) at Collins Cove and 14,391/m2 (1,377/ft2) at the site across the river.

Mean number of 0-year class soft-shell clam recruits did not vary significantly from one side of the river to the other (East: 27.0 ± 17.6 individuals/m2; West: 8.5 ± 8.0 individuals/m2; n = 80). Significant spatial variation in mean number of recruits was observed, however, on the east side vs. the west side of the river (Table 13). There was a tendency for density of recruits to increase with increasing distance away from the mouth of the river (Fig. 46)

Table 13. Analysis of variance on the mean number of 0-year class soft-shell clam recruits from benthic cores taken within netted plots at 10 sites on each side of the Harraseeket River, Freeport, Maine during the period between 4-10 November 2015. Each site had four 22-ft x 14-ft plots that were covered with a piece of flexible netting (4.2 mm aperture) that was intended to deter large predators from preying on juvenile cultured clams that were seeded into each plot in mid-April 2015 at a density of 10.5/ft2 (113 individuals/m2).

Source of Variation df SS MS F Pr > F

Side (East v. West) 1 119.025 119.025 0.99 0.3338 Site(Side) 18 2172.375 120.688 8.72 <.0001 Site – East Side 9 2096.113 229.901 16.62 <.0001 Site – West Side 9 76.262 8.474 0.61 0.7837 Net(Side x Site) 60 830.000 13.833 0.51 0.9962

Error 80 2155.000 26.938 Corrected Total 159 5276.400

The conclusion from this portion of the study from 2015 is: 1) observations made in 2014 about cultured clam survival in protected plots are not generalizable to other sites along the river; and, 2) soft-shell clam recruitment is not consistent from year-to-year – poor recruitment (as measured by seed settling into plots covered with protective netting) generally occurred on both sides of the river.

Several observations made during 2014 and 2015 about the use of predator netting in this system are important to mention because they have implications for future strategies to enhance soft- shell clam populations. 1) Nets in both years at most intertidal sites became heavily fouled with the egg masses of the mud snail, Iyanassa obsoleta (Fig. 47). Snails, reaching densities of > 500/m2, laid eggs in masses on the tops of nets that became so heavy that the Styrofoam floats failed to float the net above the sediment-water interface during tidal inundation. Cautiously, we tried to remove egg masses from nets by physical means (brushes, hands, water jets); however, none of these measures were 100% effective in removing the egg masses. In most cases, we chose to replace the net for fear that the fouling would result in a complete lack of oxygenated seawater getting to the clams in the plot below. This factor, alone, placed the cost of nets and labor to double to protect clam seed. 2) Milky ribbon worms are abundant in this system, and protective netting does not deter these worms from attacking clams in netted plots. Worms, unlike crabs, seem to prefer larger clams (i.e., those that have attained sizes > 25 mm SL), and appear to wait until clams reach a certain (relatively large) size before consuming their prey. These nemertean worms have been observed in nearly all sites we have worked along the river, and strategies to control them or deter them must be developed before any enhancement scheme can be deemed successful.

300

200

100

Mean Number of 0-Year Class Clams per m per Clams Class of0-Year Number Mean 0 1 2 3 4 5 6 7 8 9 10 11

Site on East Side of Harraseeket River

Figure 46. Spatial variation in abundance of soft-shell clam recruits along the east side of the Harraseeket River. Site 1 refers to a site close to the South Freeport docks (closest to the mouth of the river). Site 10 refers to a site near the upper portion of the river close to Porter Landing (see Fig. 38).

Figure 47. Mud snail eggs laid in masses on the top of flexible netting (4.2 mm aperture) at Staples Cove on 19 July 2014. The collective mass of eggs becomes so heavy that it acts to weigh the netting down creating areas of anoxic sediments beneath. Clams in these anoxic zones die.

V. Determine whether the use of predator deterrent netting, in combination with various densities of adult soft-shell clams, will result in an enhancement of wild clam recruits.

This objective was completed in 2014 and discussed in the Second Performance Progress Report.

VI. Train clammers and other interested fishers in the use of aquacultural techniques to improve local clam harvests.

Several projects conducted in 2014 were continued in 2015. Both were intended to provide hands-on experience in aquacultural and other techniques to improve local clam harvests. One focused on using a bivalve nursery upweller to grow cultured seed (2 mm SL) from the Downeast Institute at a commercial dock in South Freeport. One upweller was constructed in 2014 and another in 2015. As in 2014, 50,000 clams (Fig. 48) were added to each of twenty 55- gallon silos on 10 June 2014. Silos were initially cleaned on a weekly basis, but after mid-July, cleaning occurred 3-4 times per week. As we observed in 2014, clams placed in the upweller in 2015 grew rapidly during the first two months (Fig. 49), and had attained an average SL of nearly 20 mm by mid-September (Fig. 50). Once again, however, fouling from solitary and colonial tunicates, blue mussels, and hydroids made cleaning the clams difficult by that time.

The second upweller (Figs. 51-52) was deployed in mid-July, and seeded it with soft-shell clam juveniles. Overall, this particular portion of the project generated the most positive attention to the project than any other aspect. The upweller was visible on the waterfront, easily accessible to clammers and the general public, and created a lot of good publicity for the project by the print and radio media.

Another project that occurred in 2015 was initiated by the clammers who are partners in this project – Stewards of the Sea, LLC. Field experiments using juvenile soft-shell clams grown in the Freeport upweller in 2014 and overwintered using techniques discussed in Beal et al. (1995) were placed in wooden boxes on top of the mudflats were initiated at five intertidal sites in Freeport in April 2014 (Fig. 53-54). Variables assessed in the experiments are: stocking density, type of exclusion netting used on top and bottom of the boxes, and intertidal location.

One experiment was conducted at four intertidal sites (two in the Harraseeket River – sites I and VII on the western side – see Fig. 42; two outside the river). Results are presented from the two sites within the Harraseeket River as these were similar to those from outside the river that will be presented in the Final Report.

At all four sites, a 6 x 5 matrix of 4-ft x 2-ft wooden boxes (1 m space between rows and columns) was initiated in early May 2015. The bottom of each box was lined with flexible netting (4.2 mm aperture). To deter crabs, the top of each box was lined with a piece of 4.2 mm or 6.4 mm flexible netting. To assess effects of stocking density on clam growth and survival, juvenile, overwintered clams were seeded at a density of 30, 45, or 60 per square foot. The two factors, protective netting (a = 2) and stocking density (b = 3), were combined factorially to create six treatments. Treatments were assigned randomly to positions within the matrix. Boxes were filled with ambient sediments, seeded with clams, and then the top was secured. In November, two benthic cores (Area = 0.008107 m2) were taken haphazardly from each box. The contents of each sample were washed through a 1 mm sieve and all live and dead cultured clams, wild clams, and green crabs were enumerated and measured (SL for clams; CW for crabs) to the nearest 0.01 mm using digital calipers.

Figure. 48. Bivalve nursery upweller, South Freeport, Maine. July 2014.

Figure 49. 2 mm cultured soft-shell clam seed from the Downeast Institute were added to 55- gallon silos at a density of 50,000/silo on 5 May 2015.

Figure 50. Clams from the upweller on 19 July 2014. Mean SL = 13.4 ± 0.61 mm (n = 29). This represents a mean growth of ca. 11 mm in 39 days.

Figure 51. The second upweller was built during the spring of 2015 and deployed at the South Freeport dock adjacent to the upweller that was built and deployed in 2014 (See Fig. 48).

Figure 52. The 2014 upweller (rightmost) and the 2015 unit in South Freeport – July 2015.

Figure 53. A wooden box (ca 2-ft x 4-ft) that has mesh on the bottom and is filled with ambient sediments. Wild juvenile clams that had been overwintered are seeded in the box.

Figure 54. Clammers affixing predator-exclusion netting to the top of a growout box.

Results from the predator exclusion experiment at Sites I and VII on the west side of the Harraseeket River (April – November 2015)

Site I

Live cultured clams were sampled from only two of the 30 boxes (6.6%). No live clams were sampled from boxes protected with tops that were covered with the larger flexible netting aperture (6.4 mm) (Table 14). No significant effects of type of protective netting or stocking density on mean percent alive were detected (P > 0.35). Overall mean percent of live clams was 0.84 ± 1.7% (n = 30). The combined mean percent of clams dead with undamaged valves and those with chipped or crushed valves was 92.4 ± 6.6% (n = 30). Mean SL of live, dead undamaged, and dead crushed individuals was 35.4 ± 7.3 mm (n = 6), 30.8 ± 1.2 mm (n = 151), and 29.9 ± 1.6 (n = 59), respectively.

Mean number of wild soft-shell clam recruits did not vary significantly with stocking density (P = 0.5623), but did with type of mesh used to exclude predators (P = 0.0023). Approximately 15x more wild recruits were sampled from boxes protected with the smaller (4.2 mm) vs. larger (6.4 mm) aperture netting (4.2 mm: 6.1 ± 3.6 individuals/core; 6.4 mm: 0.4 ± 0.3 individuals/core). The density of wild recruits from boxes protected with the smallest aperture netting was 756.5 ± 446.5 individuals/m2 (n = 30).

Table 14. Fate of cultured soft-shell clam juveniles planted in wooden boxes (4-ft x 2-ft) at Site I (along the western shore of the Harraseeket River – see Fig. 42) in May 2015 and sampled in November 2015. Mean SL of cultured clams in May was 19.8 ± 0.32 mm (n = 260). Five boxes were deployed for each combination of top mesh aperture and stocking density.

Top Mesh Stocking Mean % Mean % Mean % Aperture Density/ft2 Alive Dead Undamaged Dead Crushed

4.2 30 0.0 ( - ) 54.8 (41.2) 35.3 (36.3) 4.2 45 0.0 ( - ) 54.6 (25.9) 35.4 (35.6) 4.2 60 5.1 (13.1) 70.8 (16.2) 24.1 (18.1)

6.4 30 0.0 ( - ) 31.7 (25.8) 48.3 (25.7) 6.4 45 0.0 ( - ) 49.3 (29.1) 50.7 (29.1) 6.4 60 0.0 ( - ) 54.2 (27.1) 45.8 (27.1)

Site VII

Live clams were sampled from seven of the 30 (23.3%), and in all but one of the treatments (mesh = 4.2 mm, stocking density = 45; Table 14). No statistically significant differences in mean number of live clams was noted across the three sources of variation (netting type: P = 0.4517; stocking density: P = 0.5069; or the interaction of these two main factors: P = 0.7774). Overall mean percent alive was 8.9 ± 6.8% (n = 30). Highest percent losses came in the dead undamaged category (Table 14) where the overall mean percent of clams recovered dead with undamaged valves was 73.3 ± 10.5% (n = 30). Mean SL of live, dead undamaged, and dead crushed individuals was 33.0 ± 2.3 mm (n = 33), 31.2 ± 1.2 mm (n = 223), and 26.5 ± 3.6 (n = 23), respectively.

Table 14. Fate of cultured soft-shell clam juveniles planted in wooden boxes (4-ft x 2-ft) at Site VII (along the western shore of the Harraseeket River – see Fig. 42) in May 2015 and sampled in November 2015. Mean SL of cultured clams in May was 19.8 ± 0.32 mm (n = 260). Five boxes were deployed for each combination of top mesh aperture and stocking density.

Top Mesh Stocking Mean % Mean % Mean % Aperture Density/ft2 Alive Dead Undamaged Dead Crushed

4.2 30 11.7 (20.2 ) 78.3 (26.9) 0.0 ( - ) 4.2 45 0.0 ( - ) 79.8 (34.6) 20.2 (34.7) 4.2 60 8.3 (17.9) 79.3 (20.4) 12.3 (22.2)

6.4 30 17.7 (42.6 ) 50.9 (44.3) 21.4 (43.8) 6.4 45 10.0 ( 27.8) 71.9 (51.9) 8.1 (15.3) 6.4 60 5.8 ( 10.1) 79.6 (23.5) 4.5 ( 7.7) Mean number of wild soft-shell clam recruits at site VII varied significantly with stocking density (P = 0.0174; Fig. 54), but not with type of mesh used to exclude predators (P = 0.0743). While the trend was towards more recruits in boxes with the smaller mesh (4.2 mm aperture: 690.8 ± 457.7 individuals/m2, n = 30) vs. the larger mesh (6.4 mm aperture: 304.3 ± 261.4 individuals/m2, n = 30), the relatively high variance from box-to-box within a given netting treatment resulted in a non-significant P-value.

1800

1600

1400

1200

1000

800

600

400

200

Mean Number of Clam Recruits per 1m per Recruits Clam of Number Mean 0 325 485 645

Stocking Density (Clams Per Square Meter)

Figure 54. Mean number of soft-shell clam recruits across stocking density treatments (325, 485, and 645 clams/m2 = 30, 45, and 60 clams/ft2, respectively) at site VII in the Harraseeket River (see Fig. 42). Significant differences occurred between stocking densities (P = 0.0174), but not types of mesh netting used to deter predators. (n = 20).

Results similar to those from sites I and VII were observed at two other sites in the Freeport area where a similar experimental design was used. Those results will be presented in the Final Report. Field trial to discourage milky ribbon worms and green crabs – Staples Cove, Freeport, Maine

Because milky ribbon worms apparently are not deterred by protective netting placed on top of the mud flats, nor are they discouraged from entering boxes seeded with cultured soft-shell clam juveniles through the bottom of the box with a 4.2 mm aperture, we designed a field trial to attempt to discourage these nemerteans from entering boxes infaunally.

On 7 May 2015, 21 boxes (4-ft x 2-ft x 6-inches deep) were deployed near the upper mid- intertidal at Staples Cove, Freeport, Maine. Boxes had one type of top: 4.2 mm flexible netting. One third of the boxes had bottoms covered with a 6.4 mm VEXAR (extruded polyethylene), one third of bottoms were covered with the 4.2 mm flexible netting (polypropylene, which was the same material as all the box tops), and one third of bottoms were covered with Pet screening (see: http://www.homedepot.com/p/Phifer-48-in-x-50-ft-Black-Pet-Screen- 3004134/100614683). Ambient sediments were used to fill each box, and then soft-shell clams ranging in size from 6-20 mm SL were scattered onto the surface sediments of each box at a density of 100 individuals/ft2. Tops were secured using rigid staples, nails, and laths. Seven replicates of each type of box bottom were used, and treatments were assigned randomly to positions within a 7 x 3 matrix (with 1 meter spacing between rows and columns).

On 10 November 2015, two benthic cores (Area = 0.008107 m2) were taken from each box, and the contents of each washed through a 1 mm sieve. All cultured clams and wild recruits were enumerated and the final SL of each recorded to the nearest 0.01 mm using digital calipers.

No live clams were recorded from boxes that were covered on the bottom with the 6.4 mm or 4.2 mm mesh (Table 15). Approximately 55% of clams in boxes with Pet screen bottoms were recovered alive at the end of the study ( x = 55.2 ± 17.6%, n = 7). In addition, only samples from boxes with Pet screen bottoms contained wild soft-shell clam recruits (0-year class individuals) (Table 15).

Table 15. Fate of cultured clams planted in 4-ft x 2-ft wooden boxes at Staples Cove from 7 May to 10 November 2015. Cultured clams (6-20 mm) were seeded in each box at a density of 100/ft2. Boxes were placed directly on top of the sediment surface and were filled with ambient sediments. Bottoms of boxes were covered in one of three types of mesh: 6.4 mm extruded (polyethylene) netting; 4.2 mm flexible (polypropylene) netting; or Pet screen. On 10 November 2015 two benthic cores (A = 0.008107 m2) were taken haphazardly from each box. # Alive, # DU, and # DC refers to number of live clams, dead with undamaged valves, and dead with chipped or crushed valves per core, respectively. % Alive = (# Alive / (#Alive + #DU + #DC). # Recruits refers to the number of wild, 0-year class soft-shell clam individuals per core. # Crabs refers to the number of green crabs, Carcinus maenas per core. # Worms refers to the number of milky ribbon worms, Cerebratulus lacteus, per core.

Bottom Rep # Alive # DU # DC %Alive # Recruits # Crabs # Worms

6.4 mm 1a 0 7 0 0.00 0 1 0 1b 0 5 0 0.00 0 0 0 2a 0 3 1 0.00 0 1 0 2b 0 7 0 0.00 0 0 0 3a 0 15 3 0.00 0 0 0 3b 0 12 4 0.00 0 0 0 4a 0 12 5 0.00 0 0 0 4b 0 8 4 0.00 0 1 0 5a 0 3 0 0.00 0 0 0 5b 0 1 1 0.00 0 0 0 6a 0 3 0 0.00 0 0 0 6b 0 7 2 0.00 0 0 0 7a 0 5 2 0.00 0 0 0 7b 0 4 1 0.00 0 0 0 ______4.2 mm 1a 0 13 0 0.00 0 0 0 1b 0 12 2 0.00 0 0 0 2a 0 4 3 0.00 0 0 0 2b 0 3 4 0.00 0 0 0 3a 0 13 0 0.00 0 0 0 3b 0 13 5 0.00 0 0 0 4a 0 13 5 0.00 0 0 0 4b 0 3 1 0.00 0 1 0 5a 0 13 1 0.00 0 0 0 5b 0 1 1 0 0.00 0 0 1 6a 0 18 0 0.00 0 0 0 6b 0 15 1 0.00 0 0 0 7a 0 5 6 0.00 0 0 0 7b 0 16 3 0.00 0 0 0 Pet Screen

1a 16 2 1 84.2 0 0 0 1b 10 3 1 71.4 0 0 0 2a 2 7 3 16.7 0 0 0 2b 5 6 3 35.7 0 0 0 3a 3 12 0 20.0 0 0 0 3b 15 8 2 60.0 3 0 0 4a 0 2 1 0.00 0 0 0 4b 3 9 0 25.0 1 0 0 5a 5 1 1 71.4 0 1 0 5b 1 0 0 100.0 0 0 1 6a 12 3 2 70.6 0 0 0 6b 9 3 0 75.0 3 0 0 7a 21 3 0 87.5 8 0 0 7b 11 9 0 55.0 0 0 0

The final field project from 2015 was deployed at each site of the Harraseeket River where the four protective nets were positioned (see Figs. 38 & 42). At each of the ten sites on each side of the river, a series of six wooden boxes (1-ft x 2-ft x 3-inches deep (Figs. 55-56) were deployed in a 2 x 3 matrix. Each box was covered on both the top and bottom with a piece of netting. Four boxes received Pet screening on the top and bottom, while two received an extruded, polyethylene mesh (6.4 mm aperture). Boxes were placed on top of the sediments in April at the same time that nets were deployed. Approximately 1000 ml of play sand was added to two of the boxes with Pet screening, while the other four received no extra sediment.

Boxes were used to examine closely natural recruitment in highly protected environments. Each box received natural sediments during the period between April and November 2015. In addition to wild recruits of soft-shell clams, other benthic organisms such as hard clams, false angel wing clams, blue mussels, dwarf surf clams, and green crabs occurred in many of the boxes. The Pet screening had small enough apertures to keep out most large predators; however, some of these predators were able to enter even these boxes as post-settled larvae. Boxes were retrieved during the November 2015 sampling, and the contents of each washed through a 1 mm sieve. The sieved contents were then frozen, and results from this study will be presented in the Final Report. The idea is that the boxes will increase information about natural recruitment at each of the 20 stations.

Figure 55. Wooden boxes (1-ft x 2-ft) either with Pet Screening on the top and bottom or VEXAR – an extruded, heavy plastic with 6.4 mm apertures. Boxes are designed to increase information about wild recruits of soft-shell clams.

Figure 56. Clam recruitment boxes that are deployed at 10 stations along both sides of the Harraseeket River, Freeport, Maine. The box on the left is protected with Pet screening on both the top and bottom while the box on the right is protected with an extruded, polyethylene mesh (6.4 mm aperture).

References

Beal, B.F. 2002. Adding value to live, commercial size soft-shell clams (Mya arenaria L.) in Maine, USA: results from repeated, small-scale, field impoundment trials. Aquaculture 210, 119-135.

Beal, B.F., Bayer, R.C., Kraus, M.G., Chapman, S.R. 1999. A unique shell marker of juvenile hatchery-reared individuals of the soft-shell clam, Mya arenaria L. Fish Bull. 97, 380-386.

Beal, B.F., Lithgow, C., Shaw, D., Renshaw, S. & Ouellette, D. 1995. Overwintering hatchery- reared individuals of the soft-shell clam, Mya arenaria L.: a field test of site, clam size, and intraspecific density. Aquaculture 130:145-158.

Beal, B.F., Parker, M.R. & Vencile, K.W. 2001. Seasonal effects of intraspecific density and Predator exclusion along a shore-level gradient on survival and growth of juveniles of the soft-shell clam, Mya arenaria L., in Maine, USA. J. Exp. Mar. Biol. Ecol. 264:133-169.

Beal, B.F. 2006. Relative importance of predation and intraspecific competition in regulating growth and survival of juveniles of the soft-shell clam, Mya arenaria L., at several spatial scales. J. Exp. Mar. Biol. Ecol. 336:1-17.

Beal, B.F., Kraus, M.G. 2002. Interactive effects of initial size, stocking density, and type of predator deterrent netting on survival and growth of cultured juveniles of the soft-shell clam, Mya arenaria L. in eastern Maine. Aquaculture 208:81-111.

Bourque, D., Miron, G., Landry, T. 2001. Predation on soft-shell clams (Mya arenaria) by the nemertean Cerebratulus lacteus in Atlantic Canada: implications for control measures. Hydrobiologia 456:33-44.