THE EFFECT OF PREDATOR NETTING ON RECRUITMENT IN BAYNES SOUND, BC WITH A SPECIAL FOCUS ON THE RESPONSE OF THE MANILA CLAM ( PHILIPPINARUM)

by

DAPHNE MARIE MUNROE

B.Sc.Hons., Simon Fraser University, 2000

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

in

THE FACULTY OF GRADUATE STUDIES

( Science)

THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 2006

© Daphne Marie Munroe, 2006 Abstract

Passive and active forces determine the patterns of settlement of larvae.

Research efforts into larval settlement have been dominated by attached and conspicuous

in hard substrate environments. Here, data on early recruitment patterns of a mobile

bivalve species from a soft-sediment habitat is provided. In particular, how intertidal clam

netting influences the distribution of settling pediveliger larvae was investigated.

Early recruitment patterns of Manila clam larvae () were examined in

relation to predator netting used in farming in British Columbia. A method for sampling

recent settlers from intertidal sediments was developed, proven effective and employed to

sample settled clams (<600 um shell length) from four sites in Baynes Sound, on the eastern side

of Vancouver Island, B.C. in 2003 and 2004. Paired netted and non-netted plots were compared

for number of early recruits. Plots with the netting and high density of adult clams experienced

lower levels of settlement. Settlement varied annually with 2003 experienceing an order of

magnitude less recruitment than 2004. In addition, laboratory tests were run using flumes to

examine the retention of competent clam larvae within flumes with netting on the bottom. No

difference in the retention of clam larvae was observed due to netting or sediment treatments.

Sediment properties (sediment grain size, organic carbon and inorganic carbon) were

also compared between netted and non-netted plots. No difference was seen in the sediment

properties measured except for slightly higher levels of organic carbon beneath nets; this was

likely due to the higher number of adult clams beneath the nets. The netting buffers temperature

at the sediment surface during tidal exposure by up to 3°C, the biological relevance of this

remains untested.

No increase in was measured beneath netting; however, decreased bivalve

settlement beneath netting was observed but only in the year when overall settlement was high.

This decrease in recruitment was not supported by the flume trials; however these were run at

ii one velocity. Trials at different velocities may produce different results. These field observations are an important contribution to understanding larval settlement of mobile species in a soft-sediment habitat. Table of Contents

ABSTRACT H

TABLE OF CONTENTS IV

LIST OF TABLES VI

LIST OF FIGURES VH

ACKNOWLEDGEMENTS XI

DEDICATION Xffl

CHAPTER 1: INTRODUCTION 1 INTRODUCTION 1 LARVAL BIOLOGY 2 Spawning and Fertilization 2 Larval Development 4 Settlement and 7 JUVENILE DISPERSAL .10 LARVAL ECOLOGY 17 History 17 Factors Influencing Settlement Patterns 19 Biological Factors 19 Chemical Factors 22 Physical Factors 26 FLUID MOTION 28 Benthic Boundary Layer 28 Reynolds Number 30 Turbulence 31 AQUACULTURE 32 Manila Clam Aquaculture 33 Clam Culture in British Columbia 35 Predator Netting 36 THESIS OUTLINE 38 REFERENCES 40 CHAPTER 2: SAMPLING RECENTLY SETTLED CLAMS FROM SEDIMENTS 56 INTRODUCTION 56 MATERIALS AND METHODS 57 RESULTS 59 DISCUSSION 61 CONCLUSIONS 63 REFERENCES 64

CHAPTER 3: THE EFFECT OF NETTING ON ESTERTIDAL SEDIMENTATION 66 INTRODUCTION 66 MATERIALS AND METHODS 68 Site 68 Clam Populations. 70 Sediment Grain Size 70 Carbon 72 Temperature 72 RESULTS 73 Clam Populations. 73 Sediment Grain Size 75 Carbon 76

iv Temperature 77 DISCUSSION 79 Clam Populations. 79 Sediment Grain Size 80 Carbon 81 Temperature 81 CONCLUSIONS 82 REFERENCES 83

CHAPTER 4: BIVALVE RECRUITMENT TO CULTURE PLOTS 86

INTRODUCTION 86 MATERIALS AND METHODS 89 RESULTS 91 DISCUSSION 97 CONCLUSIONS 102 REFERENCES 104

CHAPTER 5: SETTLEMENT OF LARVAE IN EXPERIMENTAL FLUMES Ill

INTRODUCTION Ill MATERIALS AND METHODS 113 RESULTS 117 DISCUSSION 120 CONCLUSIONS 123 REFERENCES A 124

CHAPTER 6: CONCLUSIONS AND GENERAL DISCUSSION 128

APPENDIX 1: SUMMARY TABLE OF JUVENILE BIVALVE DISPERSAL RESEARCH 131

REFERENCES APPENDLX 1 134

APPENDIX 2: COMPARISON OF METHODS FOR THE DETERMINATION OF CARBON ES ENTERTEDAL SEDIMENTS 137

INTRODUCTION 137 MATERIALS AND METHODS 139 Sample Collection and Preparation: 139 Acid-Burn: 140 LOI: 141 CHN: 142 RESULTS 143 DISCUSSION 148 CONCLUSIONS 149 REFERENCES APPENDLX 2 150

APPENDLX 3: LARVAL SETTLEMENT DATA FROM 2002 152

APPENDIX 4: FIELD SITE VELOCITY MEASUREMENTS 156

APPENDIX 5: CONSIDERATION OF TURBULENCE IN CALIBRATION OF PLASTER BLOCKS USED FOR FLOW MEASUREMENT 158

INTRODUCTION 158 MATERIALS AND METHODS 159 RESULTS 161 DISCUSSION 162 REFERENCES APPENDLX 5 163

v List of Tables

Table 1-1. Current velocities reported to cause byssal drift in post metamorphic bivalves 15

Table 2-1. Grain size components, percentage by dry weight, of each sediment type.

The size category >2000um contains both granule+ and broken shell 59

Table 3-1. Site characteristics (tidal height is reported at meters above chart datum) 69

Table 3-2. Results of tests of assumptions for paired T-test. Normality tested on the distribution of the difference between pairs, correlation calculated for linear regression of pairs 71 Table 4-1. Results of ANOVA test of factors influencing Venerupis philippinarum settlement 93

Table 4-2. Results of linear regression of Venerupis philippinarum versus larval settlement 94

Table 5-1. Lengths of Venerupis philippinarum larvae (nm ± SD) used for each trial and source batch; n = 20 for each measure 115

Table 5- 2. Summary statistics from ANOVA test for percentage of Venerupis philippinarum larvae leaving the system during the trial 117

Table 5-3. Summary statistics from ANOVA test for proportion of Venerupis

philippinarum larvae leaving in the last 30 minutes of the trial 120

Table Al-1. Summary of research on juvenile bivalve dispersal 131-

Table A2-1. Means and standard errors for each test for carbon analysis method and each value measured 145 Table A2-2. Significance values for multiple comparisons of means for each comparison of test type for organic carbon values 146

Table A2-3. Significance values for multiple comparisons of means for each comparison of test type for inorganic carbon values 147 List of Figures

Figure 1-1. The general life cycle of marine bivalves 3

Figure 1-2. General diagram of the and veliger larvae of marine bivalves. (A) trochophore ; (B) veliger larva with velum extended. Not drawn to scale 5

Figure 1-3. Modes of post-larval dispersal. A. drift; long byssus threads carry the bivalve through the . B. Climbing (from Yankson, 1986); the animal uses its ciliated foot and strong byssus to climb walls. Side branches of byssus are used to hold the animal while it probes with its foot. C. Drifting by foot protrusion (from Sorlin, 1988); the animal begins in a normal feeding position, works its way to the surface then protrudes its foot to act as a sail 13

Figure 1-4. Graphic representation of the flows in the Benthic Boundary Layer. Longer arrows represent faster flows; grey at the bottom represents the surface. Flow increases with distance from the surface and eventually reaches a rate equivalent to the free-stream 29

Figure 1-5. Annual production of mollusc aquaculture by mass shown with open squares and on left axis. Number of molluscan species in production worldwide shown with solid grey circles and on right axis. Data from FAO 2005 32

Figure 1-6. Global molluscan production by mass contribution by country. Country labels are listed on the right. For each year shown, the top eight countries are graphed, the rest of the countries for that year are pooled in "rest of world" category. Data from FAO 2005 34

Figure 1- 7. Comparison of Manila clam (Venerupis philippinarum) production from capture versus aquaculture. Capture fishery is shown with grey bars and aquaculture shown in black. Data from FAO 2005 35

Figure 1-8. Location of Baynes Sound on Vancouver Island, Canada. Inset left shows location of Vancouver Island in relation to Canada 38

Figure 2-1. Means and standard deviation for numbers of clams (Venerupis philippinarum) per sample for the three sediment types. The dashed line indicates the expected number of clams per sample (58.8) based on number of larvae placed in each tank. N = 3 for each treatment 60

Figure 3-1. Map of beach sampling sites within Baynes Sound. Each beach is marked with number and labelled with site name. Inset top right: Location of Vancouver Island within Canada. Inset bottom left: Location of Baynes Sound on Vancouver Island, British Columbia, Canada 69

Figure 3-2. Length frequencies (count) of Venerupis philippinarum (>5mm) from each site, 2003 in left column and 2004 in right. Clams measured from netted plots represented by black bars, clams from non-netted plots represented by open bars. Shell length in mm plotted along the horizontal axis, frequency on the vertical axis 74 Figure 3-3. Mean number of Venerupis philippinarum (>5mm shell length) per m2 from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n=16 75

Figure 3-4. Mean number of Nuttalia obscurata (>5mm shell length) per m2 from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n=16 75 Figure 3-5. Percent silt (<0.063 mm grain size) content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beach 4 netted plot in 2004 where n=5. Error bars represent 95% confidence interval .76 Figure 3-6. Percent gravel (>2mm grain size) content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6. Error bars represent 95% confidence interval 76 Figure 3-7. Percent inorganic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beachl netted plot in 2004 (marked above bar with N=5) where n=5. Error bars represent 95% confidence interval 77 Figure 3-8. Percent organic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6. Error bars represent 95% confidence interval .77 Figure 3-9. Daily temperature measurements at the sediment/air interface, x-axis shows hour of the day. Grey boxes connected with dashed line show the temperature at netted plots, black triangles show non-netted plots and tidal exposure is shown with the shaded grey vertical band. Left column shows the new moon, center shows first quarter moon and right shows the full moon .78 Figure 4-1. Average rates of Venerupis philippinarum settlement per m2 for each beach site (net and no-net combined). Grey circles and black line shows the average settlement rate for all sites combined. 10/11 August, 25/26 August and 7/8 September in 2003 and 10/11 August and 25/56 August in 2004 are considered "pre-settlement" 91 Figure 4-2. Average Venerupis philippinarum early recruits per m2 for 2003 (upper panels) and 2004 (lower panels) counted on non-netted (left panels) and netted plots (right panels). Sample date shown on x-axis, error bars represent 95% confidence interval. Upper panels are shown with a finer scale than lower panels to allow data to be viewed more clearly 92 Figure 4-3. Relationship of early recruit (<0.5mm shell length) density to Venerupis philippinarum biomass (shell length >5mm). Each data point represents the average early recruit density for each beach. Data from 2003 are counts from date 4, thus representing peak initial settlement and are shown with triangle markers. Data from 2004 are counts from date 3, representing initial peak settlement and are shown with square markers. White markers indicate non-netted plots and black markers indicate netted plot values. The R2 value is shown on the graph next to the corresponding trend line 94

Figure 4-4. Average Venerupis philippinarum early recruit length for each beach and plot within beach on the post-settlement date in 2003 (date 4 - shown on left) and 2004 (date 3 - shown on right). Hatched bars represent netted plots, black bars represent non-netted plots. Error bars represent 95% confidence interval. The number directly below each bar represents the sample size 95

Figure 4-5. Shell length frequencieso f Venerupis philippinarum early recruits from 2004 samples. Date 3 shown with circles and solid line, date 4 shown with squares and hatched line. Non-netted plots are shown in the left column and netted plots shown on the right. Beach 3 and Beach 4 are shown with a smaller y-axis because of lower recruitment overall to those sites. Difference between the peaks of the solid line versus the hatched line is considered to represent growth of that cohort from date 3 to date 4 96

Figure 5-1. Top view of flume dimensions. Flow is from left to right. Acoustic Doppler measurements were made at the position marked by the X. Bottom treatments were applied between the straws and the outflow 113

Figure 5-2. Larval batch spawning and splitting dates and resulting groups used in each trial 114

Figure 5-3. Percentage of total Venerupis philippinarum larvae input at time zero that left the system by the end of the trial (75 minutes). Error bars represent ± standard deviation (n=5 for each bar). Percentage of polystyrene spheres leaving the system is shown on the right side of the chart 118

Figure 5-4. Proportion of all polystyrene spheres exiting the system shown by time collected. Error bars represent ± standard deviation 118

Figure 5-5. Proportion of Venerupis philippinarum larvae (or beads in case of trial 7) exiting the flume over time. White band indicates proportion leaving in the first 15 minutes; the dotted band indicates the proportion leaving in the second 15 minutes and so on. Trial number and treatment listed along the x- axis. ** Trial 7 was conducted with polystyrene beads; all other trials shown were conducted with larvae 119

Figure A2-1. Mean values of organic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison) 144

Figure A2-2. Mean values of inorganic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison) 145 Figure A2-3. Result of multiple comparison of error variances for organic carbon values. Variances connected with underline represent variances that were not significantly different 146

Figure A2-4. Result of multiple comparison of error variances for inorganic carbon values. Variances connected with underline represent variances that were not significantly different 147

Figure A3-1. Density of settled Venerupis philippinarum larvae per m2 in 2002 at Beach 1 site. Netted plot is shown with the black hatched line, non-netted plot is shown in the grey solid line. Error bars represent the 95% confidence interval. For each point, n=24 except the October data where n=12 153

Figure A3-2. Lengths of clams (Venerupis philippinarum) sampled in 2002. Netted plots shown with hatched bars and non-netted plots shown with grey bars. Error bars represent 95% confidence interval 154

Figure A3-3. Length frequency graphs for Venerupis philippinarum early recruits collected in 2002 on Beach 1. Solid black line shows the length frequency for August 19*, the open dotted line shows September 5th. The top panel shows data from the neted plot, the bottom panel shows the non-netted plot 155

Figure A4-1. Positioning of the clod card in the sediment. A small plastic spike attached to the bottom of the clod allows it to be inserted into the sediment to keep it in place 157

Figure A4-2. Velocity measurements from field sites as estimated by clod card dissolution. Beach 1 shown in white bars, Beach 2 in light grey bars, Beach 3 in dark grey bars and Beach 4 in black bars. Bars with hatch marks represent netted plots, bars without hatching represents non-netted plots. Error bars show the 95% confidence interval, n=6 for each bar. Relationship for turbulent calibration is shown in Appendix 5 157

Figure A5-1. Dimensions of clod cards used 160

Figure A5-2. Graph of percentage mass lost from blocks over 24 hour period at flows from 0-4 cm/sec. Turbulent data points shown with black squares and solid trendline (R2=0.7085), laminar datapoints shown with grey triangles and dashed trendline (R2=0.8291) 161 Acknowledgements

This thesis could not have been completed without help from many people. Thank you to Dr. William Pennell and Brian Kingzett, who made time to meet with me in the early stages and encouraged me to take this research on. To my supervisor, Dr. R. Scott McKinley, thank you for the freedom to pursue avenues that I would otherwise not have been able to explore.

And to the rest of my committee: Dr. Neil Bourne, Dr. Douglas Bright and Dr. Murray Isman, thank you for your guidance and support throughout.

Most considerably, I am forever awestruck and grateful for the encouragement and love provided me by my best friend, Shawn Mason, who continues to sustain me through the thickest and thinnest times.

The co-operation of the shellfish growers of Baynes Sound was essential to completion of this work. In particular, thank you to Odyssesy Shellfish and Mac's for their contributions of thoughts and advice, use of sites and transport to and from. In particular, thanks to Dwayne Johnson and Rob Marshall for walking the beach and sharing their insight. Thanks also to Taylor Shellfish for donation of larvae.

I owe enormous gratitude to the staff at Malaspina University-College (MUC), who provided me with a second home for the duration of a majority of my work. I am especially grateful to Jennifer Dawson-Coates, Gordon Edmondson and Simon Yuan for their hands on help with absolutely everything, to Don Tillapaugh and Dr. Pennelope Barnes for always saying yes when I needed lab space or equipment use, and especially to the fleet of MUC undergrads who toiled with me in the field and lab: Kerry Bates, Edith Billington, Amy Hoare, Sabrina

Halvorsen, Heidi Lydersen, Amber Perkovich, Soliel Switzer, Brendon Campbell and Cameron

Robinson.

To my altruistic friend, Heidi Low, thank you for giving up so many weeks of your

summer vacations to spend time on beaches at low getting stinky and muddy. And to

xi Heidi's parents, Veronica and Bill Phaneuf, thanks for providing us with "field accommodation" and glorious meals while we worked.

An enormous number of people helped improve chapter drafts with generous comments and important suggestions for improvement of drafts of chapters. Thanks to Dr. Stefanie Duff,

Robert Marshall, Dr. Chris Pearse, Dr. Louis Gosselin, Dr. Kevin Butterworth, Dr. Terri

Sutherland, Dr. Neil Bourne, James Hill and Dr. William Pennell who all brandished the red pen at some time on my behalf. Temporary space and use of various laboratory equiptment was shared generously by Dr. Matthew Dodd of Royal Roads University and Dr. Ray Lett of the

Ministry of Energy, Mines and Petroleum Resources. Maureen Soon of the University of British

Columbia gladly provided me with guidance and patiently answered questions regarding carbon analysis and Dr. Carl Schwarz offered statistical help with some tests.

To the Gorging Dragons, I owe many hours of mental, physical and emotional escape that proved invaluable in maintaining balance through the years of work.

To my parents, Deborah and Kenneth Munroe, thanks for providing me with a foundation of tenacity, patience and the genetics to handle it all.

This research was supported by student scholarships from the Natural Science and

Engineering Research Council (NSERC) and was conducted in Baynes Sound and at the Center for Shellfish Research at Malaspina University-College. Dedication

Tor my

"So don't you sit upon the shoreline find say you're satisfied Choose to chance the rapids Jinddare to dance the tide." CHAPTER 1: Introduction

Introduction

Survival and early settlement patterns of larval bivalves in the natural environment are poorly understood, particularly for mobile species like clams (Eckman, 1990). Research efforts into larval settlement have been dominated by attached and conspicuous species in hard substrate environments (Underwood, 2000) and polychaete settlement (Qian, 1999). In this thesis, I provide data currently lacking in the literature, on early recruitment patterns of a mobile bivalve species from a soft-sediment habitat. Determination of a suitable settlement location is influenced by many factors: chemical cues from con-specifics or predators (Pawlik, 1992a;

Steinberg et al., 2002), bacterial films (Wieczorek and Todd, 1998), physical properties of the

sediment, light, temperature, salinity and hydrodynamics (Orton, 1937; Bayne, 1964a, b;

Meadows and Campbell, 1972; Butman, 1987; Ableson and Denny, 1997).

Intertidal shellfish farming practices have the potential to alter settlement patterns. At most farm sites in British Columbia, clam farming involves covering portions of the intertidal

lease area with predator netting. The netting is intended to provide protection from by

, birds, and other bivalve predators (Anderson, 1982; Toba et al., 1992). Netting placed on beaches has been shown to stabilize sediment and increase sedimentation (Spencer et

al., 1996). Evidence of increased sedimentation indicates that the nets influence local

hydrodynamic processes. This alteration of flow could direct settlement of larvae and thereby

increase the local bivalve population (Heath et al., 1992; Beal and Kraus, 2002).

The purpose of this research was to determine if there is an effect on recruitment of the

Manila clam (Venerupisphilippinarum - A. Adams and Reeve, 1850) by intertidal predator

netting used in culture of clams in British Columbia. In addressing this question I was first

interested in confirming appropriate and accurate sampling methods for making quantitative

1 measures of early bivalve settlers in soft-sediments. Secondly, I examined whether the nets were creating a small-scale depositional environment. To assess this, I measured levels of silt and organic carbon (among other parameters) at netted and non-netted plots. I was then able to ask the question: Are bivalve larvae recruiting in higher numbers to netted or non-netted plots?

Finally, in an effort to better understand the observed patterns of recruitment to field sites, I carried out controlled flume experiments designed to examine settlement patterns of clam larvae in relation to netted and non-netted bottom types. This introductory chapter discusses the importance of carrying out this research, provides evidence from the literature illustrating why this area needs to be addressed and offers an introduction to the questions answered in subsequent chapters.

Larval Biology

The general life cycle of free spawning marine bivalves involves two distinct modes of life; pelagic and benthic. The life cycle (Figure 1-1) begins with release of gametes into the water column via the exhalent of the adult. In the early stages of life, the trochophore and veliger stages, the are pelagic and can swim weakly. Through the process of settlement and metamorphosis, the pelagic larva transforms into a sedentary benthic juvenile and adult form.

Spawning and Fertilization

Spawning in most marine bivalves involves the release of a large number of eggs and sperm through their siphon into the water. The Manila clam, Venerupis philippinarum are oviparous (gametes released into the water) and dioecious (separate sexes) (Helm and Bourne,

2004). There are a number of stimuli that trigger gamete release including temperature

(Podniesinski and McAlice 1986; Devauchelle, 1990; Barber and Blake, 1991; Thompson et al.,

2 1996), physical agitation (Seed and Suchaneck, 1992), salinity, tide, solar or lunar phase

(Devauchelle, 1990; Morgan, 1995), algae blooms (Starr et al., 1990) and the presence of other gametes (Galtsoff, 1964; Thompson et al., 1996). The Manila clam will typically undergo one or two spawning events per season (one large spawning in July followed by continuous female spawning through the summer was observed in Washington - Holland, 1972); the latter of the two being the largest (Ponurovsky and Yakovlev, 1992). o D-hlnged larva 0' Pedlvellger larva Trochophore Pelagic Gametes released

Settlement and rnetamorphosls

Figure 1-1: The general life cycle of marine bivalves.

A mature female (shell length >35 mm) can produce 5-8 million eggs in a single

spawning depending upon condition and time of the year; in general, larger clams will produce more eggs (Utting and Spencer, 1991). Venerupis philippinarum releases eggs that are initially pear-shaped but become round after a brief period in seawater (Helm and Bourne, 2004).

Before fertilization can take place, shed gametes must encounter one another in the water column, a process that can be hindered by advection and dilution (Pennington 1985; Denny and

Shibata 1989; Levitan, 1995). Adaptations exist to maximize the encounter rate of gametes such as synchronous spawning (Galtsoff, 1964; Levitan, 1995; Thompson et al., 1996), sperm chemotaxis (Miller et al., 1994; Levitan, 1995) and dense aggregations of adults (Levitan et al.

1992; Levitan, 1995; Mann and Evans 1998).

Once sperm and egg meet and successful fertilization occurs, meiosis is completed in the egg (Gosling, 2003). The fertilised egg undergoes spiral cleavage and eventually becomes a ciliated, motile, trochophore larva (Kasyanov et al., 1998). Time required for the embryo to develop is dependant on the species and water temperature (Helm and Bourne, 2004).

Larval Development

Within roughly 24 hours of fertilization, most embryos become trochophore larvae

(Figure 1-2) (Gosling, 2003). The trochophore is ciliated, with 1 to 3 rows of cilia around the middle called the prototroch (Kasyanov et al, 1998). These rows of cilia are used for swimming

(in a spiral pattern) and although have been reported to have developed a mouth

(Galtsoff, 1964; Kasyanov, et al., 1998), they are not believed to feed until later stages. Anterior to the prototroch is the pretrochal region which contains the apical plate and apical tuft of cilia at the top that performs sensory functions. Below the prototroch is the posttrochal region, in some groups this area contains another crown of cilia called the telotroch (Raven, 1958).

4 Figure 1-2: General diagram of the trochophore and veliger larvae of marine bivalves. (A) trochophore larva; (B) veliger larva with velum extended. Not drawn to scale.

The trochophore becomes a veliger larva (Figure 1-2), which is more complex and has more fully developed organs than the trochophore (Raven, 1958; Kasyanov et al., 1998). The prototroch becomes the velum of the veliger, which remains ciliated and is still the swimming and feeding organ (Raven, 1958). The velum is attached to the mantle by two pairs of velar retractor muscles and can be extended or retracted by the larvae. Cilia around the periphery of the velum beat to cause swimming in a sprial motion and create water currents for collection and transfer of food particles to the mouth located in the ventral part of the velum (Kasyanov et al.,

1998; Gosling, 2003). Veligers feed on many different species of (Bayne, 1965;

Paulay et al., 1985), bacteria, detritus and dissolved organic matter (Olson and Olson, 1989;

Baldwin and Newell, 1991; Lutz and Kennish, 1992; Boidron-Metairon, 1995).

The apical plate remains in the center of the velum and when the veliger is swimming, contact with the apical flagelium causes the velum to be retracted (Kasyanov et al., 1998). The posttrochal region becomes the body of the bivalve and a shell develops, the shell is at first d- shaped and the larvae are called straight-hinge or D-hinge larvae. The shell continues to grow and starts to take on a typical bivalve shape; however the shell is primarily composed of aragonite and is thin and transparent (Kennedy et al., 1996).

5 Larval development takes place in the water column (Thorson, 1946) and although the larvae can swim, they do so slowly (approximately 1-10 mm/s - Mileikovsky, 1973, Chia and

Buckland-Nicks 1984, also summary table of multi-species speeds provided by Kennedy et al.,

1996 pg. 382). Larval development can take from three to five weeks depending upon species food and temperature (Gosling, 2003) (larval development in V. philippinarum is approximately three weeks at 25°C - Quayle and Bourne, 1972; Helm and Pellizzato 1990). Thus, for the duration of larval development, swimming speeds cannot overcome water currents and the animals are primarily passively distributed (Young, 1995). Larvae exhibit some control over horizontal advection by moving up and down in the water column (into different flow regimes) in response to various cues (Carriker, 1951; Morgan, 1995; Shanks, 1995; Young, 1995;

Carriker 2001); this vertical migration is also believed to be used additionally as predator avoidance (Gosling, 2003).

Mortality has been estimated to be extremely high during pelagic larval development

(Thorson, 1946; Morgan, 1995; Gosling, 2003). The cause of this loss remains unclear.

Johnson and Shanks (2003) have shown that predatory losses may be much lower than previously believed. The authors made in situ measurements on predation rates on near-natural assemblages in Oregon and Washington and found that observed predation rates were lower than has been formerly assumed and may only be of importance when specific predators are present.

As the veliger develops it begins to form a foot, and like the velum, pedal retractor muscles are developed which allow it to be extended beyond the shell (Carriker, 2001). At the stage when the larva has both a velum and a foot, it is called a pediveliger (Carriker, 1956). As a pediveliger the animal can use both the velum for swimming and the foot for crawling and periodically lands to crawl and test the substrate with its foot (Kasyanov et al., 1998; Zardus and

Mattel, 2002). The gills also begin to develop at this stage, but are not used in feeding until

6 after metamorphosis. The anterior adductor muscle is developed early and in the latter veliger stages the posterior adductor is developed. The digestive system is well developed by the late veliger stage. A pigmented eye spot forms in the middle of the shell in some species. At this point the larva is considered "competent", or ready to metamorphose.

Settlement and Metamorphosis

Metamorphosis involves shedding or breakdown of the pelagic larval structures and the development of benthic adult structures (Raven, 1958). Generally for bivalves this includes loss of the velum and associated musculature, development of labial palps (the apical plate becomes incorporated in the labial palps - Hickman and Gruffydd, 1971), gill growth, and dissoconch formation along the outer edge of the larval shell. The dissochonch is the adult shell; it is thicker and stronger and composed of calcite instead of aragonite (Ansell, 1962; Zardus and

Martel, 2002). During metamorphosis, feeding slows for some species because of the change in primary feeding mechanisms from velar feeding to suspension feeding with the gills (Baker and

Mann, 1994).

It was noted by Quayle (1952) that changes at metamorphosis are much more dramatic for fixed species than for burrowing bivalve species. Clams are seen as a group that has undergone less evolutionary specialization, and therefore metamorphosis involves less change.

Interestingly, this group also has the longest period of metamorphosis (Ansell, 1962), the loss of the velum happens rapidly, while growth of the foot, dissochonch and gills develops slowly

(Ansell, 1962). Siphons develop during metamorphosis, with the exhalent siphon first in most species (Quayle, 1952; Caddy, 1969). During the period before the siphons can be used to create feeding currents, some species use cilia on the foot to draw water into the mantle for feeding (Reid et al., 1992), a process called pedal feeding.

7 Shell length at metamorphosis varies little between species; most species metamorphose at around a shell length of 250 pm. The (Panopea abruptd) is one of the largest burrowing clams in the world and is larger at metamorphosis (350-400 mm - Goodwin and

Pease, 1989) than most other bivalves.

Most species are able to delay metamorphosis if suitable habitats are not available at the time that competence is reached (Mazzarelli, 1922 in Young, 1990; Thorson, 1946; Bayne,

1965). This delay allows more time to search for appropriate settlement sites; however, the longer metamorphosis is delayed, the less discriminating the larva becomes among habitats

(Bayne, 1965). Larvae in cooler water or in water with optimal salinity are able to delay for a longer period of time (Bayne, 1965). The Pacific , gigas, has been seen to delay metamorphosis up to 30 days (Coon et al., 1990), the blue , Mytilus edulis, can delay metamorphosis between 12 and 45 days (Bayne, 1965), and the geoduck P. abrupta can delay up to six weeks (King, 1986). During the period of delay, degeneration of the velum may begin and swimming ability of the larvae weakens, leading to decreased feeding in larvae who have delayed for some time. Collet et al. (1999) compared post-metamorphic growth rates of juvenile Pacific oysters that had delayed metamorphosis to those that had not delayed and found that the oysters that did not delay metamorphosis grew faster.

Metamorphosis can be induced in many species once the larvae become competent, through the use of chemical inducers. Coon and Bonar (1985) showed that oyster larvae (C. gigas) could be induced to settle and metamorphose by L-3,4-Dihydroxyphenylalanine (L-

DOPA) at a concentration of 2.5 x 10"5M; prolonged exposure or higher concentrations had negative effects. The same study also found that oyster larvae could be made to metamorphose without settlement with epinephrine or norepinephrine at a concentration of lO^M. Tan and

Wong (1995) also showed that the oyster, Crassostrea belcheri, settled and metamorphosed with exposure to gamma-amino butyric acid (GABA). Gastropods have been induced to

8 metamorphose with increased concentrations of potassium ions (K+) (Pechenik and Heyman,

1987). Urrutia et al. (2004) demonstrated that Ruditapesphilippinarum could be induced to metamorphose using treatment with acetylcholine, carbamylcholine and serotonin but not catecholamines and L-DOPA, suggesting differences in metamorphic triggers between clams versus oysters, or . Chemical cues are present in the environment that are also involved in settlement and metamorphosis; these will be discussed in greater detail in a subsequent section ("Factors Influencing Settlement Patterns: Chemical").

During metamorphosis the feeding mechanism is in transition and decreased feeding or no feeding is possible until metamorphosis is complete. This process may take days or weeks depending on the species. Consequently, the pediveliger must rely on stored energy reserves to sustain them through metamorphosis. It has been proposed (Rodriguez et al., 1990) that stored food reserves are in the form of protein. Others postulate that stored reserves are lipids (Bayne,

1965). Whyte et al. (1992) believed that either type of reserve can be utilized and that the quality and level of food reserve available is dependant on the quality of the larval diet long before metamorphosis. Still others have related the quality of food provided during conditioning of the brood stock prior to spawning to the metamorphic food reserves available. Reid et al.

(1992) showed that some species can use pedal feeding during the metamorphic transition to supplement food reserves.

For bivalve larvae to be able to test surfaces and eventually settle, they must travel through the benthic boundary layer and make contact with the surface. Directed swimming or sinking has been shown to aid in concentrating competent larvae near the bottom, increasing the likelihood of contact with the surface (Butman, 1987; Eckman, 1990; Gross, et al. 1992;

Eckman, et al. 1994). In a study using video observations in a flume, Finelli and Wethey (2003) were able to document a novel behavior of Crassostrea virginica larvae whereby the larvae contacted the bottom of the flume using abrupt, accelerated downward swimming that the

9 authors termed "dive-bombing". Hydrodynamics in the near-bed region have also been demonstrated to influence the number of competent larvae reaching the surface (see later section

"Factors Influencing Larval Settlement: Physical"). Ultimately, control over contact with the surface results from a combination of active forces by larvae and passive forces of water motion

(Butman, 1987; Underwood and Keough, 2001).

Larval settlement is often used interchangeably with the term recruitment although the two terms refer to different processes (Keough and Downes, 1982). Settlement involves the larval contact with a suitable settlement site, subsequent attachement (permanent attachement in the case of attached speces like oysters or non-permanent in the case of mobile species like clams) and metamorphosis. Recruitment is the survival of the settled larvae to an arbitraty point in time and can therefore be influenced by factors like predation, differential settlement and immigration/emigration (Keough and Downes, 1982; Olafsson et al., 1994)

Juvenile Dispersal

Although both pre-metamorphic and post-metamorphic events influence recruitment

(Keough and Downes, 1982; Woodin, 1991), evidence now indicates that the initial dispersal as veliger larvae may be as important as subsequent dispersals carried out as post-metamorphic early juveniles (Palmer, 1988; Baker and Mann, 1997; Armonies, 1996).

The presence of post-metamorphic bivalves in the water column was evident early in the previous century (Nelson, 1928; Sullivan 1948; Baggerman, 1953; Bayne, 1964a) but few

explanations were presented for how these bivalves entered or maintained their occupancy in the water column without a velum. One theory, offered by Nelson (1928), was that air bubbles

found within the shells of sampled M. edulis increased the buoyancy of the animal and allowed

it to drift. Nelson also observed that these bubbles originated from the gill area, suggesting the bubble contained oxygen much like a swim bladder of a fish.

10 Another theory on the floating behaviour seen in post-metamorphic mussels was suggested by Bayne (1964a). He observed the migration of early M. edulis juveniles in the

Menai Strait, North Wales and postulated that young mussels extended their long ciliated foot to act as a sail to catch passing currents. Although both Nelson (1928) and Bayne (1964a) noted young mytilids hanging from the water surface by thin threads, the link between the threads and entrance into the water column was not made at the time.

Post-metamorphic juveniles of M. edulis have now been shown to enter the water column by secreting a long byssus thread (Lane et al., 1985). This mode of dispersal was termed "byssus drifting" by Sigurdsson et al. (1976) who tested and found byssus drift to occur in 22 species of bivalves. Many authors have observed byssus drift behaviour (See Appendix 1 for a summary table of juvenile dispersal research). The mechanism of byssus drifting is secretion of a long thin "byssus" thread into a passing current. The increased viscous drag on the thread allows the small bivalves to be lifted by the current and carried (Figure 1-3A), much like the flight of young spiders on web strands. Wang and Xu (1997) studied drifting of the larval bivalve Sinonovacula constricta and found it could produce a byssal thread 50 times its body length from a small pore in the base of the foot and calculated that the thread increased the viscous drag 6.7 times the drag on the body alone.

Byssus drift is easily overlooked by observers because the threads are very thin and transparent (Yankson, 1986) and small bivalves are sensitive and will release the threads when disturbed (Beukema and de Vlas, 1989). Composition of these postlarval byssus threads is poorly understood (Baker and Mann, 1997). Yonge (1962) identified the presence of byssal attachment structures in adult bivalves as a neotenous condition. Although the adult byssus may be a derived form of postlarval byssus threads, it is apparent that the structure of postlarval and

adult byssus differs (Lane et al., 1985; Montaudouin, 1997). Scanning electron microscope

images taken by Lane et al. (1985) of both attachment and drift byssus of postlarval M. edulis,

11 show distinct differences. The two types of threads are similar in diameter, however the drift byssus is considerably longer (up to 11cm long) and mono filamentous compared to the fibrous attachment threads. These differences in structure may constitute evidence of functional differences (attachement versus drift). The postlarval drifting threads have been described as

"transparent, elastic, and non-sclerotized" by Yankson, 1986. Many studies indicate that the threads may be mucous-derived (Prezant and Chalermwat, 1984; Beukema and de Vlas, 1989;

Caceres-Martinez et al., 1994). Drifting gastropod molluscs have also been reported to use mucous for drifting (Martel and Chia, 1991; Olivier, 1996). Sigurdsson et al. (1976) was able to stain drift byssus with Alcian blue which indicates it contains acid mucopolysaccarides. It appears that there is a variety of forms of byssus in postlarval bivalves.

In two closely related species, Cerastoderma edule and Cerastoderma glaucum,

Yankson (1986) revealed differences in the form of byssus and how it is used by these two species. The threads of C. glaucum were thicker with more forked side branches and appeared stronger than those of C. edule. The more forked, stronger threads of C. glaucum facilitated climbing of walls in the way a rock climber does, on a single byssus with tufts of side branches where the animal stops to provide support while it probes with its foot (Figure 1-3B).

Montaudouin (1997) carried out a similar study comparing the different functions of postlarval byssus in C. edule and R. philippinarum, two less closely related species. It was found that the byssus of the clam (R. philippinarum) was better at adhesion while the cockle (C. edule) byssus was more effective for suspension and drift.

12 Figure 1-3: Modes of post-larval dispersal. A. Byssus drift; long byssus threads carry the bivalve through the water column. B. Climbing (from Yankson, 1986); the animal uses its ciliated foot and strong byssus to climb walls. Side branches of byssus are used to hold the animal while it probes with its foot. C. Drifting by foot protrusion (from Sorlin, 1988); the animal begins in a normal feeding position, works its way to the surface then protrudes its foot to act as a sail.

The number of different species that carry out byssus drift is extensive and the body size of animals using drift is variable. Byssus drifters have been recorded as small as 0.25mm

(M.edulis- Newell, 1994) to as large as 18.8mm ( - Beaumont and Barnes,

1992); however, scallops of 18.8 mm shell length are also capable of active swimming and therefore this observation of drift in animals of that size may be due to swimming and not byssus drift. Calculation of drag on the byssus in relation to the size of the animal should allow for prediction of the upper limit of the shell length possible for byssus drifting using a single byssus thread. I have come across no literature that goes through such a mathematical exercise.

A large range of species performing byssus drift has been documented (see Appendix 1 for a

summary).

In addition to byssal drifting, other methods of post-larval dispersal have been noted in the literature. Mucous drifting (Prezant and Chalermwat, 1984; Beukema and de Vlas, 1989;

Caceres-Martinez et al., 1994), where mucous is used similarly to byssus described above, to

catch passing currents and facilitate entrance into the water column. Another method, also

13 already mentioned above, is climbing (Rygg, 1970; Boozer and Mirkes, 1979; Yankson, 1986;

Armonies, 1994a) in which the foot is used to pull the animal up the wall and byssus is used to support the animal as it probes with the foot to determine direction. Drifting via foot protrusion, first discussed by Bayne (1964a), was revisited by Sorlin (1988) and documented to occur in

Macoma balthica. Sorlin (1988) provides an extensive description and diagram (Figure 1-3C) of the method of foot protrusion and swelling of the foot and successive drifting of M balthica, however, he also notes the use of byssal threads and does not test whether the foot or the threads are more important in drifting. Simply opening the valves has also been reported as a means of drifting or reducing fall velocity (Olivier et al., 1996; Montaudouin, 1997). Crawling has been noted extensively as a method of postlarval mobility (Brafield and Newell, 1961; Rygg, 1970;

Boozer and Mirkes, 1979; Yankson, 1986; Ahn et al., 1993; Caceres-Martinez et al., 1994;

Montaudoun, 1997). Crawling in young postlarvae is carried out by extension of the long foot by the heavy covering of cilia and mucous on the sole of the foot. Pedal crawling by bivalves offers a smaller range dispersal than drifting and therefore is unlikely as the means for long range dispersal.

Simple bedload transport has also been observed to cause dispersal of recently settled bivalves (Emerson and Grant, 1991; Roegner et al., 1995; Turner et al., 1997) and juvenile brooding bivalves (Sellmer, 1967; Commito et al., 1995), although unlike other forms of dispersal already discussed, this is primarily a passive process. While the process of byssal drift is seen to occur at lower current velocities (Table 1-1), bedload transport takes place at relatively higher velocities (Emerson and Grant, 1991; Roegner et al., 1995). In a study carried out in

Nova Scotia, analysis of contents showed Mya arenaria ranging from 8-15 mm in length being carried with sediments at both exposed and sheltered sites (Emerson and Grant,

1991).

14 Table 1-1: Current velocities reported to cause byssal c rift in post metamorphic bivalves. Reference Current velocity Species studied Sigurdsson et al., 1976 1 cm/sec. Mytilus edulis, Abra alba Prezant and Chalermwat, 1984 10-20 cm/sec. fluminea Laneetal., 1985 0.1 cm/sec. Mytilus edulis Sorlin, 1988 5 cm/sec. Macoma balthica Cummings et al., 1995 6 cm/sec. Macomona lilliana Montaudouin, 1997 10-24 cm/sec. Cerastoderma edule, philippinarum Wang andXu, 1997 lcm/sec. Sinonovacula constricta Hiddink et al., 2002 0.2 cm/sec. Macoma balthica

Directional movement could be accomplished by byssal drift preferentially on an ebb or flood tide depending on the intended direction (onshore/offshore). Olivier et al. (1996) reported

Abra alba drifting on the peak flood tide resulting in net movement towards adult conspecifics.

Cummings et al. (1995) provided evidence of drift in the New Zealand species Macomona lilliana, and found that it drifted most often on ebb . Post-settlement migrations of five species in the Wadden Sea were examined by Armonies (1996) and each was seen to move differently. M. balthica and americanus both initially settled at mean low tide, M. balthica then migrated to the upper intertidal while E. americanus moved to the subtidal. M. edulis initially settled in areas near adults and later moved laterally in the intertidal. C. edule was seen to distribute evenly after patchy settlement and M. arenaria simply moved randomly.

Differences in the resulting movement of these five bivalve species indicate that active processes are at work in determining their final patterns of distribution.

Byssus drifting bivalves have also been reported to show diel patterns in timing of drift

(Armonies, 1994a; Hiddink et al., 2002). Sampling of drifting juveniles in the Wadden Sea by

Armonies (1994a) showed C. edule, M. balthica and E. americanus were more abundant in plankton samples taken in the dark than in light. Hiddink et al. (2002) also found postlarvae M.

15 balthica migrated through the water column at night, and suggested that this behaviour helps with predator avoidance although the authors were unable to detect postlarvae in the stomach contents of predators. Palmer (1988) also noted that meio fauna were more likely to enter the water column at night when risks of pelagic predation by visual predators decreased.

A ten year study by Williams and Porter (1971) revealed annual patterns in the occurrence of post metamorphic bivalves in plankton samples, some species being found regularly during the summer, others were found during winter months.

The directionality, distances involved, duration and timing in post settlement dispersals leads to what some consider active migrational movement (Bayne, 1964a; Armonies, 1994a,b;

Beukema and de Vlas, 1989). Migration happens as a result of an organism choosing one habitat over another based on advantages that the new habitat offers (Hiddink et al., 2001). One of the more extensively studied postlarval migrations is that of M. balthica in the Wadden Sea,

Netherlands. Field sampling on tidal flats in Konigshafen showed that M. balthica initially settled in the lower intertidal, followed by a pelagic summer migration into the upper intertidal, then winter migrations seaward again to areas of adult populations (Armonies, 1994a,b). It was also observed that growth rate for post-settlement larvae was higher in the upper intertidal compared to the lower intertidal. Beukema and de Vlas (1989) confirmed the same migrational pattern of M balthica in the Wadden Sea. They also noted that the lower intertidal offers better adult survival, lower parasite infection and higher growth. However, this area is poorly suited as a nursery for juveniles since higher epifaunal predation, unsuitable sediments and increased exposure make growth rate and survival of juveniles low. The authors attribute the success of

M. balthica in the Wadden Sea to its adaptive migration pattern.

A second postlarval migration that has been identified in bivalves is that of M edulis in the Menai Strait (Bayne, 1964a). Sampling was carried out to track the various life stages of M. edulis from planktonic larvae to late plantigrades. Bayne reported that the larvae settled on

16 filamentous algae in early June, then migrated to adult mussel beds as platigrades (1.0-1.5 mm in length) two months later. He notes that a short growth period before the young mytilids enter into direct competition with adults for resources is advantageous.

A large body of evidence now indicates that in the typical bivalve life cycle there are two stages of dispersal. The first is as pelagic larvae and the second by recently metamorphosed juveniles. Evidence shows this second stage of dispersal is carried out worldwide by a variety of species in a variety of habitats.

Larval Ecology

The majority of marine species have a pelagic larval phase. Unlike terrestrial insects, marine species experience dispersal during the larval stage rather than as adults. As dispersing , larvae are under the control of many factors that influence their distribution and survival. The study of factors that control distribution and abundance of larvae is the field of larval ecology. Marine larval ecology is a relatively young discipline owing partly to difficulties in observing and studying larvae in the field (Young, 1990).

History

The following derives from the comprehensive review by Young (1990) documenting origins and history of larval ecology. Initial recognition of larvae as transitional forms linking embryo and adult life stages can be attributed to John Vaughn Thompson in the early 1800's.

This discovery was essentially the birth of the field of larval ecology, because without an understanding of larval the field could not exist. Through the remainder of the nineteenth century, many more key discoveries of larval linkages between mysterious larval forms and adults were made. These important connections laid the foundation necessary for larval ecology to begin. One of the earliest contributions in the field of larval ecology came from Edward Forbes in 1844 when he noted that molluscs were appearing in areas of previously uninhabited and connected this appearance with recruitment of larvae. During the early twentieth century, oyster culturists began field studies on larval ecology. Of particular note, Julius Nelson made observations of oyster larvae in the field and was able to witness larval migrations and abundance patterns. His son, Thurlow Nelson, followed the work of his father and documented predation by ctenophores on larval oysters in 1925. Predation in the plankton continues today as an important but understudied element. The concept of delay of metamorphosis has proven to be of central importance in larval ecology and is accurately attributed to Guiseppe Mazzarilli in

1922 and Theodore Mortensen in 1921 but is often misattributed to a later paper by Douglas P.

Wilson in 1932. Metamorphic delay led to thinking that larvae had more control over resultant distributions than previously believed. Victor Loosanoff studied specific invertebrate populations for an extended period and was able to determine that populations resulting from planktonic larvae show extreme temporal variability. In addition, Loosanoff made significant contributions to larval spawning and nutrition.

During the Second World War, emphasis shifted from aquaculture driven research to patterns of fouling organisms. Paul Visscher was among the scientists of the time investigating the problem and made important contributions concerning searching behaviour of competent larvae and larval responses to light. Another major contributor to larval ecology was Gunnar

Thorson whose meticulous field work provided a basis on which thorough reviews were created.

He also developed many essential field apparati that are indispensable today, for example larval traps and in situ rearing chambers.

This is not intended to be an extensive review of all contributions to the field but a brief

synopsis of some of the key advances in the field. For a detailed examination of the development of the field of larval ecology, one should consult Young's (1990) thorough review.

18 Factors Influencing Settlement Patterns

Factors influencing larval settlement patterns vary widely and can be classed as either biological or physical. Often, factors overlap and specific influences become difficult to separate and especially difficult to study (Butman, 1987; Butman and Grasle, 1992; Underwood and Keough, 2001; Crimaldi et al., 2002; Pernet et al., 2003). In addition, differential survival versus differential settlement obscures what factors play the largest roles (Woodin, 1976).

Nevertheless, I will outline briefly the physical and biological factors that have been demonstrated to influence settlement; I have included chemical factors as a separate sub-section within biological factors as this field is emerging as heavily influential in larval settlement and warrants a separate summary.

Biological Factors

The microscopic nature and expansive distribution of most planktotrophic larvae makes tracking and determining the interactions and influences of certain biological factors challenging for a given larval cohort (Underwood and Keough, 2001). Nonetheless, many biological factors have been identified as having importance to the distribution and settlement patterns of invertebrate species (Scheltema, 1974). These factors include fertilization success (Levitan,

1995), predation (Thorson, 1946), adult filter feeders (Woodin, 1976), larval nutrition (Boidron-

Metairon, 1995), and larval behaviour.

Larval supply is the original source of variability of larvae arriving at settlement sites.

This supply is initially determined by fertilization success and later modified by other processes like predation and larval behaviour. Levitan (1995) summarises factors that establish fertilization success and notes that in free-spawning species fertilization can be low, but numerous adaptations could be selected for allowing increased fertilization success rate. Most

19 studies on fertilization success have been conducted in the laboratory. Models of field situations have been based on data collected in laboratory measurements (Denny and Shibata, 1989).

Pennington (1985) was the first to conduct in situ fertilization experiments with the urchin

Strongylocentrotus droebachiensis, and revealed the importance of dilution in realistic fertilization success.

After fertilization, significant losses of larvae in the planktonic stage, a phenomenon termed by Thorson (1946) "wastage", can also strongly modify larval supply. It is believed that the primary source of this "wastage" is predation (Nelson, 1925; Thorson, 1946; Morgan, 1995;

Gosling, 2003). One of the earliest records of predatory losses of larvae was made by Nelson

(1925) who observed larval oysters in the guts of ctenophores. It is estimated that pelagic predators consume greater quantities of larvae than their benthic counterparts (Morgan, 1995); however, Johnson and Shanks (2003) made in situ observations on larval predation by planktonic predators and found that predatory losses may be much lower than previously estimated. With limited relevant data from field based research, the question of the impact of predation on pelagic larval stages remains.

One group of benthic organisms that has received recent attention for their potential to shape larval settlement patterns is adult filter feeders. They have been considered to influence recruitment by direct filtration of larvae from the water column (Woodin, 1976; Williams, 1980;

Maurer, 1983; Ambrose, 1984; Hines et al., 1989; Andre and Rosenberg, 1991; Borsa and

Millet, 1992; Mitchell, 1992; Andre et al., 1993; Olafsson et al., 1994, Beukema and Cadee,

1996; Lehane and Davenport, 2004) or by changing near bed flow patterns (Nowell and Jumars,

1984; Ertman and Jumars, 1988; Lindegarth et al., 2002). Reduced larval settlement has been

associated with high adult densities in populations of V. philippinarum (Williams,

1980), M. arenaria (Hines et al., 1989) and C. edule (Andre and Rosenberg, 1991; Andre et al.,

1993). Other studies; however, have shown no overall effect of adult filter feeding populations on settlement of larvae (Maurer, 1983; Hunt et al., 1987; Ertman and Jumars, 1988; Hines, et al.

1989; Thrush et al., 1996). This topic remains unclear on the resulting influence of filter feeding populations on larval settlement patterns.

Survival through the planktonic phase can also be influenced by larval nutrition (this does not apply to lecithotrophic larvae that are supplied by yolk and do not feed while planktonic - Levin and Bridges, 1995). Starvation is a risk if larvae are subjected to extended periods of food limitation; however, food sources are patchy in distribution and larvae are able to survive for extended periods without food (Olson and Olson, 1989; Boidron-Metairon, 1995).

Phytoplankton, dissolved organic matter, bacteria and detritus all contribute to the diets of larvae

(Olson and Olson, 1989; Baldwin and Newell, 1991; Lutz and Kennish, 1992; Boidron-

Metairon, 1995). Although they enjoy a diverse food source, limitations in timing and quality of food have been shown to reduce the number of larvae surviving metamorphosis because of compromised food reserves (Holland and Spencer, 1973; McEdward and Qian, 2001; Pernet et al., 2006).

Behavioural responses of larvae to various cues lead to larval ability to influence their own settlement patterns thus making "active choices" in settlement (Keough and Downes,

1982). Larvae swimming up and down in the water column can result in differential dispersal, particularly if the water is stratified and water in separate strata are moving independently

(Forward, 1988). The influence of large-scale circulation is summarized below in the section titled "Physical Factors". Chemical cues in the environment have a strong influence on larval behaviour resulting in reactions to biofilms and gregarious settlement. These and other chemical factors involved in larval settlement will be summarised in the following section.

21 Chemical Factors

Chemicals have been isolated that influence settlement and metamorphosis in certain larval organisms (Pawlik, 1992a). Some chemicals inhibit settlement of larvae; these are often referred to as anti-fouling chemicals (Dobretsov et al. 2006). Others encourage settlement of certain species of larvae (Steinberg et al., 2002).

The chemical nature of inhibitory and inductive settlement cues is inherently different.

Inhibitory settlement cues would be most effective when associated directly with the surface

(Steinberg et al., 2002). This way, chemicals would not be lost to the overlying water wasting metabolic production energy. There is no need for an inhibitory chemical to act in the overlying water, since there is no need to prevent settlement of a larva that is passing by the surface.

Therefore, inhibitory chemicals tend to be non-polar, surface-associated metabolites. Inducers, on the other hand, should be diffusible and operate in the overlying water, encouraging settlement of larva that would otherwise pass by the surface. Therefore, inducers tend to be water-soluble, primary metabolic compounds. Primary metabolites would be more effective as inducers because organisms are pre-adapted to have an affinity for primary metabolites since they are nutrient or internal signal type molecules (Steinberg et al., 2002).

The anti-fouling properties of the red alga, Delicea pulchra, have been well studied

(Steinberg et al, 2001). It produces a variety of nonpolar halogenated fiiranones that are held in surface vesicles able to release the chemicals onto the plant surface (Steinberg et al., 2001).

Tests with the fiiranones produced by the red algae at relevant concentrations have shown that the fiiranones are effective in preventing settlement of co-occuring larvae on the algal surface

(DeNys et al., 1998).

Sponge metabolites have also been shown to have inhibitory effects (Lee et al., 2001).

The morphology of the sponge provides channels and voids where diffused metabolites would be trapped and the effect of the chemical inhibitor would be kept near the diffusing organism.

22 Bacterially derived inhibitory chemicals have been identified, indicating that some anti-fouling characteristics of organisms may be the result of surface bio films (Lee et al., 2001; Steinberg et al., 2001).

Peptides appear to be important settlement induction chemicals (Steinberg et al., 2001).

One example of this is a dipeptide molecule from oyster shells that induces settlement behaviour in larval oysters. This molecule has not been characterised, but the tripeptide glycine-glycine- arginine (GGR) has the same effect as oyster conditioned water and a similar molecular weight

(Tamburri et al., 1996). Other water soluble peptide-based examples exist for various larvae like sand dollars, , , nudibranchs, and tube worms (Steinberg et al., 2001).

Carbohydrates have received increased attention as chemical inducers of settlement and metamorphosis of larvae. The urchin Holopneustes purpurascens, has been shown to metamorphose in response to a sugar derivative from a red algal host species. The urchin did not respond to extracts from a host species that did not contain the sugar inducer

(Williamson, et al., 2000).

Research by Pawlik and associates (Pawlik, 1992a; Pawlik and Butman, 1993) demonstrated the importance of a fatty acid cue in settlement and gregarious behaviour of the tube worm Phragmatopoma lapidus californica. The fatty acid was isolated from sand used by

adults of the species in building their tubes.

Bio films have been known for some time as an important factor in settlement of many

larval species (Scheltema, 1974; Bonar et al., 1986; Tamburri et al., 1992; Wieczorek and Todd,

1998; Hadfield and Paul, 2001; Huang and Hadfield, 2003). The composition of the biofilm in

the field is difficult, if not impossible, to re-create in the laboratory (Zhao et al., 2003). Most

experimental work on the influence of bacterial colonies on a surface on settling larvae has used

bacterial monocultures. This method provides valuable information about the influence of the

bacteria on settlement, but has limited ecological relevance to films in the field that are complex

23 polycultures and may themselves be chemically influenced by the host surface (Steinberg et al.,

2001). The bio films produce inductive chemicals that facilitate larval settlement and responses of larvae are varied (reviewed by Hadfield and Paul, 2001). Larval responses to biofilms have been shown to change as the bio film ages (Keough and Raimondi, 1996; Wieczorek et al., 1995) and as the density of the bacteria changes (Huang and Hadfield, 2003).

Some species settle exclusively on another plant or animal species, this is called associative settlement (Crisp, 1974). A good example of this is metamorphosis of the (Phestilla sibogae) larvae in response to cues from its coral (Parties) prey. The nudibranch responds to waterborne chemical inducers from coral specifically, resulting in the juvenile nudibranch living on an abundant food source (Hadfield and Paul, 2001). Some other examples of associative settlement are abalone larvae settling on crustose on which it feeds, blue settlement on , a sacoglossan that settles in response to a cue from its algal food , and opisthobranch molluscs that settle in response to prey (Hadfield and

Paul, 2001 and references therein).

Gregariousness, or settlement in the presence of conspecifics (Cole and Knight-Jones,

1939), is seen in a variety of marine organisms and is thought to be related to chemical cues presented by adults or juveniles. Tube worms (Toonen and Pawlik, 1996) barnacles (Knight-

Jones, 1953), sand dollars (Highsmith, 1982), and oysters (Cole and Knight-Jones, 1939) have

all been reported to exhibit gregariousness. Because of their commercial significance, extensive research has been done concerning oyster settlement and metamorphosis. It has been suggested that oyster larvae respond to surface chemicals from bio filmed oyster shells (Bonar et al, 1986), water conditioned with adult oysters (Crisp, 1967), ammonia released at oyster beds (also thought to be related to the microbial films on the oyster) and a peptide similar to GGR

(Tamburri et al., 1996). The larvae may respond to one or all cues at different stages of development.

24 Action of chemical inducers on settlement and metamorphosis can be mimicked by neurotransmitter compounds. GABA, or y-amino butyric acid, induces metamorphosis in abalone (Morse, 1991) and oysters (Tan and Wong, 1995). Coon and Bonar (1985) demonstrated that competent oyster larvae (C. gigas) could be forced to settle and metamorphose by L-3,4-Dihydroxyphenylalanine (L-DOPA) at a concentration of 2.5 x 10"5M, and forced to metamorphose without settlement using epinephrine or norepinephrine at a concentration of lO^M. These neurotransmitter mimics have not been proven to be associated with natural settlement in any of the organisms tested; however, involvement of these compounds in settlement and metamorphosis allows the neuroactive pathways to be investigated in more detail.

Little is known about the mechanisms by which larvae interpret chemical signals that induce or inhibit settlement and metamorphosis. Pawlik (1992b) notes two reasons for the difficulty in study of the chemosensory organs of larvae. First, the size of the larval body and the fine structure makes neurophysio logical examination difficult. Second, repeat experimentation is often impossible since sensory organs to be tested are lost or modified at metamorphosis which occurs directly after sensing the signal chemical(s). Development in this area of research has been slow, and it is not yet proven that sensory organ stimulation is required for settlement induction. In some cases, direct exposure of larvae to ions, neuroactive agents or electrical impulses has stimulated metamorphosis (Pawlik, 1992b).

Evidence exists, however, in favour of the theory that chemoreception is involved in

settlement choice in larvae. Knight-Jones (1953) showed that the antennules of cyprids

functioned in that way. The barnacle larvae use brush-like discs on their antennules to walk over the surface, and these discs may sense the adult chemical cue, arthropodin (Nott and Foster,

1969).

25 Studies on nudibranch larval sensory organs have proposed that the chemoreceptive organs are located between the velar lobes (Bonar, 1978; Chia and Koss, 1982), an area called the apical sensory system. This was confirmed by Hadfield et al. (2000) in an experiment where apical cells of the larvae (Phestilla sibogae) were irradiated. Once irradiated, the nudibranch larvae were unable to respond to cues that induce metamorphosis in larvae that have a fully functioning apical sensory system. Application of K+ and Cs+ ions to the disabled larvae still caused metamorphosis, indicating that these ions operated downstream of the initial chemical inducer. For most other organisms studied, cilia are proposed to be involved in the chemo sensory activities of the larvae.

Physical Factors

Many physical factors play a role in settlement and ultimate distribution of invertebrate larvae. Some of these factors include light (Thorson, 1946), gravity (Bayne, 1964b), wind

(Bertness et al., 1996), (Roughgarden et al., 1988; Wing et al., 1995), hydrodynamics

(Butman, 1987; Boxshall, 2000), surface contour (Eckman, 1990), and geochemistry (Butman and Grassle, 1992; Engstrom and Marinelli, 2005).

Many larvae are known to be differentially light and gravity responsive through their

larval life span. Planktotrophic larvae are known to be photopositive (swim towards light) when they are young and become increasingly photonegative as they near metamorphosis. This

behaviour is believed to increase feeding opportunities when young, and improve settlement

opportunities when competent (Thorson, 1946). Similarly, during the majority of life in the plankton, larvae are geonegative (move away from the direction of the pull of gravity) and when

competent, become geopositive again increasing contact with surfaces (Bayne, 1964b).

Large scale oceanographic circulation patterns can dictate larval dispersal and arrival at

settlement sites (Carriker, 1951). The magnitude of this influence is dependant on duration of

26 the larvae in the water column which is typically longer for planktotrophic larvae (Underwood and Keough, 2001). Currents driven by upwelling along the coast of have been shown (Wing et al., 1995) to influence distribution of settling crabs {Cancer sp.). Bertness et al.

(1996) were able to connect distribution of settling barnacle larvae (Semibalanus balanoides) and overall wind-driven circulation in a bay in Rhode Island. Shanks and Brink (2005), on the other hand, studied the movement of bivalve larvae (Tellina sp., Mulina lateralis, Spisula solidissima and Ensis directus) via upwelling and downwelling currents on the coast of North

Carolina and found that movement of larvae was dependant on vertical distribution of larvae and the species, not the upwelling or downwelling currents.

Many authors have identified bottom roughness and related changes to the near-surface hydrodynamics as factors influencing settling larvae (Crisp, 1955, 1974; Williams, 1978, 1980;

Nowell and Jumars, 1984; Wethey, 1986; Ertman and Jumars, 1988; Gallagher et al., 1983;

Eckman, 1990; Snelgrove et al., 1993; Harvey et al, 1995; Gregoire et al., 1996; Ableson and

Denny, 1997; Kohler, et al., 1999; Boxshall, 2000; Pech, at al., 2002; Crimaldi et al., 2002). In an experiment using needles to mimic the effect of animal tubes on flow, Eckman (1979) found increased recruitment of a tanaid shrimp and a sabellid polychaete near the needles. Gallagher et al. (1983) carried out a similar experiment using sticks instead of needles and found that they facilitated larval settlement in a number of species. And while trying to make observations on juvenile cockles (Cardium edule), Baggerman (1953) used iron gauze screens placed perpendicular to intertidal flow to create a "current shadow" that captured cockle settlers in the

Wadden Sea indicating that the turbulent wake created by the screen led to increases in recruitment.

Slight increases in bottom irregularity (approximately 1.5 mm) have been measured and result in changes in small scale turbulence structure near the bottom (Hendriks et al., 2006).

Turbulence structure has been highlighted by Ableson and Denny (1997) as influencing

27 settlement in a number of ways. The authors note that flow can be a settlement cue unto itself, it can help to mediate other settlement cues in the water by distributing them, and it can help place larvae in physical contact with a surface. The influence of turbulence has been confirmed by other authors who have also observed changes in settlement of larvae resulting from altered turbulence structures (Crisp, 1955; Boxshall, 2000; Pernet, et al., 2003; Fuchs et al., 2004).

Fluid Motion

The motion of water relative to surfaces and organisms, affects all marine biota and in particular with reference to this thesis, it affects bivalve gametes, larvae and juveniles. As such, it is appropriate to highlight some aspects of fluid dynamics of relevance to the dispersing and/or settling larvae.

Benthic Boundary Layer

As a fluid moves past a solid, there is a gradient of velocity created. At the surface of the solid, the "no-slip" condition dictates that the fluid in contact with the solid is stationary

(Vogel, 1994). Friction of the fluid (shear) away from the solid creates a gradient of velocity, eventually terminating in the "free-stream" or maximum velocity. The area under the influence of friction is called the benthic boundary layer (BBL) (Figure 1-4) and the thickness of this layer varies according to a number of parameters (bottom roughness, temperature, water velocity).

This variability and differing research approaches leads to a number of different definitions for the outer limit and an arbitrary nature depending on the scale of interest (Boudreau and

Jorgensen, 2001). Vogel (1994) sums up the typical biological understanding of the BBL with this statement: "(most biologists) have a fuzzy notion that it's a distinct region rather than a

distinct notion that it is a fuzzy region."

28 I: Bottom ;

Figure 1-4: Graphic representation of the flows in the Benthic Boundary Layer. Longer arrows represent faster flows; grey at the bottom represents the surface. Flow increases with distance from the surface and eventually reaches a rate equivalent to the free-stream.

Because hydrodynamic changes in the BBL are of such a fine-scale, certain challenges and hindrances exist for investigators. Recent advances in instrumentation and technology have allowed high resolution measurements to be made within the BBL (Khalili et al., 2001).

Crimaldi et al. (2002) employed a specialised instrument called a laser-Doppler anemometer

(LDA) to analyse flow and help model larval flux to the bottom in turbulence. The LDA uses

Doppler scatter from three laser beams to make detailed measurements of flows in the BBL.

Lasers are non-invasive and thus the sampling artifacts that constrain other systems are eliminated. Flumes are also an excellent method for study of the BBL and other flow related phenomena because they allow control and manipulation of any number of variables (Khalili at al., 2001). Other researchers have used flumes to test patterns of larval settlement in realistic flows (Butman, 1987; Snelgrove et al., 1993, 1999; Gregoire et al., 1996; Boxshall, 2000; Finelli and Wethey, 2003).

Bottom roughness is known to affect friction above the bottom and the BBL characteristics (Dade et al., 2001; Hendricks et al., 2006) and dispersing and settling larvae interact strongly with the BBL (Jumars et al., 2001). The influence of bottom roughness and turbulence on larval settlement patterns was discussed above in section "Factors Influencing

Settlement Patterns: Physical Factors".

Reynolds Number

Reynolds number is a unitless measure of the ratio of viscous and inertial forces. This relationship was established by Osborne Reynolds in the late 1800's while working with flow in pipes and attempting to understand the laws concerning the change in flow from laminar to turbulent (Vogel, 1994). The formula for the Reynolds number (Re) is shown here:

H v

Where /?=density, ^characteristic length (greatest length of the solid in the direction of the

flow), f/=velocity, /w=dynamic viscosity, and v=kinematic viscosity. The ratio of density and viscosity (v: the kinematic viscosity) becomes an important part of this relationship. When Re is

low, kinematic viscosity is high and flows are laminar; when Re is high, kinematic viscosity is

low and turbulence develops. The Reynolds number can be used to predict transitions from

laminar to turbulent flow, and is also useful in scaling experimental parameters to accurately

reflect a system of interest.

When the characteristic length (/) is small, as is the case for bivalve larvae, Re is small

and laminar viscous forces dominate (Vogel, 1994). Life at low Re is very different than from

what we find normal, domination of viscosity and virtual elimination of inertial forces makes

movement an entirely different matter for bivalve larvae and other plankton. With essentially no

inertia to carry it along, the swimming larvae that stops actively swimming stops moving

altogether.

30 Turbulence

Turbulence is ubiquitous in the marine environment (Denny, 1988). In a clever metaphor, Vogel (1994) compared viscous forces to "groupiness" and inertial forces to

"individuality" and in this manner, laminar flow would be an orderly march while turbulence would be a randomly strolling crowd. Turbulence results when water velocity is high enough for the inertial forces to overcome the viscous forces (Vogel, 1994). In turbulent flow, inertial forces dominate and viscous forces oppose it. Turbulence causes mixing and makes chemical signals unpredictable in space and time (Weissberg et al., 2002).

Crisp (1965) predicted that turbulent flow over a surface would effectively dilute any water-soluble cue released a small distance from the surface. Thus, any chemical cue released from a surface would only be held in sufficient quantities in the boundary layer, and in natural conditions that boundary layer would be approximately the same depth as the larvae itself.

Despite this prediction, Tamburri and colleagues (1996) demonstrated that oyster larvae

{Crassostrea virginica) were able to detect chemical cues in flowing conditions. The authors

used both adult oyster conditioned water and the artificial settlement inducer, glycyl-glycyl-L-

arginine, in flowing water to test if oyster larvae reacted differently to the cue in flow. The

larvae demonstrated the same behaviour in flowing conditions as had been observed previously

in still water.

In a test using polychaete larvae {P. lapidosa californica), Pawlik and Butman (1993)

investigated the effects of flow speed and turbulence on settlement. Larvae and passive larval-

mimics were passed over metamorphosis inducing sand and non-inducive sand at various fluid

velocities. Intermediate velocities were the most effective for settlement; the larvae swam away

at low flows and were swept away from the surface at high flows.

31 Shellfish Aquaculture

Shellfish farming has a long history, but it was not until the later part of the 19th century that modern methods were developed (Gosling, 2003). More recently, the number of species and the amount of production has grown enormously. Figure 1-5 shows the production of molluscs from aquaculture (millions of tonnes) and the number of species in production per year

since 1970 (FAO, 2005). Average annual growth in the worldwide production of clams and cockles alone in 2002 was 14.1%, growing from 2.63 million tonnes in 2000, to 3.43 million tonnes in 2002 (FAO, 2004).

14 n Global Trends in Molluscan Aquaculture 70

60 12 -o— Production

-©---Number of species ©®8>fe©®'* 73 10 50 s "O O 8 40 «, cou 5,®®®-®©®-®-® CL 30 W

CD .O + 20 E 3

2 10

1969 1974 1979 1984 1989 1994 1999 2004 Year

Figure 1-5: Annual production of mollusc aquaculture by mass shown with open squares and on left axis. Number of molluscan species in production worldwide shown with solid grey circles and on right axis. Data from FAO 2005.

Bivalves are particularly attractive for culture purposes; they feed on naturally produced

algae and need very little husbandry once they are outplanted (Folke & Kautsky 1989, 1992;

Crawford et al, 2003). Many parts of the world still rely on collection of wild settled juveniles

as a source for production; however advancing hatchery technology is making production of

32 hatchery spawned and raised seed animals more commonly available. Hatcheries have the advantage of being able to produce seed at all times of the year and in large, reliable quantities

(Helm and Bourne, 2004).

Although many countries practice shellfish aquaculture, the global production is heavily dominated by . Figure 1-6 shows the proportional contribution by mass (top eight countries each year only shown) from 1970 through 2004. China's overwhelming dominance of world molluscan production began in the early 1990's; in 2004 China produced 10.4 million tonnes of molluscs by farming, equalling 79% of the global production that year (FAO, 2005).

In comparison, Canada produced 30,000 tonnes of molluscs accounting for 0.3% of the global total in 2004 (FAO, 2005).

Manila Clam Aquaculture

Manila clams (V. philippinarum) are native to but were accidentally introduced to

British Columbia in the 1930's along with oyster seed shipments from Japan (Bourne, 1982).

The clams were first observed in Ladysmith Harbour, British Columbia and were misidentified as Paphia bifurcata (Quayle, 1938). The clams quickly spread through the southern portion of

British Columbia and into Puget Sound (Quayle, 1964). The Manila clam soon supported a strong recreational and commercial fishery (Quayle and Bourne, 1972) and in 1985, became the basis of a culture industry.

33 Global Molluscan Production by Country

1970 1975 1980 1985 1990 1995 2000 2004

Figure 1-6: Global molluscan production by mass contribution by country. Country labels are listed on the right. For each year shown, the top eight countries are graphed, the rest of the countries for that year are pooled in "rest of world" category. Data from FAO 2005.

Although the Manila clam is native to Japan and other parts of southeast Asia, it has

been introduced (both accidentally and intentionally) to many parts of the world. Manila clams

were intentionally introduced to the Hawaiian Islands in the early 1900's and in France in 1972

(Goulletquer, 1997). Subsequent introductions have occurred throughout Europe and in most

locations naturally recruiting populations have become established (Goulletquer, 1997).

The Manila clam belongs to the class , subclass , order Veneroida,

family (Coan et al., 2000). Worldwide distribution and commercial importance of the

species has led to extensive taxonomic confusion. This species has been referred to using 34

different scientific ( and species) names in the literature and has over 22 common names

(Ponurovsky and Yakovlev, 1992; Goulletquer, 1997).

34 The Manila clam is a high value world crop. In 2004, worldwide production of the

Manila clam was 2.9 million tonnes, valued at 2.2 million US$ (FAO, 2005). The capture fishery was once the only source of Manila clams, but aquaculture production has been rapidly increasing since the mid-1980's and quickly outpaced the capture fishery. Manila clam aquaculture today produces nearly 50 times the amount produced by the capture fishery (Figure

1-7).

2,500

World Production Tapes philippinarum

2,000

o ? 1,500

CO cCD c H Capture Fishery o • Aquaculture •= 1,000

500

1950 1955 1960 1965 1970 1975 1980 1985 1990 1995 2000

Figure 1-7: Comparison of Manila clam (Venerupis philippinarum) production from capture fishery versus aquaculture. Capture fishery is shown with grey bars and aquaculture shown in black. Data from FAO 2005.

Clam Culture in British Columbia

In British Columbia, shellfish aquaculture production totalled 9,324 tonnes with a value

of $15 million in 2004; 17% of this production (1,528 tonnes) and nearly half of the value ($7

million Cdn $) were farmed clams (farmgate values - Statistics Canada, 2005). Clam farming in

British Columbia has been traced to pre-European contact when aboriginal people manipulated

35 and managed intertidal areas to make "clam gardens" as a consistent food source (Harper et al.,

2006). Today, clam farming exists in a different, more industrial form.

Farmers in British Columbia typically obtain seed clams from hatcheries. In the hatchery, broodstock adults are conditioned then spawned to obtain larvae. The larvae are fed cultured algae and raised in the hatchery until they metamorphose. Once metamorphosed, the

small seed can be moved to a nursery system where it is grown to a suitable size for planting out on intertidal plots. These nursery systems come in a variety of forms; for a summary see Helm

and Bourne, 2004. Young clams (5-10 mm in shell length) are spread on intertidal plots at a

density of roughly 400-600 individuals per meter2. The details of seed size and density are often

site specific and each grower will make slight adjustments based on what is best suited to the

beach where culture takes place. At most farms, these plots are protected by predator nets (see

next section for more detail). The clams take from two to four years to reach marketable size on

most beaches and once that size is reached, digging crews visit the beach and harvest using

hand-rakes.

Predator Netting

During grow-out, intertidal clams are vulnerable to predators such as moon (eg.

Euspira lewisii - Toba et al., 1992) crabs (eg. Cancer productus - Quayle and Bourne, 1972)

and diving ducks (eg. Melanitta deglani, M. perspincillata andM. nigra - Bourne, 1984).

Efforts to reduce mortality by predation were undertaken in the early 1980's by researchers at

the University of Washington who tested the efficacy of nets placed on the surface of the

sediment (Anderson, 1982). The netting proved effective at reducing predatory losses of clams

and the use of nets soon became common practice for clam farmers (Toba et al., 1992). Use of

nets has since been tested and proven effective in in NW (Cigarria and

Fernandez, 2000) and Maine, USA (Beal and Kraus, 2002). In addition to protecting clams from predation, nets have the potential to stabilize sediments, interfere with local hydrodynamic processes, and increase silt and organic matter deposition (Spencer et al., 1996). Stabilization of beach sediments may lead to a more favourable habitat for the clams and consequently a larger, healthier population. Alternatively, it could lead to changes in sediment characteristics that are important to the benthos, and nutrient and gas exchange at the sediment-water interface (Driscoll, 1975, Bartoli et al., 2001).

Nugues et al. (1996) noted small changes in the benthic community associated with increases in organic carbon and silt content of sediments beneath intertidal oyster trestles in the River Exe , England. The trestles altered water flow in the area around the cultivation site resulting in a decrease in the depth of the oxygenated sediment layer and reduced abundance of benthos below culture structures.

The most commonly used nets in British Columbia at the time of writing this thesis are plastic with a mesh size of 1 -2 cm aperture although some farmers use heavier cotton nets. The nets are secured by pinning down the edge with rebar staples pushed into the ground, digging the edges into the sediment or placing rocks around the periphery. The nets are left in place until harvesting; when the nets are removed for digging (this can be done in one day) then they are replaced (Toba et al., 1992). Depending on the farm location and time of year, the nets can become fouled with algae. When fouling is heavy, the clams below can be at risk of anoxia, so

in those cases the nets are removed and de-fouled before replacement (Toba et al., 1992).

The Baynes Sound region of British Columbia is the source of roughly 50% of the farmed shellfish products in British Columbia. The study sites chosen for research described in this thesis are located in this region. The intertidal area of Baynes Sound is approximately 1,530 hectares; it was estimated that 32% of that area (493 ha) was under tenure for beach culture and

5% (approximately 76 ha.) was covered with clam netting in 2002 (Ministry of Sustainable

Resource Management, 2002).

37 Figure 1-8: Location of Baynes Sound on Vancouver Island, Canada. Inset left shows location of Vancouver Island in relation to Canada.

Thesis Outline

Some settling larvae can actively select settlement locations on a small scale and in some cases turbulence influences that selection. Small changes to the roughness of the sediment can

influence turbulence in near-bottom flows. Predator netting applied to the surface of intertidal clam farms presents a unique scenario to test, in a natural environment, whether distribution of recently settled larvae is affected by netting. Given recent increases in clam farming worldwide,

it is important to understand the potential influences of predator netting on settling larvae.

As a prerequisite to sampling recently settled larvae in the field, I first evaluated a field

sampling method to establish the recovery rate and ensure that differences in sediment grain size

would not bias estimates of recruits. Chapter 2 describes the field sampling method devised

and the evaluation of the method in a laboratory trial using four different sediment types.

38 If netting influences intertidal turbulence, then it follows that the sediments beneath the nets should also display changes beneath netting compared to adjacent plots without netting.

Based on results of other research I predicted that the netting would interrupt intertidal flow such that it would create a more depositional environment in which larvae could potentially settle out in higher numbers. In Chapter 3 I examine the influences of clam netting on sediment grain size, organic and inorganic carbon content as well as examining temperature at netted versus non-netted plots.

Evidence from other research (Glock, 1978; Mitchell, 1992) suggests that clams (>5 mm) recruit at a higher rate to intertidal areas covered with netting. I propose that this higher observed rate of recruitment of adult clams could be explained by greater deposition of larvae if the larvae are acting as passive particles. To test this hypothesis, I measured the density of early recruits on netted and non-netted plots over two years. Chapter 4 describes recruitment rate of recently settled larval clams to plots with netting and without to determine if there is a pattern related to nets on farmed beaches.

The preceding chapters describe results of sampling from field sites where there is limited ability to control certain variables such as predation rate, desiccation, larval influx etc.

Observed patterns of early recruits (described in Chapter 4) were potentially confounded with biotic factors also measured at the field sites. To test the influence of netting on settlement of clams in the absence of biotic factors, laboratory flume trials were used. In Chapter 5 I describe the results of laboratory flume trials comparing settlement of competent, hatchery- raised larvae in flumes with sediment and nets used as crossed treatment factors.

In conclusion, Chapter 6 provides a summary of the results, a brief discussion and recommendations for future research.

39 References

Abelson, A., and M. Denny. 1997. Settlement of marine organisms in flow. Annual Review of Ecology and Systematics, 28: 317-339.

Ahn, I., G. Lopez and R. Malouf. 1993. Effects of the gem clam Gemma gemma on early post- settlment emigration, growth and survival of the Mercenaria mercenaria. Marine Ecology Progress Series, 99: 61-70.

Ambrose Jr, W.G., 1984. Influence of residents on the development of a marine soft-bottom community. Journal of Marine Research. 42: 633-654.

Anderson, G. J. 1982. Intertidal culture of the Manila clam, Tapes philippinarum, using large netting enclosures in Puget Sound, Washington. MSc. Thesis, University of Washington, Seattle. 100pp.

Andre, C and R. Rosenberg. 1991. Adult-larval interactions in the suspension-feeding bivalves Cerastoderma edule and Mya arenaria. Marine Ecology Progress Series. 71: 227-234.

Andre, C, P. R. Jonsson, and M. Lindegarth. 1993. Predation on settling bivalve larvae by benthic suspension feeders: the role of hydrodynamics and larval behaviour. Marine Ecology Progress Series. 97: 183-192.

Ansell, A. D. 1962. The functional morphology of the larva, and the post-larval development of Venus striatula (Da Costa). Journal of the Marine Biological Association of the U.K., 42:419-443.

Armonies, W. 1994(a). Drifting meio - and macrobenthic on tidal flats in Konigshafen: a review. Helgolander Meeresuntersuchungen, 48:299-320.

Armonies, W. 1994(b). Turnover of postlarval bivalves in sediments of tidal flats in Konigshafen (German Wadden Sea). Helgolander Meeresuntersuchungen, 48:291-297.

Armonies, W. 1996. Changes in distribution patterns of 0-group bivalves in the Wadden Sea: byssus-drifting releases juveniles from the constraints of hydrography. Journal of Sea Research 35: 323-334.

Baggerman, B. 1953. Spatfall and transport of Cardium edule. Archives Neerlandaises do Zoologie, 10:315-342.

Baker, P., and R. Mann. 1997. The postlarval phase of bivalve molluscs: a review of functional ecology and new records of postlarval drifting of Chesapeake Bay bivalves. Bulletin of Marine Science, 61:409-430.

Baldwin, B. S., and R. I. E. Newell. 1991. Omnivorous feeding by planktonic larvae of the Crassostrea virginica. Marine Ecology Progress Series, 78: 285-301

40 Barber, B. J. and N. J. Blake. 1991. Reproductive physiology. In: Scallops: Biology, Ecology and Aquaculture. Ed. S. E. Shumway, pp. 377-428. Elsevier Science Publishers B. V., Amsterdam. 2016pp.

Bartoli, M., D. Nizzoli, P. Viaroli, E. Turolla, G. Castaldelli, E. A. Fano, and R. Rossi. 2001. Impact of Tapes philippinarum farming on nutrient dynamics and benthic respiration in the SaccadiGoro. Hydrobiologia, 455:203-212.

Bayne, B. L. 1964a. Primary and secondary settlement in Mytilus edulis L. (). Journal of Animal Ecology, 33:513-523.

Bayne, B. L. 1964b. The responses fo the larvae of Mytilus edulis L. to light and gravity. Oikos, 15: 162-174.

Bayne, B. L. 1965. Growth and delay of metamorphosis of the larvae of Mytilus edulis (L.). Ophelia, 2: 1-47.

Beal, B.F. and G. M. Kraus. 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.

Bertness, M. D., S. D. Gaines, and R. A. Wahle. 1996. Wind driven settlement patterns in the acorn barnacle Semibalanus balanoides. Marine Ecology Progress Series, 137: 103-110.

Beaumont, A. R., and D. A. Barnes. 1992. Aspects of veliger larval growth and byssus drifting of the spat of Pecten maximus and Aequipecten (Chlamys) opercularis. ICES Journal of Marine Science, 49: 417-423.

Beukema, J. J. and J. de Vlas. 1989. Tidal-current transport of thread-drifting postlarval juveniles of the bivalve Macoma balthica from the Wadden Sea to the North Sea. Marine Ecology Progress Series, 52: 193-200.

Beukema, J.J. and G.C. Cadee. 1996. Consequences of the sudden removal of nearly all mussels and cockles from the Dutch Wadden Sea. PSZN I: Marine Ecology. 17: 279-289.

Boidron-Metairon, I. F. 1995. Larval nutrition, pp. 223-248. In: L. McEdward (ed) Ecology of Marine Invertebrate Larvae. CRC Press, Inc., Boca Raton.

Bonar, D. B. 1978. Ultrastructure of a cephalic sensory organ in larvae of the gastropod Phestilla sibogae (Aeolidacea, Nudibranchia). Tissue and Cell, 10: 153-165.

Bonar, D. B., R. M. Weiner, and R. R. Colwell. 1986. Microbial-invertebrate interactions and the potential for biotechnology. , 12: 101-110.

Boozer, A. C. and P. E. Mirkes. 1979. Observations on the fingernail clam, Musculium partumeium (Pisidiidae), and its association with the introduced Asiatic clam, . The , 93:73-83.

41 Borsa, P., and B. Millet. 1992. Recruitment of the clam Ruditapes decussatus in the of Thau, Mediterranean. Estuarine, Coastal and Shelf Science, 35: 289-300.

Boudreau, B. P. and B. B. J0rgensen. 2001. Introduction, pp. 1-3. In: The Benthic Boundary Layer. Eds. B. P. Bourdreau and B. B. J0rgensen. Oxford University Press, New York.

Bourne, N. 1982. Distribution, reproduction and growth of Manila clam, Tapes philippinarum (Adams and Reeve), in British Columbia. Journal of Shellfish Research, 2: 47-54.

Bourne, N. 1984. Clam predation by scoter ducks in the Strait of Georgia, British Columbia, Canada. Canadian Technical Report of and Aquatic Sciences No. 1331. 17 pp.

Boxshall, A. J. 2000. The importance of flow and settlement cues to larvae of the abalone, Swainson. Journal of Experimental and Ecology, 254: 143-167.

Brafield, A. E. and G. E. Newell. 1961. The behaviour of Macoma balthica (L.). Journal of the Marine Biological Association of U.K., 41: 81-87.

Butman, C. A. 1987. Larval settlement of soft-sediment invertebrates: the spatial scales of pattern explained by active habitat selection and the emerging role of hydrodynamical processes. Oceanography and Marine Biology: An Annual Review, 25: 113-165.

Butman, C. A., and J. P. Grassle. 1992. Active habitat selection by Capitella sp. larvae. Two- choice experiments in still water and flume flows. Journal of Marine Research, 50: 669- 715.

Caceres-Martinez, J., J. A. F. Robledo, and A. Figueras. 1994. Settlement and post-larvae behaviour of Mytilus galloprovincialis: Field and laboratory experiments. Marine Ecology Progress Series, 112: 107-117.

Caddy, J.F. 1969. Development of mantle organs, feeding, and locomotion in postlarval Macoma balthica (L.) (Lamellibranchiata). Canadian Journal of Zoology 47: 609-617.

Carriker, M. R. 1951. Ecological observations on the distribution of oyster larvae in New Jersey estuaries. Ecological Monographs, 21:19-37.

Carriker, M. R. 1956. Biology and propagation of young hard clams, Mercenaria mercenaria. Journal of Elisha Mitchell Science Society, 72: 57-60.

Carriker, M. R. 2001. Embryogenesis and organogenesis of veligers and early juveniles, pp. 77-115. In: Biology of the Hard Clam. Developments in aquaculture and . Eds: J. Krauter and M. Castagna. Elsevier Science, Amsterdam, Netherlands. 772 pp.

Carriker, M. R. 2001. Functional morphology and behaviour of shelled veligers and early juveniles, pp. 283-303. In: Biology of the Hard Clam. Developments in aquaculture and fisheries science. Eds: J. Krauter and M. Castagna. Elsevier Science, Amsterdam, Netherlands. 772 pp.

42 Chia, F. S., and R. Koss. 1982. Fine structure of the larval rhinophores of the nudibranch, Rostanga pulchura, with emphasis on sensory receptor cells. Cell Tissue Research , 225: 235-248.

Chia F.S., and J. Buckland-Nicks. 1984. Locomotion of marine invertebrate larvae: a review. Canadian Journal of Zoology 62: 1205-1222.

Cigarria, J, and J.M Fernandez. 2000. Management of Manila clam beds I. Influence of seed size, type of substratum and protection on initial mortality. Aquaculture, 182: 173-182.

Coan, E. V., P. V. Scott, and F. R. Bernard. 2000. Bivalve Seashells of Western Marine bivalve molluscs from Arctic Alaska to Baja California. Santa Barbera Museum of Natural History. 764 pp.

Cole, H. A., and E. W. Knight-Jones. 1939. Some observations and experiments on the setting behariour of larvae of . J. Cons. Int. Explor. Mer. 14: 86-105.

Collet, B., P. Boudry, A. Thebault, S. Heurtebise, B. Morand and A. Gerard. 1999. Relationship between pre- and post-settlement growth in the Crassostrea gigas (Thunberg). Aquaculture 175: 215-226.

Commito, J. A., C. A. Currier, L. R. Kane, K. A. Reinsel, and I. M. Ulm. 1995. Dispersal dynamics of the bivalve Gemma gemma in a patchy environment. Ecological Monographs, 65: 1-20.

Coon, S. L., and D. B. Bonar. 1985. Induction of settlement and metamorphosis of the Pacific oyster, Crassostrea gigas (Thunberg), by L-DOPA and catecholamines. Journal of Experimental Marine B io logy and Eco logy, 94: 211 -221.

Coon, S. L., W. K. Fitt, and D. B. Bonar. 1990. Competence and delay of metamorphosis in the Pacific oyster Crassostrea gigas. Marine Biology, 106:379-387.

Crawford, C. M., C. K. Macloed, and I. M. Mitchell. 2003. Effects of shellfish farming on the benthic environment. Aquaculture, 224: 117-140.

Crimaldi, J. P., J. K. Thompson, J. H. Rosman, R. J. Lowe, and J. R. Koseff. 2002. Hydrodynamics of larval settlement: The influence of turbulent stress events at potential recruitment sites. and Oceanography, 47:1137-1151.

Crisp, D. J. 1955. The behaviour of barnacle cyprids in relation to water movement over a surface. Journal of Experimental Marine Biology, 32: 569-590.

Crisp, D. J. 1965. Surface chemistry, a factor in the settlement of marine invertebrate larvae. Botanica Gotenburgensia III Proceedings of the fifth marine biological symposium. Goteborg 1965: 51-65.

Crisp, D. J. 1967. Chemical factors inducing settlement in Crassostrea virginica (Gmelin). Journal of Animal Ecology, 36: 329-335.

43 Crisp, D. J. 1974. Factors influencing the settlement of marine invertebrate larvae. In: P.T. Grant and A. M. Mackie (eds.) Chemoreception of marine organisms. New York: Academic Press, New York. pp. 177-265.

Cummings, V. J., R. D. Pridmore, S. F. Thrush, and J. E. Hewitt. 1995. Post-settlement movement by intertidal benthic macro invertebrates: Do common New Zealand species drift in the water column? New Zealand Journal of Marine and Freshwater Research, 29: 59-67.

Dade, W. B., A. J. Hogg, and B. P. Bourdeau. 2001. Physics of flow above the sediment-water interface. In: The Benthic Boundary Layer. Eds. B. P. Bourdreau and B. B. J0rgensen. pp. 4-43. Oxford University Press, New York.

Denny, M. W. 1988. Biology and the Mechanics of the Wave-Swept Environment. Princeton University Press, Princeton, New Jersey. 329 pp.

Denny, M. W., and M. F. Shibata. 1989. Consequences of surf-zone turbulence for settlement and external fertilization. American Naturalist, 134: 859-889.

De Nys, R, S. A. Dworjanyn, and P. D. Steinberg. 1998. A new method for determining surface concentrations of marine natural products on . Marine Ecology Progress Series, 162:79-87.

Devauchelle, N. 1990. Sexual development and maturity of Tapes philippinarum. In: G. Alessandra (ed.) Tapes philippinarum biologia e sperimentazione. Ente Svillupo Agricolo Veneto, Venice, , pp 49-62.

Dobretsov, S., H. U. Dahms and P. Y. Qian. 2006. Inhibition of biofouling by and their metabolites. Biofouling, 22: 43-54.

Driscoll, E. G. 1975. Sediment-animal-water interaction, Buzzards Bay, Massachusetts. Journal of Marine Research, 33: 275-302.

Eckman, J. E. 1979. Small-scale patterns and processes in a soft-substratum, intertidal community. Journal of Marine Research, 37: 437-457.

Eckman, J.E. 1990. A model of passive settlement by planktonic larvae onto bottoms of differing roughness. Limnology and Oceanography, 35: 887-901.

Eckman, J. E., F. E. Werner, and T. F. Gross. 1994. Modeling some effects of behavior on larval settlement in a turbulent boundary layer. Deep Sea Research II, 41: 185-208.

Emerson, C. W., and J. Grant. 1991. The control of soft-shell clam (Mya arenarid) recruitment on intertidal sandflats by bedload sediment transport. Limnology and Oceanography, 36: 1288-1300.

44 Engstrom, S. J., and R. L. Marinelli. 2005. Recruitment responses of benthic infauna to manipulated sediment geochemical properties in natural flows. Journal of Marine Research, 63: 407-436.

Ertman, S. C, and P. A. Jumars. 1988. Effects of bivalve siphonal currents on the settlement of inert particles and larvae. Journal of Marine Research, 46: 797-813.

FAO, 2004. The state of the world fisheries and Aquaculture (SOFIA) 2004. FAO Publishing, Rome, Italy. 153 pp.

FAO, 2005 Fisheries Global Information System (FIGIS), Global Aquaculture Production 1950-2004. Last updated May 3 2006. Data retrieved May 15 2006 from:_http://www.fao.org/figis/servlet/TabLandArea?tb_ds=Aquaculture&tb_mode=TAB LE&tb_act=SELECT&tb_grp=COUNTRY

Finelli, C. M., and D. S. Wethey. 2003. Behaviour of oyster {Crassostrea virginica) larvae in boundary layer flows. Marine Biology, 143: 703-711.

Folke, C, and N. Kautsky. 1989. The role of ecosystems for a sustainable development of aquaculture. Ambio, 18: 234-243.

Folke, C, and N. Kautsky. 1992. Aquaculture with its environment: Prospects for sustainability. Ocean and Coastal Management, 17:5-24.

Forward, R. B. 1988. - zooplankton photobiology and behaviour. Oceanography and Marine Biology, 26: 361-393.

Fuchs, H. L., L. S. Mullineaux, and A. R. Solow. 2004. Sinking behavior of gastropod larvae {Ilyanassa obsoleta) in turbulence. Limnology and Oceanography, 49: 1937-1948.

Gallagher, E. D., P. A. Jumars, and D. D. Trueblood. 1983. Facilitation of soft-bottom benthic succession by tube builders. Ecology, 64:1200-1216.

Galtsoff P. S. 1964. The American oyster Crassostrea virginica Gmelin. Fisheries Bulletin, 64: 1- 480.

Glock, J. W. 1978. Growth, recovery and movement of Manila clams {Venerupis philippinarum) planted under protective devices and on open beaches at Squaxin Island, Washington. MSc. Thesis, University of Washington.

Goodwin, C. L., and B. Pease. 1989. Species profiles: Life histories and environmental requirements of coastal and invertebrates (Pacific Northwest) - Pacific geoduck clam. U.S. Fish. Wildl. Serv. Biol. Rep. 82(11.120). US Army Corps of Engineers, TR EL-82-4. 14pp.

Gosling, E. 2003. Bivalve mollusks, biology ecology and culture. Fishing News Books, Blackwell Science. Maiden Massachusetts, 443 pp.

45 Goulletquer, P. 1997. A bibliography of the Manila clam, Tapes philippinarum. IFREMER, RIDRV-97.02/RA/La Tremblade, France. 122 pp.

Gregoire, Y., E. Bourget, and J. L. Verrette. 1996. Deposition of mimics of planktonic invertebrate larvae on simple and complex substrata in flume flows. Marine Ecology Progress Series, 135: 89-100.

Gross, T.F., F. E. Werner, and J. E. Eckman. 1992. Numerical modeling of larval settlement in turbulent bottom boundary layers. Journal of Marine Research, 50: 611-642.

Hadfield, M. G., E. A. Meleshkevitch, and D. Y. Boudko. 2000. The apical sensory organ of a gastropod veliger is a receptor for settlement cues. Biological Bulletin, 198: 67-76.

Hadfield, M. G., and V. J Paul. 2001. Natural chemical cues for settlement and metamorphosis of marine-invertebrate larvae. In: J.B McClintock and B. J. Baker (eds.) Marine Chemical Ecology. CRC Press, Florida, pp. 431-461.

Harper, J. R., R. Bouchard, A. Dick, D. Kennedy, M. Morris, D. Mitchell, D. Sewid-Smith and K. Recalma-Clutesi. 2006 Clam gardens of British Columbia. Canadian Archaeologist Association, in press.

Harvey, M., E. Bourget, and R. G. Ingram. 1995. Experimental evidence of passive accumulation of marine bivalve larvae on filamentous epibenthic structures. Limnology and Oceanography, 40: 94-104.

Heath, W. A., T Broadley, I. Sutherland, and L. Clayton. 1992. A biological assessment of culturing the manila clam in British Columbia. Bulletin of the Aquaculture Association of Canada, 92: 33- 37.

Helm, M. M., and M. Pellizzato. 1990. Hatchery breeding and rearing of the Tapes philippinarum species. In: G. Alessandra (ed.) Tapes philippinarum biologia e sperimentazione. Ente Svillupo Agricolo Veneto, Venice, Italy, pp 117-140.

Helm, M. M., and N. Bourne. 2004. Hatchery culture of bivalves. A practical manual. FAO Fisheries Technical Paper. No. 471. Rome, FAO. 177 pp.

Hendriks, I. E., L. A. van Duren, and P. M. J. Herman. 2006. Turbulence levels in a flume compared to the field: Implications for larval settlement studies. Journal of Sea Research, 55: 15-29.

Hickman, R. W., and L.L. Gruffydd. 1971. The histology of the larva of Ostrea edulis during metamorphosis. In: Crisp D. J. (ed) 4th European Marine Biology Symposium. Cambridge University Press, p 281-294.

Hiddink, J. G., R. P. Kock, and W. J. Wolff. 2002. Active pelagic migrations of the bivalve Macoma balthica are dangerous. Marine Biology, 140: 1149-1156.

Highsmith, R. C. 1982. Induced settlement and metamorphosis in of sand dollar (Dendraster excentricus) larvae in predator-free sites: adult sand dollar beds. Ecology, 63: 329-337.

46 Hines, A. H., M. H. Posey, and P. J. Haddon. 1989. Effects of adult suspension- and deposit- feeding on recruitment of estuarine infauna. Veliger, 32: 109-119.

Holland, D. A. 1972. Various aspects of the reproductive cycle of the Manila clam (Venerupis japonica). M.Sc. Thesis, University of Washington. 60 pp.

Holland, D.L., and B. E. Spencer. 1973. Biochemical changes in fed and starved oysters, Ostrea edulis L. during larval development, metamorphosis and early spat growth. Journal of the Marine Biological Association U.K., 53: 287-298.

Huang, S. and M G. Hadfield. 2003 Composition and density of bacterial biofilms determine larval settlement of the polychaete Hydroides elegans. Marine Ecology Progress Series, 260: 161-172.

Hunt, J. H., W. G. Ambrose, and C. H. Peterson. 1987. Effects of the gastropod, Ilyanassa odsoleta (Say), and the bivalve, Mercenaria mercenaria (L.), on larval settlement and juvenile recruitment of infauna. Journal of Experimental Marine Biology and Ecology, 108: 229-240.

Johnson, K. B. and A. L. Shanks. 2003. Low rates of predation on planktonic marine invertebrate larvae. Marine Ecology Progress Series, 248: 125-139.

Jumars, P. A., J. E. Eckman, and E. Koch. 2001. Macroscopic animals and plants in benthic flows. In: The Benthic Boundary Layer. Eds. B. P. Bourdreau and B. B. Jergensen. pp. 320-347. Oxford University Press, New York.

Kasyanov, V.L., G. A. Kryuchkova, V. A. Kulikova, V.A., and L. A. Medvedeva. 1998. Larvae of marine bivalves and . Oxonian Press Pvt. Lid., New Delhi.

Kennedy, V. S., R. I. Newell, and A. F. Eble. 1996. The Eastern oyster Crassostrea virginica. Maryland SeaGrant College, Maryland USA. 734 pp.

Keough, M. J., and B. J. Downes. 1982. Recruitment of : The role of active larval choices and early mortality. Oecologia 54: 348-352.

Keough, M. J., and P. T. Raimondi. 1996. Responses of settling invertebrate larvae to bioorganic films: effects of large-scale variation in films. Journal of Experimental Marine Biology and Ecology 207: 59-78.

Khalili, A., M. Huetell, and W. Merzkirch. 2001. Fine-scale flow measurements in the benthic boundary layer. In: The Benthic Boundary Layer. Eds. B. P. Bourdreau and B. B. Jorgensen. pp. 44-77. Oxford University Press, New York.

King, J. J. 1986. Juvenile feeding ontogeny of the geoduck Panope abrupta (Bivalvia: Saxicavacea), and comparative ontogeny and evolution of feeding in bivalves. M.Sc. Thesis, University of Victoria, Victoria.

47 Knight-Jones, E. W. 1953. Laboratory experiments on the gregariousness during setting in Balanus balaoides and other barnacles. Journal of Experimental Biology 30: 584-599.

Kohler, J., P. D. Hansen, and M. Wahl. 1999. Colonization patterns at the substratum-water interface: how does surface microtopography influence recruitment patterns of sessile organisms? Biofouling, 14: 237-248.

Lane, D. J. W., A. R. Beamont, and J. R. Hunter. 1985. Byssus drifting and the drifting threads of the young post-larval mussel Mytilus edulis. Marine Biology, 84:301-308.

Lee, Y. K., J. H Lee, and H. K. Lee. 2001. Microbial symbiosis in marine sponges. Journal of Microbiology, 39: 254-264.

Lehane, C. and J. Davenport. 2004. Ingestion of bivalve larvae by Mytilus edulis: experimental and field demonstrations of larviphagy in farmed blue mussels. Marine Biology. 145: 101-107.

Levin,L. A., and T. S. Bridges. 1995. Pattern and diversity in reproduction and development. In: L. McEdward (ed) Ecology of Marine Invertebrate Larvae. CRC Press, Inc., Boca Raton, pp. 1-48.

Levitan, D.R. 1995. The Ecology of Fertilization in Free-Spwaning Invertebrates. In: L. McEdward (ed) Ecology of Marine Invertebrate Larvae. CRC Press, Inc., Boca Raton, pp. 123-156.

Levitan, D.R., M.A. Sewell, and F. S. Chia. 1992. How distribution and abundance influence fertilization success in the sea urchin Strongylocentrotus franciscanus. Ecology, 73: 248- 25.

Lindegarth, M., P. R. Jonsson, and C. Andre. 2002. Physical and numerical modeling of the role of hydrodynamic processes on adult-larval interactions of a suspension-feeding bivalve. Journal of Marine Research, 60:499-516.

Lutz, R. A., and M. J. Kennish. 1992. Ecology and morphology of larval and early post-larval mussels. In: E. M. Gosling (ed). The Mussel Mytilus: Ecology, physiology, genetics and culture. Elsevier Science Publishers, B. V. Amsterdam, pp. 53-85.

Mann, R., and D. A. Evans. 1998. Estimation of oyster, Crassostrea virginica, standing stock, larval production and advection loss in relation to observed recruitment in the James River, Virginia. Journal of Shellfish Research, 17: 239-253.

Mattel, A., and F. Chia. 1991. Drifting and dispersal of small bivalves and gastropods with direct development. Journal of Experimental Marine Biology and Ecology, 150: 131-147.

Maurer, D. 1983. The effect of an infaunal suspension feeding bivalve Mercenaria mercenaria (L.) on benthic recruitment. Marine Ecology, 4: 263-274.

48 McEdward, L.R., and P. Y. Quian. 2001. Effects of the duration and timing of starvation during larval life on the metamorphosis and initial juvenile size of the polychaete Hydroides elegans (Haswell). Journal of Experimental Marine Biology and Ecology, 261: 185-197.

Meadows, P. S. and J. I. Campbell. 1972. Habitat selection by aquatic invertebrates. Advances in Marine Biology. 10: 271-382.

Mileikovsky, S. A. 1973. Speed of active movement of pelagic larvae of marine bottom invertebrates and their ability to regulate their vertical position. Marine Biology 23: 11-17.

Miller, R. L., J. J. Mojares, and J. L. Ram. 1994. Species-specific sperm attraction in the , Dreissena polymorpha, and the quagga mussel, Dreissena bugensis. Canadian Journal of Zoology, 72: 1764-1770.

Ministry of Sustainable Resource Management, British Columbia, Coast and Marine Planning Branch. 2002. The Baynes Sound coastal plan for shellfish aquaculture. British Columbia Ministry of Sustainable Resource Management. Victoria, Canada. 79 pp.

Mitchell, D. 1992. Effect of seed density on Manila clam growth and production at a British Columbia clam farm. Bulletin of the Aquaculture Association of Canada 92: 29-32.

Montaudouin, X de. 1997. Potential of bivalves' secondary settlement differs with species: a comparison between cockle (Cerastoderma edule) and clam (Ruditapes philippinarum) juvenile resuspension. Marine Biology, 128: 639-648.

Morgan, S.G. 1995. Life and Death in the Plankton: Larval Mortality and Adaptation. In: L. McEdward (ed) Ecology of Marine Invertebrate Larvae, Boca Raton, p 279-321.

Morse, A.N. 1991. How do planktonic larvae know where to settle? American Scientist, 79: 154-167.

Nelson, T. C. 1925. On the occurrence and food habits of ctenophores in New Jersey inland coastal waters. Biological Bulletin, 48:92-111.

Nelson, T. C. 1928. Pelagic dissoconchs of the common mussel, Mytilus edulis, with observations on the behaviour of the larvae of allied genera. Biological Bulletin, 55: 180- 192.

Newell, C. R. 1994. Estuary-scale dispersal of post-larval mussels, Mytilus edulis, among eelgrass ( marina) meadows and subsequent recruitment to planted live and mussel shell cultch. Journal of Shellfish Research, 13: 281.

Nott, J. A., and B. A. Foster. 1969. On the structure of the antennular attachment organ of the cypris larva of Balanus balanoides (L.) Philosophical Transactions of the Royal Society of London Series B - Biological Sciences, 256: 115-133.

49 Nowell, A. R. M., and P. A. Jumars. 1984. Flow environments of aquatic benthos. Annual Review of Ecology and Systematics, 15: 303-328.

Nugues, M. M., M. J. Kaiser, B. E. Spencer, and D. B. Edwards. 1996. Benthic community changes associated with intertidal oyster cultivation. Aquaculture Research, 27: 913-924.

Olafsson, E. B., C. H. Peterson, and W. G. Ambrose. 1994. Does recruitment limitation structure populations and communities of macro-invertebrates in marine soft-sediments: the relative significance of pre and post-settlement processes. Oceanography and Marine Biology: An Annual Review, 32: 65-109.

Olivier, F., C. Vallet, J. Dauvin, and C. Retiere. 1996. Drifting in post-larvae and juveniles in an Abra alba (Wood) community of the eastern part of the bay of Seine (English Channell). Journal of Experimental Marine Biology and Ecology, 199: 89-109.

Olson, R. R., and M. H. Olson. 1989. Food limitation of planktotrophic marine invertebrate larvae: does it control recruitment success? Annual Review of Ecological Systems, 20: 225-247.

Orton, J. H. 1937. Some interrelations between bivalve spatfalls, hydrography and fisheries. Nature, 140: 505-506.

Palmer, M. A. 1988. Dispersal of marine meifauna: a review and conceptual model explaining passive transport and active emergence with implications for recruitment. Marine Ecology Progress Series, 48: 81-91.

Paulay, G., L. Boring, and R. R. Strathmann. 1985. Food limited growth and development of larvae: experiments with natural seawater. Journal of Experimental Marine Biology and Ecology, 93: 1-10.

Pawlik, J. R. 1992a. Chemical ecology of the settlement of benthic marine invertebrates. Oceanography and Marine Biology Annual Review. 30: 273-335.

Pawlik, J. R. 1992b. Induction of marine invertebrate larval settlement: evidence for chemical cues. In: V. J. Paul (ed.), Ecological roles of marine natural products. Cornell University Press, pp. 189-236.

Pawlik, J. R., and C. A. Butman. 1993. Settlement of a marine tube worm as a function of current velocity: interacting effects of hydrdynamics and behaviour. Limnology and Oceangraphy 38:1730-1740.

Pech, D., P. L. Ardisson, and E. Bourget. 2002. Settlement of a tropical marine epibenthic assemblage on artificial panels: Influence of substratum heterogeneity and complexity scales. Estuarine, Coastal and Shelf Sciences, 55: 743-750.

Pechenik, J. A. and W. D. Heyman. 1987. Using KCl to determine size at competence for a marine gastropod, Crepidula fornicate (L). Journal of Experimental Marine Biology and Ecology, 112:27-38.

50 Pennington, J. T. 1985. The ecology of fertilization of echinoid eggs: The consequence of sperm dilution, adult aggregation, and synchronous spawning. Biological Bulletin, 169: 417-430.

Pernet, F., R. Tremblay and E. Bourget. 2003. Settlement success, spatial pattern and behavior of mussel larvae Mytilus spp. in experimental 'downwelling' systems of varying velocity and turbulence. Marine Ecology Progress Series, 260: 125-140.

Pernet, F., V. M. Briselj and S. Cartier. 2006. Lipid class dynamics during larval ontogeny of sea scallops, , in relation to metamorphic success and response to antibiotics. Journal of Experimental Marine Biology and Ecology, 329: 265- 280.

Podniesinski, G.S., and B. J. McAlice. 1986. Seasonality of , Mytilus edulis L. larvae in the Damariscotta River Estuary, Maine. Fishery Bulletin, 84: 995-1001.

Ponurovsky, S. K. and Y.M. Yakovlev. 1992. The reproductive biology of the Japanese Littleneck clam, Tapes philippinarum (A. Adams and Reeve, 1850) (Bivalvia: Veneridae). Journal of Shellfish Research, 11: 265-177.

Prezant, R. S. and K. Chalermwat. 1984. Floatation of the bivalve Corbicula Jluminea as a means of dispersal. Science, 225: 1491-1493.

Qian, P. Y. 1999. Larval settlement of polychaetes. Hydrobiologia, 402: 239-253.

Quayle, D. B. 1938. Paphia bifurcata, a new molluscan species from Ladysmith Harbour, BC. Journal of the Fisheries Research Board of Canada, 4: 53-54.

Quayle, D.B. 1952. Structure and biology of the larvae and spat of Venerupis pullustra (Montagu). Transactions of the Royal Society of Edinburgh, 62: 255-297.

Quayle, D. B. 1964. Distribution of introduced marine mollusca in British Columbia waters. Journal of the Fisheries Research Board of Canada, 21: 1155-1181.

Quayle, D. B. and N. Bourne. 1972. The clam fisheries in British Columbia. Fisheries Research Board of Canada, No. 179. 70 pp.

Raven, C. P. 1958. Morphogenesis: the analysis of molluscan development. Pergamon Press, London. 311 pp.

Reid, R. G. B., R. F. McMahon, D. O Foighil, and R. Finnigan. 1992. Anterior inhalant currents and pedal feeding in bivalves. The Veliger, 35:93-104.

Roegner, C, C. Andre, M. Lindegarth, J. Eckman, and J. Grant. 1995. Transport of recently settled soft-shell clams (Mya arenaria L.) in laboratory flume flow. Journal of Experimental Marine Biology and Ecology, 187: 13-26.

Rodriguez, J. L., F. J. Sedano, L. O. Garcia-Martin, A. Perez-Camacho, and J. L. Sanchez. 1990. Energy metabolism of newly settled Ostrea edulis spat during metamorphosis. Marine Biology. 106: 109-111.

51 Roughgarden, J., S. Gaines, and H. Possingham. 1988. Recruitment dynamics in complex life cycles. Science, 241: 1460-1466.

Rygg, B. 1970. Studies on Cerastoderma edule (L.) and Cerastoderma glaucum (Poiret). Sarsia, 43:65-80.

Scheltema, R. S. 1974. Biological interactions determining larval settlement of marine invertebrates. Thalassia Jugoslovica, 10:263-269.

Seed, R., and T. H. Suchanek. 1992. Population and community ecology of Mytilus. In: The Mussel Mytilus: Ecology, physiology, genetics and culture. Ed. E. M. Gosling, pp. 87- 169. Elsevier Science Publishers B. V., Amsterdam.

Sellmer, G. P. 1967. Functional morphology and ecological life history of the gem clam, Gemma gemma (Eulamellibranchia: Veneridae). Malacologia, 5: 137-223.

Shanks, A. L. 1995. Mechanisms of Cross-Shelf Dispersal of Larval invertebrates and Fish. In: L. McEdward (ed) Ecology of Marine Invertebrate Larvae. CRC Press, Inc., Boca Raton, pp.323-367.

Shanks, A. L., and L. Brink. 2005. Upwelling, downwelling, and cross-shelf transport of bivalve larvae: test of a hypothesis. Marine Ecology Progress Series, 302: 1-12.

Sigurdsson, J. B., C. W. Titman, and P. A. Davies. 1976. The dispersal of young post-larval bivalve molluscs by byssus threads. Nature, 262: 386-387.

Snelgrove, P. V. R., C. A. Butman, and J. P. Grassle. 1993. Hydrodynamic enhancement of larval settlement in the bivalve Mulinia lareralis (Say) and the polychaete Capitella sp. I in microdepositional environments. Journal of Experimental Marine Biology and Ecology, 168: 71-109.

Snelgrove, P.V.R., J. Grant, and C. A. Pilditch. 1999. Habitat selection and adult-larvae interactions in settling larvae of soft-shell clam Mya arenaria. Marine Ecology Progress Series 182: 149-159.

Sorlin, T. 1988. Floating behaviour in the tellinid bivalve Macoma balthica (L.). Oecologia 77: 273-277.

Spencer, B. E., M. J. Kaiser, and D. B. Edwards. 1996. The effect of Manila clam cultivation on an intertidal benthic community: the early cultivation phase. Aquaculture Research, 27: 261-276.

Starr, M., J. H. Himmelman, and J. C. Therriault. 1990. Direct coupling of marine invertebrate spawning with phytoplankton blooms. Science, 247:1071-1074.

Statistics Canada. 2005. Aquaculture statistics 2004. Catalogue no. 23-222-XIE. Ministry of Industry, Ottawa, Ontario, Canada.

52 Steinberg, P. D., R. DeNys, and S. Kjelleberg. 2001. Chemical mediation of surface colonisation. In: J.B McClintock and B. J. Baker (eds.) Marine Chemical Ecology. CRC Press, Florida, pp.355-387.

Steinberg, P. D., R. DeNys, and S. Kjelleberg. 2002. Chemical cues for surface colonisation. Journal of Chemical Ecology, 28: 1935-1951.

Sullivan, C. M. 1948. Bivalve larvae of Malpeque Bay, P.E.I. Fisheries Research Board of Canada Bulletin 77. 59pp.

Tamburri, M. N. R. K. Zimmer-Faust, and M. L. Tamplin. 1992. Natural sources and properties of chemical inducers mediating settlement of oyster larvae: a re-examination. Biological Bulletin, 183:327-338.

Tamburri, M. N., C. M. Finelli, D. S. Wethey, and R. K. Zimmer-Faust. 1996. Induction of larval settlement behavior in flow. Biological Bulletin, 191: 367-373.

Tan, S., and T. Wong. 1995. Induction of settlement and metamorphosis in the tropical oyster, Crassostrea belcheri (Sowerby), by neuroactive compounds. Journal of Shellfish Research, 14:435-438.

Thompson, R.J., R.I.E. Newell, V.S. Kennedy, and R. Mann. 1996. Reproductive processes and early development. In: V.S. Kennedy, R.I.E. Newell A.F. Eble (eds.). The Eastern Oyster: Crassostrea viginica. Maryland Sea Grant College, College Park, MD. pp 335-370.

Thorson, G. 1946. Reproduction and larval development of Danish Marine Bottom Invertebrates, C.A.Reitzels Forlag; Bianco Lunos Bogtrykkeri A/S, Kabenhavn.

Thrush, S. F., J. E. Hewitt, R. D. Pridmore, and V. J. Cummings. 1996. Adult/juvenile interactions of infaunal bivalves: contrasting outcomes in different habitats. Marine Ecology Progress Series, 132: 83-92.

Toba, D. R., D. S. Thompson, K. K. Chew, G. J. Anderson and M. B. Miller. 1992. Guide to Manila Clam Culture in Washington. Washington Sea Grant Program, University of Washington. 80 pp.

Toonen, R. J., and J. R. Pawlik. 1996. Settlement of the tube worm Hydroides dianthus (Polychaeta: Serpulidae): cues for gregarious settlement. Marine Biology, 126: 725-733.

Turner, S. J., J. Grant, R. D. Pridmore, J. E. Hewitt, M. R. Wilkinson, T. M. Hume, and D. J Morrisey. 1997. Bedload and water-column transport and colonization processes by post- settlement benthic macro fauna: Does infaunal density matter? Journal of Experimental Marine Biology and Ecology 216: 51-75.

Underwood, A. 2000. Experimental ecology of rocky intertidal habitats: what are we learning? Journal of Experimental Marine Biology and Ecology, 250: 51-76.

53 Underwood, A., and K. Keough 2001. Supply-side ecology: The nature and consequences of variations in recruitment of intertidal organisms,. In: M. Bertness, S. Gaines, and M. Hay (eds.), Marine Community Ecology. Sinauer. pp. 183-200.

Urrutia, P. M., K. Oramoto, and N. Fusetani. 2004. Acetylcholine and serotonin induce larval metamorphosis of the Japanese short-neck clam Ruditapes philippinarum. Journal of Shellfish Research, 23: 93-100.

Utting SD, Spencer BE. 1991. The hatchery culture of bivalve mollusc larvae and juveniles. Laboratory Leaflet, MAFF Directorate of Fisheries Research, Lowestoft (68): 31pp.

Vogel, S. 1994. Life in moving fluids: the physical biology of flow. 467 pp. Princeton University Press, USA.

Wang, W. X., and Z. Z. Xu. 1997. Larval swimming and postlarval drifting behaviour in the infaunal bivalve Sinonovacula constricta. Marine Ecology Progress Series, 148: 71-81.

Weissberg, M. J., M. C. Ferner, D. C. Pisut, and D. L. Smee. 2002. Ecological consequences of chemically mediated prey perception. Journal of Chemical Ecology, 28: 1953-1970.

Wethey, D.S. 1986. Ranking of settlement cues by barnacle larvae: influence of surface contour. Bulletin of Marine Science, 39: 393-400.

Whyte, J. N. C, N. Bourne, N. G. Ginther, and C. A. Hodgson. 1992. Compositional changes in the larva to juvenile development of the Crassadoma gigantea (Gray). Journal of Experimental Marine Biology and Ecology, 163: 13-29.

Wieczorek, S. K, A. S Clare, and C. D. Todd. 1995. Inhibitory and facilitatory effects of microbial films in settlement of Balanus amphitrite larvae. Marine Ecology Progress Series, 119:221-228.

Wieczorek, S. K. and C. D. Todd. 1998. Inhibition and facilitation of settlement of epifaunal marine invertebrate larvae by microbial biofilm cues. Biofouling, 12: 81-118.

Williams, A. B., and H. J. Porter. 1971. A ten-year study of in North Carolina estuaries: occurrence of postmetamorphal bivalves. Chesapeake Science, 12: 26-32.

Williams, J. G. 1978. The influence of adults on the settlement, growth and survival of spat in the commercially important clams, Tapes japonica (Deshayes). PhD. Thesis, University of Washington.

Williams, J. G. 1980. The influence of adults on the settlement of spat of the clam, Tapes japonica. Journal of Marine Research, 38:729-741.

Williamson, J. E., R. DeNys, N. Kumar, D. G. Carson, and P. D. Steinberg. 2000. Induction of metamorphosis in the sea urchin Holopneustespurpurascens by a metabolite complex from the algal host Deliseapulchra. Biological Bulletin, 198: 332-345.

54 Wing, S.R., L. W. Botsford, J. L. Largier, and L. E. Morgan. 1995. Spatial structure of relaxation events and crab settlement in the northern California upwelling system Marine Ecology Progress Series, 128: 199-211.

Woodin, S. A. 1976. Adult-larval interactions in dense infaunal assemblages: Patterns of abundance. Journal of Marine Research. 34:25-41.

Woodin, S. A. 1991. Recruitment of infauna: positive or negative cues? American Zoologist, 31: 797-807.

Yankson, K. 1986. Observations of byssus systems in the spat of Cerastoderma glaucum and C. edule. Journal of the Marine Biological Association of U.K., 66: 277-292.

Yonge, C. M. 1962. On the primitive significance of the byssus in the Bivalvia and its effects in evolution. Journal of the Marine Biological Association of U.K., 42: 113-125.

Young, C. M. 1990. Larval ecology of marine invertebrates - A sesquicentennial history. Ophelia, 32: 1-48.

Young, C. M. 1995. Behavior and locomotion during the dispersal phase of larval life. In: L. McEdward (ed) Ecology of Marine Invertebrate Larvae, pp. 250-270.

Zardus, J. D., and A. L. Mattel. 2002. Phylum mollusca: bivalvia. In: C. M. Young (ed.), Atlas of marine invertebrate larvae. Academic Press, San Diego California, pp. 289-329.

Zhao, B., S. Zhang and P. Y. Qian. 2003. Larval settlement of the silver or goldlip pearl oyster Pinctada maxima (Jameson) in response to natural biofilms and chemical cues. Aquaculture, 220: 883-901.

55 CHAPTER 2: Sampling Recently Settled Clams from Sediments*

Introduction

The Manila clam (Venerupis philippinarum, A. Adams and Reeve, 1850) is of major importance to both the wild fishery and aquaculture industry worldwide. In British Columbia,

Canada, this species is non-native and is thought to have been introduced along with Pacific oyster seed from Japan in the 1930s (Bourne, 1982). Since its introduction, V. philippinarum has become an important species economically and is the basis of the current clam culture industry (Jones et al., 1993). Conditions in British Columbia are favorable for the Manila clam and it has become well established throughout the southern coastline (Quayle, 1974).

In bivalves, a pelagic larval stage is followed by metamorphosis, during which the swimming organ or velum is lost, and the bivalve transforms into the benthic or epibenthic juvenile form. The post-metamorphic juvenile stage of the Manila clam is found in the upper layers of the sediment; however, sampling juveniles from these environments presents many problems. The clams are similar in colour and size to the fine sediments. Consequently, few studies have focussed on this life stage. When sampling has been carried out and early juveniles in the sediment are counted, (Jones, 1974; Glock, 1978; Williams, 1980) the work is extremely time consuming and prone to error. Rarely is reference made in published studies of bivalve settlement to the accuracy of the sorting and counting methods used. A simple, consistent and effective method for separation of post-settlement bivalves from the sediments would allow for more studies to be carried out and more insight gained into recruitment patterns of the Manila clam and other valuable clam species.

" A version of this chapter has been published in: Munroe, D. M., D. Bright and S. McKinley. 2004. Separation of recently settled Manila clams (Tapes philippinarum A. Adams and Reeve, 1850) from three sediment types using sucrose density solution. Journal of Shellfish Research, 23: 89-92.

56 The density of juvenile post-settlement clams and cockles was estimated to be 1.036 -

1.076 g/mL in a study by Montaudouin (1997), and 1.1 g/mL in a study by Jonsson et al. (1991).

Minerals are denser, typically with a specific weight of 2.5 g/mL and higher (Denny, 1993).

Density gradients of silica sols have been used to separate lighter meiofauna from higher density sediment fractions (Burgess, 2001; Schwinghamer, 1981; Nichols, 1979). It has been shown that high density sucrose solutions can also be used to separate meiofauna from muddy organic sediment (Heip et al., 1974). The technique explored herein involves wet serving to isolate the size fraction of the sediment containing the bivalves, then allowing that size fraction to settle through a high density sucrose solution (1.9 g/mL) to isolate the meifauna and allow for easier counting of the bivalves. This technique was tested using 3 different sediment types to determine efficacy of the method on various sediments.

The juvenile clams were expected to float in a solution with a density of 1.9 g/mL.

However, the high concentration of sucrose increases the osmotic pressure on the animal cells causing them to dehydrate, thereby increasing the density of the animals and causing them to sink (Price et al., 1978; Bowen et al., 1972). Although the juveniles become more dense and sink, they do so slowly in relation to the higher density mineral components of the sample that are found in the top layer of the sediment once it has settled out. This process of isopycnic

sedimentation at one solute density to separate particles of different densities is called "rho

spectrometry" by Price et al. (1977).

Materials and Methods

Three different sediment types were obtained from intertidal beaches north of Nanaimo,

British Columbia. Nine tanks were prepared with each of the three different sediment types

(treatments) randomly assigned to three tanks per sediment type. The sediment types were cobble/mud, cobble/sand/shell and mud/sand according to their properties, and a subsample

57 (approximately 20 cm3) of each sediment type was analysed for grain size components, by wet sieving using methods adapted from Komar (1998). Organic material was not removed prior to wet sieving. Briefly, the sediment was wet-sieved, dried at 60°C (Boyd and Tucker, 1992) for

30 minutes, and weighed.

Sediments used in the experiment were autoclaved prior to placement in each aquarium.

Each aquarium was filled to a depth of 5 cm (surface area of each tank was 800 cm2) with the assigned sediment type, then filled with 15 L seawater (filtered to 1 um and sterilized with UV radiation). All aquaria were aerated and warmed to 20°C before addition of competent, hatchery reared Manila clam larvae. A total of 2400 clams were added to each of the nine tanks.

Tanks were maintained at 20°C and clams were fed a combination of 50:50 meullerii and Isochrysis spp (Tahitian strain) at a combined concentration of 20,000 - 30,000 cells/ mL (Jones et al., 1993). Clams were left in the tanks for 11 days to settle and metamorphose. The tanks were then drained to simulate low tide sampling conditions, and core samples of the sediment were taken. Four sediment cores were taken from each of the nine tanks (36 samples total). A small corer made of PVC pipe (5 cm internal diameter) was inserted

1 cm into the sediment and a thin metal lid was slid under the pipe to prevent the sediment from falling out. Samples were placed in a plastic sample bag, labelled and frozen for later counting.

Freezing was chosen as a method of sample preservation to ensure consistency between lab methods and field methods described in Chapter 4.

For enumeration, samples were thawed then placed in 0.01% phloxine B dye for at least

20 minutes (Williams, 1978). Samples were then washed through a series of sieves; the fraction

of sediment from 125 - 500 [im was placed into a high density sucrose solution (1.9 g/mL) in 30

mL test tubes. Tubes were inverted to mix the sediments, to avoid -particle interactions

(Price et al., 1977) then left to settle out by gravity (minimum of 25 minutes). The top layer of

sediment was pipetted off the surface of the settled sediments in the test tube. The amount of sediment pipetted off the top was approximately 1 - 2 mL which is less than 10% of the original core sample volume. The stain coloured the tissue of the clams and made them highly visible when viewed under a microscope and the number of clams in each sample was counted at 60X magnification. Statistical analysis of variance was calculated using JMP statistical software.

Results

The grain size for each sediment type is shown below in Table 2-1. The cobble/mud sediment contained the largest fraction of the >2000 pm size category with 82% by weight. The cobble/sand/shell also contained mostly >2000 pm sized components; however, it should be noted that in this case over half of this size category was comprised of broken shell while the

>2000 pm fraction in the cobble/mud was entirely small cobble. The mud/sand sediment was mostly comprised of medium sand (70% dry weight); with coarse sand and fine sand making up another 18% dry weight. The components >2000 pm were only broken shell in the mud/sand sediment.

TABLE 2-1: Grain size components, percentage by dry weight, of each sediment type. The size category >2000um contains both granule+ and broken shell.

Size class Size Range (u.m) % overall weight cobble/mud cobble/sand/shell mud/sand Shell (>2000) ~ 43 4 Granule + (>2000) 82 30 Very Coarse Sand (1000-2000) 3 8 3 Coarse Sand (500-1000) 4 7 11 Medium Sand (125-500) 7 9 70 Fine Sand (75-125) 2 2 7 Silt (<75 ) 2 1 5_

2400 clams were added to each tank and each tank had a sediment surface area of 800 cm2 on which to settle; therefore, the expected average density of clams in each tank was 3

59 clams/cm2. The surface area of each core was 19.7 cm2, so the expected average number of clams per core was 58.8. Means and 95% confidence limits for number of clams recovered from coring the three sediment types is shown in Figure 2-1. Comparison of mean numbers of clams per core among the three sediment types was done to test if all means were equal regardless of

sediment type. Mean values for clams per core were 60.0 for cobble/sand/shell, 53.1 for cobble/mud, and 57.9 for mud/sand. Based on analysis of variance, there was no significant

difference in the number of juvenile clams recovered from each of the different sediment types

and the expected value 58.8 (n =3, prob. ranged from 0.70 to 0.91). Nor was there a significant

difference between treatments (n =3, prob. = 0.90). Individual t-tests were also run to determine

if the recovery of clams from coring of any of the experimental units (9 tanks) departed

significantly from the expected value based on larval seeding density. There was no statistically

significant difference between the observed and expected values for any of the experimental

units (n=4, prob. ranged from 0.30 to 0.75).

80

70

60 k , am O o 50 a. 40 JS O 30 c co 0)

20 2 Ifllll^pl^^

10

0 Cobble/Mud Cobble/Sand/Shell Mud/Sand

Figure 2-1: Means and standard deviation for numbers of clams (Venerupis philippinarum) per sample for the three sediment types. The dashed line indicates the expected number of clams per sample (58.8) based on number of larvae placed in each tank. N= 3 for each treatment.

60 Discussion

Analysis of grain size components (Table 2-1) shows that there were large differences between the compositions of the three sediment types used in this experiment. This was important since I was testing the accuracy of the sampling methods for extraction and counting of post-settlement juveniles in different types of sediments. For example, the mud/sand sediment was comprised of 70% medium sand, which means 70% of the entire sample was the same size class in which the bivalves are found; therefore initial physical separation with sieving would only eliminate a small volume of sediment from the sample. This has the potential to lead to difficulty and inaccuracy in extraction of the clams from this large sediment fraction. In this experiment, sample composition of 70% medium sand did not create additional inaccuracies in counts: The numbers of clams counted per core did not differ in the three types of sediment.

Further, for all three sediment types, the number of clams counted in each sample did not differ from the value expected per sample based on the number of clams placed in tanks initially. This means that with this method there was nearly 100% recovery of bivalves from sediment regardless of sediment type.

Individual tanks within each sediment type were tested to ensure that mean numbers of clams counted per sample did not differ statistically from one tank to the next. Large standard

errors were seen in data from some individual tanks. These were overcome when all samples of

each sediment type were analyzed together. This may be interpreted as a result of the patchy

settlement of clam larvae (Williams, 1980), especially in coarser and heterogeneous substrates,

and implies that the sampling effort may need to be increased for such sediment types.

The recovery of 100% of the bivalves that were placed in tanks also suggests 100%

survival from the time of introduction of the larvae to the time of recovery of post-settlement

clams. Survival rates through metamorphosis for V. philippinarum in a hatchery generally vary

61 from 50 to 90% depending on larval quality (Utting and Spencer, 1991). The recovery of an estimated 100% of added larvae (had the entire sediment surface been sampled) is probably due to the relatively short duration of the study. Some of the post-settlement clams, in fact, may have been non-viable or dead at the time of sampling, but freezing and subsequent staining would not distinguish recently deceased clams from live ones. In other circumstances, it might be expected that some mortality would occur prior to settlement, so a failure to account for 100% of the introduced larvae might not be attributable to the sorting techniques in other studies.

Separation and counting of live clams prior to freezing was not attempted leaving some question about whether separation as described herein would be equally effective for live specimens or those preserved using other methods such as formalin fixation. Schwinghamer

(1981) conducted tests on live separation of benthos from mud and sediments using centrifugation in sorbitol and Percoll and found it to be an effective separation method that allowed for proper identification and observation of sampled benthos.

In a study by Burgess (2001), density separation of meiofauna from sediment was carried out using Ludox®. Sediment samples were mixed with Ludox® then centrifuged to separate the meifauna. Using this method, Burgess was able to recover 95.9% of the bivalves in the sample.

Jonge and Bouwman (1977) also found use of density separation of nematodes and from sediment and detritus to be more effective and accurate than hand-sorting decantation methods. Both Burgeses (2001), and Jonge and Bouwman (1977) noted that a potential shortcoming of the density separation method is that animals may attach to sediments and therefore sink with them. Post-metamorphic bivalve juveniles have the ability to attach to larger sediments using a byssus. In this study, the sediment fractions larger than 500 um were not examined to look for attached juveniles; however, recovery was estimated at 100% in the size fraction examined, so few, if any, clams were likely to have been found in larger fractions. It is

62 possible that if there were any clams attached to sediments by byssal threads, that the threads were released when the sediment samples were frozen.

Conclusions

Use of the sucrose-density separation method described here is effective for counting newly settled juvenile clams from sediment. Use of the high density sucrose solution to isolate the lower density animals increases sampling efficiency by decreasing the time to sort through

sediment; which in turn increases sampling accuracy since less physical and psychological variance is introduced (Price et al., 1977). This decrease in sorting time would be especially

important for sediments like the mud/sand sediment used here, where sieving would result in

retention of the majority of the sediments along with the bivalves and, thus, hand sorting and

counting would be quite tedious and prone to error. These results show that these methods can

be used in the field with the confidence to count recently settled clams in sediment samples

involving a variety of sediment types.

63 References

Bourne, N. 1982. Distribution, reproduction and growth of Manila clam, Tapes philippinarum (Adams and Reeve), in British Columbia. Journal of Shellfish Research, 2: 47-54.

Bowen, R.A., J.M. St. Onge, J.B. Colton, and CA. Price. 1972. Density-gradient centrifugation as an aid to sorting planktonic organisms, I. Gradient Materials. Marine Biology, 14: 242- 247.

Boyd, C. E., and C. S. Tucker. 1992. Water quality and soil analysis for aquaculture. Alabama Aqricultural Experiment Station, Auburn University. 183 pp.

Burgess, R. 2001. An improved protocol for separating meiofauna from sediments using colloidal silica sols. Marine Ecology Progress Series, 214:161-165.

Denny, M.W. 1993. Air and water The biology and physics of life's media. Princeton University Press, Princeton New Jersey, 342 pp.

Glock, J.W. 1978. Growth, recovery and movement of Manila clams, Venerupis japonica, planted under protective devices and on open beaches at Squaxin Island, Washington. Unpublished M. S. Thesis, Univ. Washington, Seattle. 69 pp.

Heip, C, N. Smol, and W. Hautekiet. 1974. A rapid method of extracting meiobenthic nematodes and copepods from mud and detritus. Marine Biology, 28: 79-81.

Jones, CR. 1974. Initial mortality and growth of hatchery-reared Manila clams, Venerupis japonica, planted in Puget Sound, Washington beaches. Unpublished M.S. thesis, Univ. Washington, Seattle. 90 pp.

Jones, G. G., CL. Sanford, and B. L. Jones. 1993. Manila clam hatchery and nursery methods. Innovative Aquaculture Products Ltd. and Science Council of British Columbia. 73 pp.

Jonge, V. N. de, and L. A. Bouwman. 1977. A simple density separation technique for quantitative isolation of using the colloidal silica Ludox®. Marine Biology, 42: 143-148.

Jonsson, R., C. Andre and M. Lindegarth. 1991. Swimming behaviour of marine bivalve larvae in a flume boundary-layer flow: evidence for near bottom confinement. Marine Ecology Progress Series, 79: 67-76.

Komar, P.D. 1998. Beach processes and sedimentation Second edition. Prentice Hall, New Jersey. 544 pp.

Montaudouin, X de. 1997. Potential of bivalves' secondary settlement differs with species: a comparison between cockle (Cerastoderma edule) and clam (Ruditapes philippinarum) juvenile resuspension. Marine Biology, 128: 639-648.

64 Nichols, J.A. 1979. A simple floatation technique for separating meibenthic nematodes from fine-grained sediments. Transactions of the American Microscopic Society, 98: 127-130.

Price, C.A., J.M. St. Onge-Burns, J.B. Coulton and J.E. Joyce. 1977. Automatic sorting of zooplankton by isopycnic sedimentation in gradients of silica: Performance of "rho spectrometer". Marine Biology, 42:225-231.

Price, C.A., E.M. Reardon and R.R.L. Guillard. 1978. Collection of and other marine by centrifugation in density gradients of a modified silica sol. Limnology and Oceanography, 23: 548-553.

Quayle, D. B. 1974. The intertidal bivalves of British Columbia. British Columbia Provincial Museum. Handbook No. 17.

Schwinghamer, P. 1981. Extraction of living meiofauna from marine sediments by cetrifugation in a silica sol-sorbitol mixture. Canadian Journal of Fisheries and Aquatic Sciences, 38:476-478.

Utting, S. D., and B. E. Spencer. 1991. The hatchery culture of bivalve mollusc larvae and juveniles. Lab. Leafl. No. 68., MAFF Fish. Res. Lowenstoft. 31 pp.

Williams, J.G. 1978. The influence of adults on the settlement, growth, and survival of spat in the commercially important clam, Tapes japonica Deshayes. Unpublished PhD. Thesis, Univ. Washington, Seattle. 59 pp.

Williams, J.G. 1980. Growth and survival in newly settled spat of the Manila clam, Tapes japonica. Fisheries Bulletin, 77: 891-900.

65 CHAPTER 3: The effect of netting on intertidal sedimentation "

Introduction

Although clam culture dates back several centuries in China (Pillay, 1993), recent developments in hatchery technology and grow-out methods have increased production substantially. Average annual growth in the worldwide production of clams and cockles in 2002 was 14.1%, increasing from 2.63 million tonnes in 2000, to 3.43 million tonnes in 2002 (FAO,

2004). In British Columbia, shellfish aquaculture production totalled 9,324 tonnes in 2004; 17% of this production consisted of farmed clams (Statistics Canada, 2005). Baynes Sound, located between Denman Island and Vancouver Island in southern British Columbia, Canada, is a highly productive area with large gravel and sand intertidal zones, ideal for clam culture. For almost a century, shellfish have been an important part of the local economy, and nearly one half of the cultured clams and oysters grown in British Columbia come from farms in this region (Ministry of Sustainable Resource Management, 2002). Of the total intertidal area in Baynes Sound, it was estimated that 32% was under tenure for beach culture and 4.9% was covered with clam netting in 2002 (Ministry of Sustainable Resource Management, 2002).

The swift growth of this industry has increased awareness of possible risk posed to the environment by shellfish aquaculture such as local phytoplankton depletion (Ogilvie et al., 2000;

Zhou et al., 2006), increased biodeposition (Dahlback and Gunnarsson, 1981; Baudinet et al.,

1990; Bartoli, et al., 2001; Jie et al., 2001) and ecosystem changes (Inglis and Gust, 2003;

Beadman et al, 2004). Grow-out of clams is performed on suitable intertidal areas. Intertidal soft-bottom ecosystems are dynamic and subject to a wide range of environmental conditions that create habitats for a vast diversity of species (Lenihan and Micheli, 2001). Although, many

* A version of this chapter has been submitted for publication in: Munroe, D. M. and R. S. McKinley. (2006) Commercial Manila clam (Tapesphilippinarum) tenures in British Columbia, Canada: the effects of anti-predator netting on intertidal sediment characteristics. Estuarine, Coastal and Shelf Science. Submitted May 2006.

66 studies have examined the environmental impacts of bivalve culture, most have focused on oyster and mussel cultivation (Dahlback and Gunnarsson, 1981; Kaspar et al., 1985; Castel et al., 1989; Baudinet et al., 1990; Chamberlain et al., 2001; Caldow et al, 2003; Crawford et al.,

2003; Mazouni, 2004; Harstein and Stevens, 2005) while fewer have examined how clam culture affects intertidal systems (Spencer et al., 1996; Bartoli et al., 2001; Jie et al., 2001).

During grow-out, intertidal clams are vulnerable to predators such as moon snails (eg.

Euspira lewisii - Toba et al., 1992) crabs (eg. Cancer productus - Quayle and Bourne, 1972) and diving ducks (eg. Melanitta deglani, M. perspincillata andM. nigra - Bourne, 1984). To reduce mortality by predation, nets are placed on the surface of the sediment over the small seed clams (Spencer et al, 1992). This practice significantly decreased mortality of small clams

(10.4-34 mm shell length) in estuaries in NW Spain (Cigarria and Fernandez, 2000), and in

Maine, USA, Beal and Kraus (2002) found that netting enhanced collection of wild Mya arenaria spat, resulting from decreased predation and/or increased spatfall. Nets were also proven effective protection from crab and fish predation on beaches in Washington (Anderson,

1982).

In addition to protecting clams from predation, nets have the potential to stabilize

sediments, interfere with local hydrodynamic processes, and increase silt and organic matter

deposition. This could lead to changes in sediment characteristics that are important to the benthos, and nutrient and gas exchange at the sediment-water interface (Driscoll, 1975, Bartoli

et al., 2001). Nugues et al. (1996) noted small changes in the benthic community associated

with increases in organic carbon and silt content of sediments beneath intertidal oyster trestles in the River Exe estuary, England. The trestles altered water flow in the immediate area around the

cultivation site resulting in a decrease in the depth of the oxygenated sediment layer and reduced

abundance of benthos below culture structures. Netting applied to the sediment surface could alter the bottom roughness and alter the sediment properties below. I propose that the netting

67 could cause increased levels of silt and organic matter beneath the netting and in this chapter I examine these effects and test this prediction.

Materials and Methods

Site

Four active Manila clam (Venerupisphilippinarum) aquaculture sites were selected within the Baynes Sound area on the east coast of Vancouver Island, BC, Canada (Figure 3-1 - sites are called Beach 1-4 as labelled in the figure). These beaches are typically seeded with clams (5-10 mm shell length at a density of approximately 400 individuals per m2) each year and are continuously harvested as stock grows into harvestable sizes (>38 mm shell length), although practices vary slightly in relation to beach and operational circumstances. At each beach, measurements were made on paired netted and non-netted plots located directly adjacent to one another. Adjacent paired plots were used to minimise the influence of beach to beach variability. At all four beaches, the nets (mesh size 2 cm x 2 cm) were positioned by shellfish growers as part of regular farm practice prior to initiation of sampling and in all cases the netting had been in place for at least 1 year prior to initiation of sampling. In British Columbia it is common for netting to be left in place year round unless significant fouling requires nets to be removed. None of the nets used in this study experienced notable fouling. Experimental plots were treated as part of regular farm practices as outlined above and the beaches used were representative of the farmed plots in Baynes Sound.

Each of the four beaches used was approximately 0.2 hectares (thus each plot was approximately 0.1 hectare) and had a low slope with sandy/cobble beach substrate. No samples were taken within 1 meter of the edges of the designated beach area to minimize edge effects.

Characteristics including latitude and longitude, tidal height, slope and aspect of each site are

68 summarised in Table 3-1. All sampling (described in subsequent sections) was done during daytime low tide (spring tides) in 2003 and repeated again in 2004.

1 V

V Den man \ Island vancoiPer~-^X. IsJand Vancouver y - Island ^—^—^ w v

^rr,- ',/*N \i_r[Vancouver

Baynes Sound ^" |

Figure 3-1: Map of beach sampling sites within Baynes Sound. Each beach is marked with number and labelled with site name. Inset top right: Location of Vancouver Island within Canada. Inset bottom left: Location of Baynes Sound on Vancouver Island, British Columbia, Canada.

Table 3-1: Site characteristics (tidal height is reported at meters above chart datum).

Character Beach 1 Beach 2 Beach 3 Beach 4

Latitude N49°30'55.2" N49°31'16.1" N49°27'30.9" N49°27'24.6M

Longitude W124°49'28.1" W124°49'31.2" W124°44'50.1" W124°44'37.0"

Tidal Height (m) 2 2.4 1.8 2.5

Slope 1.0% 1.3% 1.3% 2.2 %

Aspect Southeast Northeast Northeast North

69 Clam Populations

Clam populations were sampled at each beach, and each plot within each site on 10-13

August 2003 and 26-29 August 2004. Sixteen large core (15 cm diameter by 15 cm depth) samples were randomly taken at each plot. All cores were sieved to 1 mm in situ and clams retained on the sieve were counted and shell length measured to the nearest mm using vernier callipers. The majority of clams observed were V. philippinarum although small numbers of

Nuttalia obscurata (Varnish clam) were also observed; these two species will be reported here since they were the only two species to occur in high enough densities to be examined.

Clam density (mean individuals/m2) was calculated for each plot in both years (n=16).

These mean densities were used in paired t-tests (netted and non-netted plot on each beach was paired) to compare V. philippinarum and N. obscurata density between netted and non-netted plots. Normality of the difference between the paired data and correlation of the pairs was tested; results of tests are shown in Table 3-2. Density differences of Venerupis philippinarum were normally distributed (p=0.64), however, N. obscurata differences were not normal

(p=0.016) therefore a log-transformation was done to normalize the data (p=0.055). Length frequency of V. philippinarum (the dominant species) was tabulated and tests for normality

showed that the data were not normally distributed therefore non-parametric tests were used to compare the length distribution by beach (4 groups, therefore a Kruskal Wallis test was used), net (2 groups, tested with Kolmogorov - Smirnov test) and year (2 groups, tested with

Kolmogorov - Smirnov test).

Sediment Grain Size

Within each plot, six core samples (5 cm diameter by 1 cm depth) were taken on 7-8

September 2003 and 9-10 September 2004 then stored frozen in labelled plastic bags. For analysis, samples were thawed then dried in a drying oven for 24 hours at 60°C and

70 subsequently homogenized by hand with a mortar and pestle to break up particles aggregated by the drying process. Dried, homogenised samples were placed in the top of a sieve stack sequentially containing sieves with mesh 2mm, 1mm, 0.5mm, 0.25mm, 0.125mm, 0.063mm, and a bottom pan, following the geometric Wentworth grade (Buchanan, 1984). The stack was placed in an automated sieve shaker and agitated for 15 minutes. Sediment retained on each sieve was weighed on an analytical balance.

Table 3-2: Results of tests of assumptions for paired T-test. Normality tested on the distribution of the difference between pairs, correlation calculated for linear regression of pairs.

Pair Tested N Normality test result (p)** Correlation (R2) Venerupis philippinarum 8 0.64 0.002 density Nuttalia obsurata density* 8 0.055 0.703

% Silt 8 0.507 0.512

% Gravel 8 0.825 0.087

% Inorganic carbon 8 0.452 0.356

% Organic carbon 8 0.553 0.813

*data were normalised with log transformation ** Shapiro - Wilk test

Data from the six cores were pooled to avoid pseudoreplication (Hurlburt, 1984) resulting in two paired values (one from the netted plot, the other from the non-netted plot) for

% silt and % gravel for each combination of beach and year (n=8). The mean of the six cores

from each plot was used in a paired T-test to compare the percentage silt (<0.063mm) and percentage gravel (>2mm) on netted and non-netted plots. For proper application of a paired T- test, the difference of the two paired values must be normally distributed and the pairs must be

correlated. Results of tests for normality of the difference of pairs and correlation (R2) are listed

in Table 3-2.

71 Carbon

Cores were taken in the same manner described for sediment grain size above. Thawed samples were dried in a drying oven for 24 hours at 60°C then milled to <0.063mm in a Swing

Mill (Rocklabs Limited, Auckland, New Zealand).

The methods described below for measurement of organic and inorganic carbon were tested to ensure accuracy when employed on intertidal sediments. The methods were compared to two other commonly used carbon assessment methods and the results of these tests can be found in Appendix 2. To measure inorganic carbon, the milled sample was stirred to ensure complete mixing and a 30 mg subsample placed in sample tubes in a coulometer. The air lines of the coulometer system were purged for 1 minute to eliminate contamination by atmospheric

CO2. Subsequently, the sample was injected with 20% HCL with a CM5130 Acidification

Module and the CO2 gas evolved from the sample titrated using a CM5014 Coulometer, UIC

Inc. (Huffman, 1977). Once the titration endpoint was reached, the concentration of inorganic carbon in the sample was recorded.

Measurement of total carbon was achieved by flash combustion of a 20 mg sample of milled sediment prior to elemental analysis using a Carlo Erba NA-1500 Analyzer following the methods outlined in Verardo et al. (1990). Organic carbon was calculated by subtracting

inorganic carbon from total carbon values obtained above. Comparisons of organic and

inorganic carbon at netted and non-netted plots were again tested using pooled data in a paired

T-test as described in the previous sections. Results of normality and correlation tests are listed

in Table 3-2.

Temperature

Temperature was monitored at each plot using Boxcar® Tidbit temperature loggers.

Each logger was attached to a 10 cm long spike that was pushed into the sediment at each plot

72 leaving the data logger flush with the sediment surface. On netted plots they were placed flush with the sediment surface beneath the netting 2 meters from edges of the net to avoid possible edge effects. The loggers recorded temperature every minute for one half of a lunar cycle (2 weeks, new moon through full moon) in early September 2005.

Results

Clam Populations

Length frequencies for netted and non-netted plots are shown in Figure 3-2. It should be noted that the population of V. philippinarum in both netted and non-netted plots derives from both seeded clams (initially seeded beneath netting but may move from nets to the adjacent non- netted plot) and wild settlement from previous years. Clams (V. philippinarum >5mm shell

length) from the netted sites measured 32 mm on average (±8 mm S.D.). Non-netted plots contained slightly smaller clams on average (mean=22 mm ±10 mm S.D.) and demonstrated a more even length frequency distribution over size classes with the exception of Beach 2 in 2004, which shows a high frequency of small clams. Length frequency distribution differed between netted and non-netted plots (pO.OOOOl), between beaches (p=<0.0001), and between years

(p=0.004) at Bonferroni adjusted a=0.0167 (a=0.05 adjusted for 3 tests).

Density of Venerupis philippinarum was significantly higher (p=0.001) in netted plots,

although at Beach 1 in 2003 and Beach 2 in 2004, there appears to be little difference (Figure 3-

3). I failed to detect a difference (p=0.108) in the density of Nuttalia obscurata (>5mm shell

length) between netted and non-netted plots (Figure 3-4).

73 Beach 1 2003 Beach 1 2004 • No Net • No Net • Netted • Netted

ill ll J Pp,,,P,l,«n|ll.[J,X 20 25 : 20 25 30 Size Class (mm) Size Class (mm)

20 l Beach 2 2003 Beach 2 2004 18-1 • No Net 16 • Netted 14

§10 • M lilt, 20 25 30 20 25 30 Size Class (mm) Size Class (mm)

20 20 Beach 3 2003 Beach 3 2004 16 18 • No Net • No Net 16 16 • Netted • Netted 14 14 &12 &12 110 §10 • 8 • 8 * 6 4 .III 2 0 x) limn j, Hi Anita : 1111!taUi 11: 20 25 30 20 25 30 Size Class (mm) Size Class (mm)

20 Beach 4 2003 Beach 42004 18 • No Net • No Net 16 • Netted • Netted 14 £12 &12 §10 §10

i. Hi ,TI,M y , .mi20 l 25 30 10 20 25 30 Size Class (mm) Size Class (mm) Figure 3-2: Length frequencies (count) of Venerupis philippinarum (>5mm) from each site, 2003 in left column and 2004 in right. Clams measured from netted plots represented by black bars, clams from non- netted plots represented by open bars. Shell length in mm plotted along the horizontal axis, frequency on the vertical axis.

74 Figure 3-3: Mean number of Venerupis philippinarum (>5mm shell length) per m2 from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n=16.

400 350 6 300 | 250 | 200 5 150

| 50 jafeu 0 et | NoNst Nat j NoNet et | NoNet et | No Net Net | NoNet Net \ ND Net Net | NoNet Net | NoNet

Beach 1 Beach 2 Beach 3 Beach 4 Beach 1 Beach 2 Beach 3 Beach 4 Figure 3-4: Mean number of Nuttalia obscurata (>5mm shell length) per m from sites in 2003 (left) and 2004 (right). Netted samples represented with hatched bars, non-netted plots represented with grey bars. Error bars represent 95% confidence interval. For each bar, n= 16.

Sediment Grain Size

Percentage of silt (grain size <0.063mm) from sediment samples for each site and plot is shown in Figure 3-5. There was no significant difference detected between percentage of silt between netted and non-netted plots (p=0.129). Percentage of gravel (grain size >2mm) from samples at each site and plot is shown in Figure 3-6; percentage gravel was not significantly different between netted and non-netted plots (p=0.723).

75 25

2

(0 w # 1 # 1

0.5 0.5

Net | isbNet Net | to Net Net | NoNet Net I NoNet et | NoNet et | NjNet et | NoNet et | NoNet Beach 2 Beach 3 Beach 4 Beach 1 Beach 2 Beach 3 Beach 4 Beach 1 Figure 3-5: Percent silt (<0.063 mm grain size) content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beach 4 netted plot in 2004 where n=5. Error bars represent 95% confidence interval.

2003

i50 oC 40 j.30 20

Net | NoNet Net | No Met Net | NoNet m Nohfet Beach 2 Beach 3 Be.ac h 4 Figure 3-6: Percent gravel (>2mm grain size) content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6. Error bars represent 95% confidence interval.

Carbon

The percentage of inorganic carbon in sediments at each site and plot are shown in

Figure 3-7. Inorganic carbon was not significantly different between netted and non-netted plots

at a=0.05 (p=0.07); however organic carbon (Figure 3-8) was significantly (p=0.014) higher at

netted plots.

76 2004

£ 12 s , 3 1 o o 0.8 C 0.8 « ff 0.6 10.6 | 0.4 SS 0.2 Jfl J±L et | NoNet et | NoNet krt f N°Net fit | NoNet

Beach 1 Beach 2 Beach 3 Beach 4

Figure 3-7: Percent inorganic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non-netted plots. For each bar, n=6 except for Beachl netted plot in 2004 (marked above bar with N=5) where n=5. Error bars represent 95% confidence interval.

a06 S o.s 2. 0.5 | 0.4 I. <>•< O 03 £ 0 2 0.1

kit | NoNet Net | NoNet krt | NoNet kit | NoNet Net No Net Net No Net Net No Net Net No Net

Beach 1 Beach 2 Beach 3 Beach 4 Figure 3-8: Percent organic carbon content of samples from each site and plot. Data from 2003 shown in left panel, 2004 shown in right panel. Hatched bars represent netted plots, grey bars represent non- netted plots. For each bar, n=6. Error bars represent 95% confidence interval.

Temperature

Temperature was measured over one tidal cycle during September 2005. Graphs of temperatures measured over 24 hours on the new moon, first quarter moon and full moon

(September 2, 9 and 16, respectively) at each beach and plot are shown in Figure 3-9. The presence of netting appeared to have no effect on the water temperature. However during periods when these plots were exposed by low tide the temperature at the sediment/air interface

appeared to be affected by the presence of netting. These periods correspond to times when the tidal level drops below approximately 2.5m (shown with shaded vertical bar). The temperature

at the sediment/air interface during the low tide events either drops during night time low tides,

or increases during daytime low tides. The plots without netting experience wider temperature

extremes during low tides compared to plots with nets.

77 New Moon - September 2 2005 First Quarter - September 9 2005 Full Moon - September 16 2005 40 40 35 Beach 4 35 Beach 4 30 30 25 25 20 20 15 15 10 10 5 5 40 40 35 35 30 Beach 3 30 25 25 20 20 15 15 10 ID —A—NoNet 10 —*—NoNet 5 Netted 5 - - -Q- - - Netted 40 40 35 35 Beach 2 Beach 2 30 30 25 25 20 20 15 15 10 10 5 5 40 40 35 Beach 1 35 30 30 25 25 20 20 15 15 10 10 5 5 —i—i—i—i—i—i—i—i • •. 1—i i i r Iw^wlfvclfP?—1—1—1—1 1 1 0:00 4:00 8:00 12:66 16:00 20:00 0:00 4:00 8:00 12:00 16:00 20:00 0:00 4:00 8:00 TitoO "1*6:00 20:00

Figure 3-9: Daily temperature measurements at the sediment/air interface, x-axis shows hour of the day. Grey boxes connected with dashed line show the temperature at netted plots, black triangles show non-netted plots and tidal exposure is shown with the shaded grey vertical band. Left column shows the new moon, center shows first quarter moon and right shows the full moon. —i oo Discussion

Clam Populations

The appearance of greater densities of large clams beneath nets may be the result of higher survival due to the presence of nets. Alternatively, the nets may provide substrate stabilisation or offer better feeding opportunities leading to faster growth or less emigration from beneath nets. These alternate hypotheses were not examined here. The length frequency distribution appears to indicate that there is a difference in the population structure of Venerupis philippinarum between the netted and non-netted plots at Beach 2 in 2004; however, there was no significant difference in density of V. philippinarum (> 5mm) measured. The netted plot contains mostly larger clams between 35 and 40 mm shell length, while the non-netted plot contains mostly smaller clams between 10 and 15 mm shell length. This may be a result of movement of seeded clams from adjacent areas either by passive (for example bulk transport) or active (for example competition avoidance) mechanisms.

Varnish clams (N. obscurata >5mm shell length) appeared in higher density at two of the beaches on non-netted plots. Currently the Varnish clam is not a valued commercial product in

British Columbia; however, it has recently begun to be harvested from beaches. Higher densities observed outside of netted plots may be due to recent intense harvesting of those clams on netted plots that, unlike the Manila clam, are not replaced by seeding.

The Manila clam, Venerupis philippinarum, is a suspension feeder and feeding may cause biodeposition at sites of high population density. Jie et al. (2001) calculated the mean clearance rate of V. philippinarum at 0.90 ± 0.34 L.hr"1 per individual and biodeposition rate at

0.06 g.hr"1 per individual (shell length not reported) and found that biodeposition rates at farm

sites (high population density) tended to be higher than non-farm sites. In contrast, Kanaya et al. (2005) found no effect of either Manila clams or the facultative deposit feeder N. olivacia on surface nitrogen, carbon or silt content to estuarine sediments in Japan. This study demonstrated

79 significantly higher densities of V. philippinarum (>5mm shell length) in the netted plots compared to non-netted plots possibly leading to increased biodeposition at these plots. At two sites, I found higher densities of N. obscurata (>5mm shell length). N. obscurata is both a

suspension feeder and deposit feeder and therefore has the potential to contribute to biodeposition or reduction in carbon on these non-netted plots depending on the mode of feeding.

Sediment Grain Size

Placement of netting on intertidal plots has the potential to obstruct overlying water flow

and thus cause sediment deposition and an increase in silt beneath netting. In addition, higher

densities of filter feeding clams beneath netted plots may also alter sediment characteristics

through biodeposition (Jie et al., 2001). However, no difference in % silt or % gravel content in

samples from netted and non-netted plots was observed in this study. In contrast, Mojica and

Nelson (1993) observed higher levels of % silt in sediments for the hard clam (Mercenaria

mercenaria) at an intertidal grow-out site compared with two adjacent control sites; however,

the authors only reported on a single farm site that utilised an alternative farming method where

the clams were placed in net bags on the beach, rather than large nets over the substrate as was

used here.

In a manipulative experiment examining the effects of intertidal V. philippinarum culture

in England, Spencer et al. (1996) observed a four fold increase in sedimentation on netted plots

compared to non-netted plots. In their study, sedimentation rate was measured using sediment

traps. In addition to sedimentation rate, Spencer et al. (1996) also measured the percentage of

silt in sediments and found significantly lower silt (<63um) in the treatment plot with netting

and clams compared to adjacent control plots, seemingly in contrast to the results seen for

sedimentation. Goulletquer et al. (1999) also observed increased silt levels at a clam farm site

using netting to protect V. philippinarum farm plots in France. The authors account the

80 significantly higher levels of silt on the netted plot to the increased sedimentation caused by netting.

Carbon

I observed no significant effect of netting or beach site on inorganic carbon; however organic carbon was significantly higher in netted plots. Goulletquer et al. (1999) observed only a slightly significant difference in organic carbon in sediments due to netting ataV. philippinarum rearing site in France. Additionally, DeGrave et al. (1998) reported no increase in organic carbon at an intertidal site in Ireland where trestles were used for intertidal oyster seed production. Conversely, Mojica and Nelson (1993) and Spencer et al. (1996) observed increased organic carbon in samples taken at clam farm sites (both on M. mercenaria bag culture and netted V. philippinarum culture) compared to adjacent control sites. However, the observed

increase was attributed to high levels of Enteromorpha fouling on nets over the period of study.

It appears that impacts of intertidal farm practices on carbon in sediments are dependant on site specific ecology and oceanography. For the locations observed here, it is likely that the elevated density of V. philippinarum in netted plots contributed to increased organic carbon

levels there through biodeposition (Jie et al., 2001). It is also possible that populations ofN. obscurata found in two of the four non-netted plots (approximately 250 individuals per m2)

could have contributed to reduced organic carbon levels there by deposit feeding. The factor of

netting is confounded with the distribution of clam populations and because clam density was

not manipulated and crossed with netting, I was unable to separate the influence of the two.

Temperature

There was no difference in water temperature between netted and non-netted plots during

tidal immersion; however I did observe a difference in temperature at the sediment/air interface

between netted and non-netted plots during low tide events. The netted plots showed "buffered"

temperature changes during low tides by up to 3°C. During night low tides, the sediment/air

81 interface temperature reduction was less in netted plots compared to non-netted plots, and during day low tides, the sediment/air interface temperature increase was also lower at netted plots.

This is likely due to water retention by nets thus creating insulation at the sediment/air interface.

To my knowledge, this is the first observation of such an effect of netting in the literature.

Conclusions

Based on this study, it appears that netting and clam farming, as it is currently practiced in Baynes Sound British Columbia, has limited effects on the sediment. I was unable to detect a significant impact of netting on levels of silt, gravel or inorganic carbon in sediments. I did observe elevated levels of organic carbon in netted plots relative to non-netted plots although this is likely due to distribution of clam populations in the plots. The netting provides a temperature buffer during tidal exposure by up to 3°C but this effect is unlikely to be biologically significant as most organisms are adapted to tolerate extreme temperature fluxes during tidal exposure.

This study reports results that are inconsistent with those reported elsewhere particularly with reference to the effect of netting on (Mojica and Nelson, 1993; Spencer et al.,

1996; DeGrave et al., 1998; Goulletquer et al., 1999). These discrepancies highlight the importance of understanding the specific effects of aquaculture practices within the ecosystem in which they are being applied.

82 References

Anderson, G. J. 1982. Intertidal culture of the Manila clam, Tapes philippinarum, using large netting enclosures in Puget Sound, Washington. MSc. Thesis, University of Washington, Seattle. 100pp.

Bartoli, M., D. Nizzoli, P. Viaroli, E. Turolla, G. Castaldelli, E. A. Fano, and R. Rossi. 2001. Impact of Tapes philippinarum farming on nutrient dynamics and benthic respiration in the SaccadiGoro. Hydrobiologia. 455:203-212.

Baudinet, D., E. Alliot, B. Berland, C. Grenz, M. Plante-Cuny, R. Plante, and C. Salen-Picard. 1990 Incidence of mussel culture on biogeochemical fluxes at the sediment-water interface. Hydrobiologia 207: 187-196.

Beadman, H. A., M. J. Kaiser, M. Galanidi, R. Shucksmith, and R. I. Willows. 2004. Changes in species richness with stocking density of marine bivalves. Journal of , 41:464-475.

Beal, B.F., and G. M. Kraus. 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.

Bourne, N. 1984. Clam predation by scoter ducks in the Strait of Georgia, British Columbia, Canada. Canadian Technical Report of Fisheries and Aquatic Sciences No. 1331. 17 pp.

Buchanan, J.B. 1984. Sediment analysis. In: Holme, N.A., and A.D. Mclntyre (Eds.). Methods for the study of marine benthos. Blackwell Scientific Publications, Oxford, pp. 41-65.

Caldow, R.W.G., H. A. Beadman, S. McGrorty, M. J. Kaiser, J. D. Goss-Custard, K. Mould and A. Wilson. 2003. Effects of intertidal mussel cultivation on bird assemblages. Marine Ecology Progress Series, 259: 173-183.

Castel, J., P. Labourg, V. Escaravage, I. Auby and M. E. Garcia. 1989. Influence of beds and oyster parks on the abundance and biomass patterns of meio- and in tidal flats. Estuarine, Coastal and Shelf Science, 28:71-85.

Chamberlain, J., T. F. Fernandes, P. Read, T. D. Nickell and I. M. Davies. 2001. Impacts of biodeposits from suspended mussel (Mytilus edulis L.) culture on the surrounding surficial sediments. ICES Journal of Marine Science, 58: 411-416.

Cigarria, J, and J.M Fernandez. 2000. Management of Manila clam beds I. Influence of seed size, type of substratum and protection on initial mortality. Aquaculture. 182:173-182.

Crawford, C. M., C.K. A. Macleod, and I.M. Mitchell. 2003. Effects of shellfish farming on the benthic environment. Aquaculture. 224:117-140.

Dahlback, B., and L. A. H. Gunnarsson. 1981. Sedimentation and sulfate reduction under a mussel culture. Marine Biology, 63: 269-275.

83 DeGrave, S., S.J. Moore, and G. Burnell. 1998. Changes in benthic macro fauna associated with intertidal oyster, Crassostrea gigas (Thunberg) culture. Journal of Shellfish Research, 17: 1137-1142.

Driscoll, E. G. 1975. Sediment-animal-water interaction, Buzzards Bay, Massachusetts. J. Marine Research, 33: 275-302.

FAO, 2004. The state of the world fisheries and Aquaculture (SOFIA) 2004. FAO Publishing, Rome, Italy. 153 pp.

Goulletquer, P., R. Robert, and G. Trut. 1999. Manila clam Tapes philippinarum culture: Sediment clam interactions. Aquatic Living Resources, 12: 45-56.

Harstein, N.D., and CL Stevens. 2005. Deposition beneath long-line mussel farms. Aquacultrue Engineering, 33: 192-213.

Huffman, E. W. D. 1977. Performance of a new automatic carbon dioxide coulometer. Microchemistry Journal, 22: 567-573.

Hurlbert, S. H. 1984. Pseudoreplication and the design of ecological field experiments. Ecological Monographs, 54: 187-211.

Inglis, G. J., and N. Gust. 2003. Potential indirect effects of shellfish culture on the reproductive success of benthic predators. Journal of Applied Ecology., 40: 1077-1089.

Jie, H., Z. Zhinan, Y. Zishan, and J. Widdows. 2001. Differences in the benthic-pelagic particle flux (biodeposition and sediment erosion) at intertidal sites with and without clam (Ruditapes philippinarum) cultivation in eastern China. Journal of Experimental Marine Biology and Ecology, 261: 245-261.

Kanaya, G., E. Nobata, T. Toya, and E. Kikutchi. 2005. Effects of different feeding habits of three bivalve species on sediment characteristics and benthic abundance. Marine Ecology Progress Series, 299: 67-78.

Kaspar, H. F., P. A. Gillespie, I. C. Boyer, and A. L. MacKenzie. 1985. Effects of mussel aquaculture on the nitrogen cycle and benthic communities in Kenepuru Sound, Marlborough Sounds, New Zealand. Marine Biology, 85:127-136.

Lenihan, H. S., and F. Micheli. 2001. Soft-sediment communities. In: Bertness, M. D., S. D. Gaines and M. E. Hay (Eds.). Marine community ecology. Sinauer Associates Inc., Sunderland Massachusetts, USA. pp. 253-287.

Mazouni, N. 2004. Influence of suspended oyster cultures on nitrogen regeneration in a coastal lagoon (Thau, France). Marine Ecology Progress Series, 276: 103-113.

Ministry of Sustainable Resource Management, British Columbia, Coast and Marine Planning Branch. 2002. The Baynes Sound coastal plan for shellfish aquaculture. British Columbia Ministry of Sustainable Resource Management. Victoria, Canada. 79 pp.

84 Mojica, R., and W. G. Nelson. 1993. Environmental effects of a hard clam {Mercenaria mercenaria) aquaculture site in the Indian River Lagoon, Florida. Aquaculture. 113:313- 329.

Nugues, M. M., M. J. Kaiser, B. E. Spencer, and D. B. Edwards. 1996. Benthic community changes associated with intertidal oyster cultivation. Aquaculture Research, 27: 913-924.

Ogilvie, S. C, A. H. Ross, and D. R. Schiel. 2000. Phytoplankton biomass associated with mussel farms in Beatrix Bay, New Zealand. Aquaculture 181: 71-80.

Pillay, T.V.R. 1993. Aquaculture: Principles and Practices. Fishing News Books, Oxford. 575 pp.

Quayle, D.B. and N. Bourne. 1972. The shellfisheries of British Columbia. Fisheries Research Board of Canada Bulletin, 179: 70 pp.

Spencer, B. E. D. B. Edwards, and P. F. Millican. 1992. Protecting Manila clam (Tapes philippinarum) beds with plastic netting. Aquaculture. 105: 251-268.

Spencer, B.E., M. J. Kaiser, and D. B. Edwards. 1996. The effect of Manila clam cultivation on an intertidal benthic community: the early cultivation phase. Aquaculture Research, 27: 261-276.

Statistics Canada. 2005. Aquaculture statistics 2004. Catalogue no. 23-222-XIE. Ministry of Industry, Ottawa, Ontario, Canada.

Toba, D. R., D. S. Thompson, K. K. Chew, G. J. Anderson and M. B. Miller. 1992. Guide to Manila Clam Culture in Washington. Washington Sea Grant Program, University of Washington. 80 pp.

Verardo, D. J., P. N. Froelich, and A. Mclntyre. 1990. Determination of organic carbon and nitrogen in marine sediments using the Carlo Erba NA-1500 Analyzer. Deep Sea Research, 37: 157-165.

Zhou, Y., H. Yang, T. Zhang, P. Qin, X. Xu, and F. Zhang. 2006. Density-dependent effects on seston dynamics and rates of filtering and biodeposition of the suspension-cultured scallop Chlamys farreri in a eutrophic bay (northern China): An experimental study in semi-in situ flow-through systems. Journal of Marine Systems, 59: 143- 158.

85 CHAPTER 4: Bivalve recruitment to culture plots*

Introduction

Supply-side ecology has been highlighted as an important determinant of ecological communities (Lewin, 1986; Roughgarden et al., 1988; Underwood and Fairweather, 1989;

Young, 1990) and emphasis has been placed on factors that determine settlement patterns of

larval life stages (Gaines and Roughgarden, 1985; Keough, 1998; Olivier et al, 2000; Toonen and Pawlik, 2001; Huang and Hadfield, 2003). Many benthic invertebrates have complex life histories involving a pelagic larval stage that terminates with settlement and metamorphosis into the benthic juvenile form (Thorson, 1950). To better understand larval influx as a factor in

community dynamics, it is important to also understand the conditions that dictate larval

settlement and recruitment (Hadfield, 1998; Crimaldi et al., 2002).

Current understanding maintains that both behaviour and physical processes influence

recruitment patterns of marine invertebrates (Butman, 1987; Butman and Grasle, 1992;

Underwood and Keough, 2001; Crimaldi et al., 2002; Pernet et al., 2003). Large scale

observations of larval settlement tend to support the hypothesis that larvae are largely distributed

passively (Bousfield, 1955; Gaines and Bertness, 1992; Borsa and Millet, 1992; Noda, 2004),

while small scale observations tend to indicate behavioural control over settlement (Meadows

and Campbell, 1972; Woodin, 1976; Pawlik, 1992; Rittschof et al., 1998).

Due to substantial time investment, relatively few studies have examined initial

settlement patterns to soft bottom intertidal systems in situ compared to rocky intertidal or

systems involving attached juvenile and adult stages (Baggerman, 1953; Ishii et al., 2001),

although some exceptions exist (Williams, 1978, 1980; Chicharo and Chicharo, 2001b;

* A version of this chapter has been submitted for publication in: Munroe, D. M., and R. S. McKinley, 2006. Effect of predator netting on recruitment and growth of wild clam (Tapes philippinarum) spat on soft-bottom intertidal plots in British Columbia, Canada. Marine Biology - submitted May 2006.

86 Fraschetti et al., 2003). In addition, once sampled and counted, recently settled bivalves are extremely difficult to identify and few reliable taxonomic resources exist (Quayle, 1951;

Loosanoff et al., 1966, Ishii, et al., 2001). Because of the inherent challenges, the bulk of larval behaviour research has been accomplished in lab settings which can be argued to have limited relevance to the natural environment (Butman et al., 1988; Engstrom and Marinelli, 2005).

Settlement of individual larvae is not synchronous and is therefore effectively not measurable on the population level at any one point in time with the tools at our disposal currently. Measurement of settlement is most often estimated by measuring recruitment, a process that incorporates settlement and post-settlement events like immigration, emigration and mortality (Keough and Downes, 1982; Olafsson et al., 1994). As time elapses between settlement and measurement of recruitment, potential for erroneous estimation of settlement grows (Pomerat and Reiner, 1942). The larvae of the Manila clam (Venerupis philippinarum) settle at approximately 220 pm (Helm and Bourne, 2004). Here, I have attempted to measure recruitment of V. philippinarum at a postlarval size (200-600 um) that would most closely reflect the magnitude of settlers to the benthos (Butman, 1987) and will thus call these "early recruits" in an effort to reflect the discrepancy between settlement and recruitment.

It has been suggested by many authors (Crisp, 1955, 1974; Williams, 1978, 1980;

No well and Jumars, 1984; Ertman and Jumars, 1988; Eckman, 1990; Harvey et al, 1995;

Ableson and Denny, 1997; Boxshall, 2000; Pech, at al., 2002; Crimaldi et al., 2002) that

sediment surface rugosity and related alteration of near-bottom flows can influence transmission of chemical cues, physical contact with the bottom and ultimately retention of settled larvae. In an experiment where needles were stuck into sediments to mimic the effect of animal tubes on

flow, Eckman (1979) found increased recruitment of a tanaid shrimp and a sabellid polychaete

in the immediate vicinity of the needles. In a similar experiment where sticks were used in place

of needles, Gallagher et al. (1983) found that sticks facilitated larval settlement in a number of

87 species. The Japanese have been reported to use a variety of methods to slow water currents and

increase recruitment of Manila clams such as bamboo fences and placing sticks vertically throughout the tidal flats (a procedure called "brushing") (Cahn, 1951). And finally, iron gauze

screens placed perpendicular to intertidal flow created a "current shadow" on study plots in the

Wadden Sea which resulted in elevated levels of cockle (Cardium edule) settlement

(Baggerman, 1953).

The practice of clam farming in many countries worldwide involves applying large nets to intertidal surfaces suitable for clam production to protect valuable crops of cultured clams from predation (Spencer et al., 1992; Toba et al., 1992; Spencer, 2002). Use of these nets has the potential to alter flow patterns near the sediment surface and thus influence recruitment patterns of shellfish larvae to those areas (Heath et al., 1992; Beal and Kraus, 2002).

Adult filter feeders have also been predicted to modify larval settlement through direct

filtration of the potential recruits from the water column (Woodin, 1976; Lehane and Davenport,

2004) or alteration of near bed flow patterns (Nowell and Jumars, 1984; Ertman and Jumars,

1988; Lindegarth et al., 2002). Correlation of decreased larval settlement with high adult filter

feeder densities has been observed for populations of V. philippinarum (Williams, 1980), Mya

arenaria (Hines et al., 1989) and Cerastoderma edule (Andre and Rosenberg, 1991; Andre et

al., 1993). However, other studies have shown no overall effect of adult filter feeding

populations on settlement of larvae (Maurer, 1983; Hunt et al., 1987; Thrush et al., 1996).

In this study, I measured early recruitment of clam larvae (V. philippinarum) to farmed

plots with and without netting and at differing levels of clam (>5 mm shell length) density. It

was my intention to (1) determine if netting alters recruitment patterns of early post-larvae, and

(2) examine the relationship between filter feeder density and early recruitment levels in the

field.

88 Materials and Methods

Four active Manila clam (V. philippinarum) aquaculture sites were selected within

Baynes Sound on the east coast of Vancouver Island, BC, Canada (Figure 3-1). Baynes Sound is a highly productive area with large gravel/sand intertidal regions ideal for clam culture. These sampling sites will be referred to as beaches with numbers assigned as shown in Figure 3-1.

Each beach was considered a block and netting was applied as a treatment to one half of the beach (each beach was approximately 0.2 hectares). At all four beaches, the nets had been applied by shellfish growers as part of regular farm practice prior to initiation of sampling. The beaches were large, low energy and low slope with sand/cobble beach substrate, typical of the

Baynes Sound area. Characteristics such as latitude and longitude, tidal height, slope and aspect of each site are listed in Table 3-1.

At each plot (to which net or no-net was applied) within each beach, sixteen small core samples (5 cm diameter by 1 cm depth) were taken at randomly determined locations by hand with a plastic corer. Sampling was repeated in this manner on four different daytime low tides

(spring tides) each year between August and October of 2003 and 2004 to coincide with peak V. philippinarum larval settlement (Neil Bourne, DFO Nanaimo, British Columbia, Canada, personal communication; Williams, 1978). Each sampling event was carried out over two

consecutive days and the sampling dates in both 2003 and 2004 were as follows: 10/11 August,

25/26 August, 7/8 September, and 7/8 October. All samples were stored frozen (-20°C), then

each was enumerated for recently settled clams (200-600 pm shell length) following the

methods outlined in Munroe et al. (2004). All clams counted were photographed using a Zeiss

Stemi SV 11 dissecting microscope (Carl Zeiss Inc., Oberkochen, ) with a CoolSnap

Pro digital camera (Media Cybernetics Inc., Silver Springs, Maryland, USA) attached. These

89 images were used to measure shell length with ImagePro Plus 4.5.1 image processing software

(Media Cybernetics Inc., Silver Springs, Maryland, USA).

Sixteen samples from each plot were counted to compensate for the patchy nature of

larval settlement (Muus, 1973; Hall et al., 1992) and the small size required for sample cores.

These sixteen samples were averaged to avoid pseudoreplication (Hurlbert, 1984) resulting in one value for mean settlement for each combination of beach-net-year-date (n=63 due to loss of one data point prior to testing). The data were log-transformed to normalise the data prior to analysis. Normality was assessed using the Shapiro-Wilk test and homogeneity of variances was assessed with Levene's test.

Settlement rates were compared using a univariate ANOVA with a split-plot in time model using SPSS statistical software. Sample dates prior to settlement (low settlement rates) were considered "pre-settlement"; these were 10/11 August, 25/26 August, and 7/8 September in

2003 and 10/11 August and 25/26 August in 2004. Dates after the peak of settlement were

considered "post-settlement". Settlement trends demonstrating peak settlement events are

shown in Figure 4-1. In the ANOVA model, netting and year were considered fixed factors,

settlement (pre-settlement and post-settlement as described above) was considered a covariate

and beach was a block (therefore a random factor).

Species identification at such early developmental stages is challenging and few keys

exist to aid in identification (Loosanoff et al., 1966; LePennec, 1980; Goodsell et al., 1992;

Sakai and Sekiguchi, 1992; Evseev et al., 2001). Due to time constraints, I did not identify all

animals to species. However, random samples were examined for species composition and it

was verified that > 95% of the clams were V. philippinarum. The predominant populations on

the beaches sampled were V. philippinarum, and sampling coincided with known settlement

periods for this species, therefore it is highly probable that counts reflect the early recruitment of

V. philippinarum.

90 Pre-Set | 20000 2004 18000 I Beach 1 16000 E • Beach 2 14000 • Beach 3 i • Beach 4 12000 1 o 10000 re o 8000 « o> 6000

> 4000 < 2000

10/11 Aug

Figure 4-1: Average rates of Venerupis philippinarum recruitment per m2 for each beach site (net and no-net combined). Grey circles and black line shows the average settlement rate for all sites combined. 10/11 August, 25/26 August and 7/8 September in 2003 and 10/11 August and 25/56 August in 2004 are considered "pre-settlement".

Results

Density (early recruits/m2) of early recruits was measured for each treatment plot at each beach (Figure 4-2). In 2003 the density ranged from 509 - 748 early recruits/m2and in 2004 from 4,396 - 5,720 early recruits/m2 (95% C.I. for each year, all beaches, netted and non-netted combined). Results of the Shapiro-Wilks test showed the post-settlement data were normal

(p=0.296) however the pre-settlement data were not normal (p-0.01) and Levene's test showed marginal equality of variances (p=0.059). Analysis of variance showed that year (p<0.001) was a significant factor in determining recruitment, and netting was not significant at a=0.05

(p=0.061). The interaction of year and net was also significant (p=0.037) indicating that in each year the effect of netting on settlement changed (Table 4-1). The covariate, settlement status, was also highly significant in the model (p<0.00001).

91 Non-Netted Plots 2003 Netted Plots 2003 5000 A 5000

B Beach 1 H Beach 1 E 4000 E 4000 co • Beach 2 ID • Beach 2 Beach 3 • Beach 3 £ 3000 Beach 4 £ 3000 Beach 4

o 2000 a 2000 u> o E £ o > ^ 1000

10/11 Aug. 25/26 Aug. 7/8 Sept. 7/8 Oct. 10/11 Aug. 25/26 Aug. 7/8 Sept. 7/8 Oct.

40000 i 40000 n Non-Netted Plots 2004 Netted Plots 2004 35000

IS Beach 1 f= 30000 o) • Beach 2 g 25000 S Beach 3 o £ • Beach 4 >. 20000 •rac 0) £

§ 10000

5000

10/11 Aug. 25/26 Aug. 7/8 Sept 7/8 Oct. 10/11 Aug. 25/26 Aug. 7/8 Sept. 7/8 Oct Sample Date

Figure 4-2: Average Venerupis philippinarum early recruits per m2 for 2003 (upper panels) and 2004 (lower panels) counted on non-netted (left panels) and netted plots (right panels). Sample date shown on x-axis, error bars represent 95% confidence interval. Upper panels are shown with i finer scale than lower panels to allow data to be viewed more clearly. Table 4-1: Results of ANOVA test of factors influencing Venerupis philippinarum settlement. Source d.f. SS F P

Beach 3 0.344 2.40 0.246

Net 1 0.394 8.25 0.064

Settlement 1 3.182 162.3 0.00000

Year 1 1.770 67.19 0.0001

Year x Net 1 0.191 7.15 0.037

Error 46 0.902

There were significantly more V. philippinarum (>5 mm shell length) in netted plots compared to non-netted plots and the length frequency structure of the populations varied among plots and years. V. philippinarum populations (clams with shell length >5 mm) were sampled and measured for each plot in both 2003 and 2004 (see Chapter 3 for methods and results). I used length frequency data from the clam survey to estimate biomass densities of filter feeders and compared early recruits to biomass density using linear regression (Figure 4-

3). On non-netted plots there was low biomass and a negative correlation between biomass and early recruit density; however in netted plots there was higher biomass and a slightly positive relationship (Table 4-2).

There was no significant difference in mean lengths of early recruits found between beaches (pO.OOOOl), years (pO.OOOOl) or between netted and non-netted plots (p=0.0002)

(Figure 4-4). On average, the early recruits counted measured 310 pm (± 96, n=5465) in length.

Analysis of length frequency distributions in 2004 showed a larval settlement peak (mode) at a size of 223 pm (± 69, n=2828) on date 3 while on date 4 the peak has shifted to 382 nm (± 81, n=1316) (Figure 4-5). There were 30 days between date 3 and 4 and I therefore estimate growth rate of early settlers at 5.25 pm shell length per day. However, I was unable to estimate immigration, emigration and mortality here, and therefore estimates of growth may be

93 inaccurate. This estimated growth rate remained consistent between all 4 sites and did not differ with the presence of netting (Figure 4-5). I was unable to estimate growth rate for 2003 because settlement occurred on date 4 and no subsequent samples were taken.

35000

A2003-NO Net 30000 • • 2004-No Net

A2003-Netted CM 25000 - E • 2004-Netted

Q a 20000 \ \ R2 = 0.2433 15000 - li t Densit y 10000 a \ R

• 2

5000 R = 0.1606 2 • R = Q.4194 A Earl y Recr u

•

n XA i 1 1 U i I i l i <3 1000 2000 3000 4000 5000 6000 7000 Tapes philippinamm Biomass (g/m2)

Figure 4-3: Relationship of early recruit (<0.5mm shell length) density to Venerupis philippinarum biomass (shell length >5mm). Each data point represents the average early recruit density for each beach. Data from 2003 are counts from date 4, thus representing peak initial settlement and are shown with triangle markers. Data from 2004 are counts from date 3, representing initial peak settlement and are shown with square markers. White markers indicate non-netted plots and black markers indicate netted plot values. The R2 value is shown on the graph next to the corresponding trend line.

Table 4-2: Results of linear regression of Venerupis philippinarum biomass versus larval settlement.

Year Net R2 Slope (B)

Netted 0.419 0.4 2003 NoNet 0.161 -3.4

Netted 0.58 1.5 2004 NoNet 0.243 -16.6

94 Figure 4-4: Average Venerupis philippinarum early recruit length for each beach and plot within beach on the post-settlement date in 2003 (date 4 - shown on left) and 2004 (date 3 - shown on right). Hatched bars represent netted plots, black bars represent non-netted plots. Error bars represent 95% confidence interval. The number directly below each bar represents the sample size. No Net Net

Figure 4-5: Shell length frequencies of Venerupis philippinarum early recruits from 2004 samples. Date 3 shown with circles and solid line, date 4 shown with squares and hatched line. Non-netted plots are shown in the left column and netted plots shown on the right. Beach 3 and Beach 4 are shown with a smaller y-axis because of lower recruitment overall to those sites. Difference between the peaks of the solid line versus the hatched line is considered to represent growth of that cohort from date 3 to date 4.

96 Discussion

Modelling of flow has been used to make predictions about larval supply to sites in an effort to account for spatial variation in levels of settlement (Borsa and Millet, 1992; Chicharo and Chicharo, 2001a; Lundquist et al., 2004; Arnold et al., 2005). I observed higher densities of early recruits at beaches 1 and 2 versus beaches 3 and 4. A report by Hay and Company (2003) described the circulation patterns of Baynes Sound in detail in an effort to determine shellfish carrying capacity. This report indicated that flushing at the south end of Denman Island is greater than in the north end, therefore retention of larvae at northern beaches (1 and 2) should be greater than at southern beaches (3 and 4), consistent with my observations. Also mean and rms (root mean square) currents from September 2002 (Hay and Company, 2003) show that there is inflow from the North and South end of Denman Island that may hold larvae and allow greater settlement in Baynes Sound in general; perhaps resulting in higher recruitment there compared to other areas of the coast (other areas were not measured in the present study). The

Baynes Sound region contains higher population densities than other locations which would produce a greater local larval input making this theory difficult to test.

Overall settlement densities of early recruits observed at these study beaches was on average slightly lower than densities observed in both Ariake Sound, Japan (Ishii et al., 2001) and Puget Sound, Washington (Williams, 1980). I observed settlement of new recruits at densities of 509 - 748 early recruits/m2 in 2003 and 4,396 - 5,720 early recruits/m2 in 2004

(95% C.I.). In Ariake Sound, Japan, where the Manila clam is within its native range and is commercially cultivated, Ishii et al. (2001) observed settlement rates of 13,000 - 27,000 early recruits/m2 at two tidal flats. In Puget Sound, Washington, where, as in British Columbia, the

Manila clam was introduced nearly 80 years ago (Bourne, 1982), Williams (1980) reported density of Manila clam settlement at 18,600 - 31,200 early recruits/m2. I observed notably

97 higher densities of settlers than Chicaro and Chicaro (2001b) from the Ria Formosa Lagoon in

Portugal. After observing densities of early settlers for one year at one site, they reported 197 early recruits/m2 during the peak settlement event.

Our results demonstrated that year had the largest effect on variability in level of settlement. The covariate, settlement status was also highly significant in the model

(p>0.00001). Given that settlement status represents whether settlement has occurred or not, it is expected it to be important and thus included it as a covariate. Annual variability was remarkable with settlement density in 2004 being an order of magnitude higher than in 2003.

Williams (1978) also noted large annual variation in settlement rates of Manila clams in Puget

Sound, Washington; he reported a large set in 1976 but essentially none in 1977. In a 7 year study on Ruditapes philippinarum, Musculista senhousia, and Nuttalia olivacea populations,

Miyawaki and Sekiguchi (1999) also reported large fluctuation in annual rate of early settlers to tidal flats in Japan.

I also observed a marginally significant effect of netting on density of early recruits, netted plots receiving fewer settlers than non-netted plots. As noted earlier, this effect may be due to the interference of netting with benthic boundary layer flows carrying competent larvae to

settlement sites. Such interference with flows could potentially also contribute to increased

sedimentation and higher organic carbon beneath netting (Mojica and Nelson, 1993; Spencer et

al., 1996). In laboratory flume trials, Butman et al. (1988) tested settlement of M. mercenaria

larvae in still and flowing water and found greater retention of larvae in still water on glass

beads compared with mud suggesting a preference for substrates with lower organic matter.

These results were, however, not supported in further tests (Snelgrove et al., 1998). Nosho and

Chew (1972) saw no difference in distribution of recently settled V. philippinarum recruits (shell

length >0.5 mm, therefore larger than those observed in the present study) to plots with different

combinations of sand, gravel and shell. I tested sediments from beaches used here for levels of

98 silt and percent organic matter (see Chapter 3). These results showed that netting did not change the levels of either on the study beaches and therefore conclude that silt and percent organic matter are unlikely to account for the settlement patterns observed here.

Netting may increase turbulence at the sediment-water interface and alteration of turbulence structure is known to influence larval settlement patterns (Ableson and Denny, 1997;

Boxshall, 2000). Fuchs et al. (2004) studied the sinking behaviour of the gastropod larvae

Ilyanassa obsoleta in a variety of turbulent conditions. Their results demonstrated that the reacted to increased turbulence by ceasing to swim and therefore may use turbulence as a cue for settlement. Pernet et al. (2003) studied settlement of mussel larvae (Mytilus spp.) in downwellers and also found a positive correlation between turbulence and settlement. Pearce et al. (1998) observed the opposite effect with scallop (Placopecten magellanicus) larval settlement; in laboratory mesocosms, scallop larvae showed lower settlement with increased turbulence. Here, I observed higher density of recruits to non-netted plots where turbulence may be lower.

Constant motion of the nets as tidal flows pass over them may create a more frequently disturbed sediment environment beneath the netting. It has recently been demonstrated that larvae of some species are less likely to settle in sediments that have been disturbed (Woodin et al., 1995, 1998). Marinelli and Woodin (2004) used polychaete (Capitella sp.) and bivalve (M. mercenaria) early juveniles to test their response to disturbed and undisturbed sediments. Their tests showed that early juveniles avoided disturbed sediment and they attribute this to small- scale geochemical changes associated with sediment disturbance. In this study, I did not make small-scale measurements of geochemical properties of study sites; however, it is possible that the motion of the netting during tidal flux could agitate the sediment below and create

"disturbed" patches that are less suitable for settlement. Alternatively, it is possible that net motion causes smothering or otherwise kills early recruits thus influencing post-settlement

99 survival, I did not collect data concerning survival and therefore cannot evaluate these two possibilities. Further research to investigate this possibility is warranted.

The interaction of netting and year was also significant in the ANOVA model (p=0.037).

Higher recruitment observed in 2004 correlated with a greater difference between numbers of early recruits counted at netted and non-netted plots. This is possibly a result of differential survival at netted versus non-netted plots in years with higher competition pressure (higher recruitment years). It may also be related to condition of the larvae at the time of settlement.

Presumably, in a high recruitment year, larval survival in the plankton is high (evidenced by large numbers surviving to settlement) indicating adequate food supply and generally better environmental conditions. If the larvae are arriving at settlement sites in better condition, they may be more selective in choice of settlement site (Rittschof et al., 1984; Snelgorve et al., 1993).

Higher numbers of filter feeders beneath netting on the study beaches may lower the number of recruits at those sites. Many studies have suggested that the presence of filter feeders can decrease recruitment of larvae through direct filtration of the larvae from the overlying water (Woodin, 1976; Williams, 1980; Maurer, 1983; Ambrose, 1984; Hines et al., 1989; Andre and Rosenberg, 1991; Borsa and Millet, 1992; Mitchell, 1992; Andre et al., 1993; Olafsson et al., 1994, Beukema and Cadee, 1996; Lehane and Davenport, 2004). Williams (1980) measured settlement of clam larvae in relation to clam populations and found that dense populations decreased, but did not prevent larval settlement. However, results of many field studies investigating the effect of suspension feeders on larval settlement have failed to support the hypothesis that there is a negative relationship between filter feeder density and larval settlement (Maurer 1983; Hunt et al, 1987; Ertman and Jumars, 1988; Hines, et al. 1989; Thrush et al., 1996). I found that at low filter feeder densities, here associated with non-netted plots, there was a negative correlation between biomass and larval settlement. However, these data

showed that with higher density clam populations (around 450/m2), there is no effect, or even an

100 increase in larval settlement with increases in clam biomass. Due to the nature of the sites I examined and limited replication, I lack sufficient data from intermediate levels of clam biomass. Nevertheless, this is a yet unresolved result and may explain some of the discrepancies in the literature. It is possible that at low densities of filter feeders there is a decrease in larval settlement as predicted (Woodin, 1976), but once filter feeder densities reach a certain threshold, the effect on larval settlement changes and larvae are no longer hindered from settling or are even entrained as proposed by Ertman and Jumars (1988).

Neither the presence of netting nor extremely high clam density prevented the recruitment of bivalve larvae completely, and in plots with both nets and high numbers of clams there remained a strong level of recruitment of bivalves. Miyawaki and Sekiguchi (2000) found that density of early recruits did not influence the success of that cohort for survival; however, they did find a positive relationship between density of early recruits and density of clams of that cohort. In other words, a large settlement did not necessarily mean that cohort would survive, but when it did survive it tended to grow into a higher density group. Thus, I can not expect that higher recruitment to non-netted plots corresponds with increased survival of that cohort compared to netted plots. According to Miyawaki and Sekiguchi (2000), the success of the cohort is most closely related to "unpredictable environmental disturbances". In this study I did not measure survival of the early recruits beyond the four sample dates discussed and therefore can not make predictions on survival rates through the periods of high mortality noted by others (Gosselin and Qian, 1997; Stoner, 1990; Miyawaki and Sekiguchi, 2000; Nabu et al.,

2005).

Clam length is often omitted in studies concerning settlement and recruitment meaning that those studies cannot be accurately placed in the context of early post-larval development or

compared to other research. High levels of mortality immediately following larval settlement

can quickly change recruitment patterns (Stoner, 1990; Gosselin and Qian, 1997; Snelgrove,

101 1999; Miyawaki and Sekiguchi, 2000; Nabu et al., 2005). The early recruits considered here all measured within the range of early post-settlement (mean length = 310 pm) in an effort to measure recruitment as close in size to settlement and minimize in influence of predation.

Growth rate of early settlers has rarely been measured in the field. I used length frequency of early settlers to estimate growth rate in the field post-settlement. Based on the difference between the peaks from subsequent sampling dates of the length frequency plot, I estimated a growth rate of 5 pm/day averaged over the first 30 days of benthic life. This rate can be compared with growth under optimal hatchery conditions of 10 - 12 pm/day (Utting and

Spencer, 1991) for the same postsettlement stage. Data from Beach 1 in 2002 also allowed estimation of growth rates under field conditions in the same manner noted above (see Appendix

3 for summary of 2002 data). From 2002 data, larval settlement occurred approximately one month earlier and the growth rate was estimated to be 7.6 pm/day.

Conclusions

These results demonstrated that intertidal recruitment of clams varies both spatially and annually. I observed an order of magnitude difference in density of settlers between 2003 and

2004. I also noted a large amount of variability in timing and density of settlement in one year compared to another; density also varied spatially. Collectively, this highlights the importance for future studies concerning settlement patterns of bivalves to utilize adequate replicate sites, multiple years and replicate samples to ensure an accurate reflection of the overall patterns of settlement.

The presence of nets and high densities of clams correlated with lower settlement of bivalve larvae. Because clam abundances were not manipulated it is impossible to separate the effect of the nets and the filter feeders, however, it appears that both lead to lower levels of settlement to intertidal plots. These results confirmed that increasing densities of clam biomass

102 correlated with lower densities of early recruits, however that relationship did not hold at higher densities suggesting that the relationship is more complicated than first predicted, particularly in dense populations. Netting and filter feeders did not have any effect on the timing of settlement, the size at settlement or the growth rate observed for early recruits indicating that these environments were not unsuitable for settlement.

It continues to be important to observe larval settlement patterns in situ and understand the factors that influence recruitment success. Here, I demonstrated that annual and spatial variability are high in bivalve larval settlement patterns, but that netting and clam populations also alter the level of recruitment. Further research is needed to determine whether the magnitude of the alteration will translate into changes in adult population patterns.

103 References

Abelson, A. and M. Denny. 1997. Settlement of marine organisms in flow. Annual Review of Ecology and Systematics 28: 317-339.

Ambrose Jr, W.G., 1984. Influence of residents on the development of a marine soft-bottom community. Journal of Marine Research 42: 633-654.

Andre, C and R. Rosenberg. 1991. Adult-larval interactions in the suspension-feeding bivalves Cerastoderma edule and Mya arenaria. Marine Ecology Progress Series. 71: 227-234.

Andre, C, P. R. Jonsson, and M. Lindegarth. 1993. Predation on settling bivalve larvae by benthic suspension feeders: the role of hydrodynamics and larval behaviour. Marine Ecology Progress Series. 97: 183-192.

Arnold, W.S., G. L. Hitchcock, M. E. Frischer, R. Wanninkhof, and Y. P. Sheng. 2005. Dispersal of an introduced larval cohort in a coastal lagoon. Limnology and Oceanography. 50: 587-597.

Baggerman, B. 1953. Spatfall and transport of Cardium edule. Archives Neerlandaises do Zoologie, 10: 315-342.

Beal, B.F., and G. M. Kraus. 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.

Beukema, J.J., and G.C. Cadee. 1996. Consequences of the sudden removal of nearly all mussels and cockles from the Dutch Wadden Sea. PSZN I: Marine Ecology. 17: 279-289.

Borsa, P., and B. Millet. 1992. Recruitment of the clam Ruditapes decussatus in the Lagoon of Thau, Mediterranean. Estuarine, Coastal and Shelf Science, 35: 289-300.

Bourne, N. 1982. Distribution, reproduction and growth of Manila clam, Tapes philippinarum (Adams and Reeve), in British Columbia. Journal of Shellfish Research. 2:47-54.

Bousfield, E. L. 1955. Ecological control of the occurrence of barnacles in the Miramichi Estuary. National Museum of Canada Bulletin Biology Serial, 137:1-65.

Boxshall, A. J. 2000. The importance of flow and settlement cues to larvae of the abalone, Haliotis rufescens Swainson. Journal of Experimental Marine Biology and Ecology. 254: 143-167.

Butman, C. A. 1987. Larval settlement of soft-sediment invertebrates: the spatial scales of pattern explained by active habitat selection and the emerging role of hydrodynamical processes. Oceanography and Marine Biology: An Annual Review, 25: 113-165.

Butman, C. A. J. P. Grassle and C. M. Webb. 1988. Substrate choices made by marine larvae settling in still water and in a flume flow. Nature. 333:771-773.

104 Butman, C. A., and J. P. Grassle. 1992. Active habitat selection by Capitella sp. larvae. Two- choice experiments in still water and flume flows. Journal of Marine Research, 50: 669- 715.

Cahn, A. R. 1951. Clam culture in Japan. US Dept. Int., USFWS, Leaflet FL-399. 103 pp.

Chicharo, L., and M. A. Chicharo. 2001a. A juvenile recruitment prediction model for Ruditapes decussates L. (Bivalvia: Mollusca). Fisheries Research, 53: 219-233.

Chicharo, L., and M. A. Chicharo. 2001b. Effects of environmental conditionson planktonic abundances, benthic recruitment and growth rate of the bivalve mollusc Ruditapes decussates in a Portuguese coastal lagoon. Fisheries Research, 53: 235-250.

Crimaldi, J. P., J. K. Thompson, J. H. Rosman, R. J. Lowe, and J. R. Koseff. 2002. Hydrodynamics of larval settlement: The influence of turbulent stress events at potential recruitment sites. Limnology and Oceanography, 47:1137-1151.

Crisp, D. J. 1955. The behaviour of barnacle cyprids in relation to water movement over a surface. Journal of Experimental Marine Biology, 32: 569-590.

Crisp, D. J. 1974. Factors influencing the settlement of marine invertebrate larvae, p. 177-265. In: P. T. Grant and A. M. Mackie (Eds.) Chemoreception in marine organisms. Academic Press, London.

Eckman, J. E. 1979. Small-scale patterns and processes in a soft-substratum, intertidal community. Journal of Marine Research, 37: 437-457.

Eckman, J. E. 1990. A model of passive settlement by planktonic larvae onto bottoms of differing roughness. Limnology and Oceanography, 35: 887-901.

Engstrom, S. J., and R. L. Marinelli. 2005. Recruitment responses of benthic infauna to manipulated sediment geochemical properties in natural flows. Journal of Marine Research, 63: 407-436.

Ertman, S. C. and P. A. Jumars. 1988. Effects of bivalve siphonal currents on the settlement of inert particles and larvae. Journal of Marine Research, 46: 797-813.

Evseev, G. A., N. K. Kolotukhina, and O. Y. Semenikhina. 2001. Shell morphogenesis of several venerid bivalves. Journal of Shellfish Research, 20: 1279-1284.

Fraschetti, S., A. Giangrande, A. Terlizzi, and F. Boero. 2003. Pre- and post-settlement events in benthic community dynamics. Oceanologica Acta, 25: 285-295.

Fuchs, H. L., L. S. Mullineaux, and A. R. Solow. 2004. Sinking behavior of gastropod larvae (Ilyanassa obsoleta) in turbulence. Limnology and Oceanography, 49: 1937-1948.

Gaines, S. D. and M. D. Bertness. 1992. Dispersal of juveniles and variable recruitment in sessile marine species. Nature, 360: 579-580.

105 Gaines, S., and J. Roughgarden. 1985. Larval settlement rate - A leading determinant of structure in an ecological community of the marine . Proceedings of the National Academy of Sciences of the United States of America, 82: 3707-3711.

Gallagher, E. D., P. A. Jumars, and D. D. Trueblood. 1983. Facilitation of soft-bottom benthic succession by tube builders. Ecology, 64: 1200-1216.

Goodsell, J. G., S. C. Fuller, A. G. Eversole, M. Castagna, and R. A. Luts. 1992. Larval and early postlarval shell morphology of several venerid clams. Journal of the Marine Biological Association of U.K., 72: 231-255.

Gosselin, L.A., Qian, P.Y., 1997. Juvenile mortality in benthic marine invertebrates. Marine Ecology Progress Series. 146: 265-282.

Hadfield, M. G. 1998. The D. P. Wilson lecture. Research on settlement and metamorphosis of marine invertebrate larvae: past, present and future. Biofouling, 12: 9-29.

Hall, S.J., Raffaelli, D., Thrush, S.F., 1992. Patchiness and disturbance in shallow water benthic assemblages. In: Hildrew, A., Raffaelli, D.G., Giller, P.S. (Eds.), Aquatic Ecology, Scale Pattern and Process. Blackwell Science, pp. 333-375.

Harvey, M., E. Bourget, and R. G. Ingram. 1995. Experimental evidence of passive accumulation of marine bivalve larvae on filamentous epibenthic structures. Limnology and Oceanography, 40: 94-104.

Hay and Company Consultants. 2003. Baynes Sound Carrying Capacity Study: Technical Report. Unpublished manuscript prepared for the BC Ministry of Agriculture, Food and Fisheries and Environment Canada. 24pp + Figures.

Heath, W. A., T Broadley, I. Sutherland, and L. Clayton. 1992. A biological assessment of culturing the manila clam in British Columbia. Bulletin of the Aquaculture Association of Canada 92: 33- 37.

Helm, M. M., and N. Bourne. 2004. Hatchery culture of bivalves. A practical manual. FAO Fisheries Technical Paper. No. 471. Rome, FAO. 177 pp.

Hines, A. H., M. H. Posey, and P. J. Haddon. 1989. Effects of adult suspension- and deposit- feeding on recruitment of estuarine infauna. Veliger, 32: 109-119.

Huang, S. Y. and M. G. Hadfield. 2003. Composition and density of bacterial biofilms determine larval settlement of the polychaete Hydroides elegans. Marine Ecology Progress Series, 260: 161-172.

Hunt, J. H., W. G. Ambrose, and C. H. Peterson. 1987. Effects of the gastropod, Ilyanassa obsoleta (Say), and the bivalve, Mercenaria mercenaria (L.), on larval settlement and juvenile recruitment of infauna. Journal of Experimental Marine Biology and Ecology. 108: 229-240.

106 Hurlbert, S. H. 1984. Pseudoreplication and the design of ecological field experiments. Ecological Monographs, 54: 187-211.

Ishii, R., H. Sekiguchi, Y. Nakahara and Y. Jinnai. 2001. Larval recruitment of the Manila clam Ruditapesphilippinarum in Ariake Sound, southern Japan. Fisheries Science, 67: 579-591.

Keough, M. J. 1998. Responses of settling invertebrate larvae to the presence of established recruits. Journal of Experimental Marine Biology and Ecology, 231: 1-19.

Keough, M. J., and B. J. Downes. 1982. Recruitment of marine invertebrates: The role of active larval choices and early mortality. Oecologia 54: 348-352.

Lehane, C. and J. Davenport. 2004. Ingestion of bivalve larvae by Mytilus edulis: experimental and field demonstrations of larviphagy in farmed blue mussels. Marine Biology, 145: 101-107.

LePennec, M. 1980. The larval and post-larval hinge of some families of bivalve mollusks. Journal of the Marine Biological Association of U.K., 60:601-617.

Lewin, R. 1986. Supply-side ecology. Science, 234:25-27.

Lindegarth, M., P. R. Jonsson, and C. Andre. 2002. Physical and numerical modeling of the role of hydrodynamic processes on adult-larval interactions of a suspension-feeding bivalve. Journal of Marine Research, 60: 499-516.

Loosanoff, V., H. Davis, and P. Chanley. 1966. Dimensions and shapes of larvae of some marine bivalve mollusks. Malacologia, 4: 351-435.

Lundquist, C. J., S. F Thrush, J. W. Oldman, and A. K. Senior. 2004. Limited transport and recolonization potential in shallow tidal estuaries. Limnology and Oceanography, 49: 386- 395.

Marinelli, R.M. and S.A. Woodin. 2004. Disturbance and rectuirment: A test of solute and substrate specificity using Mercenaria mercenaria and Capitella sp. 1. Marine Ecology Progress Series, 269: 209-221.

Maurer, D. 1983. The effect of an infaunal suspension feeding bivalve Mercenaria mercenaria (L.) on benthic recruitment. Marine Ecology, 4: 263-274.

Meadows, P. S. and J. I. Campbell. 1972. Habitat selection by aquatic invertebrates. Advances in Marine Biology, 10: 271-382.

Mitchell, D. 1992. Effect of seed density on Manila clam growth and production at a British Columbia clam farm. Bulletin of the Aquaculture Association of Canada, 92: 29-32.

Miyawaki, D., and H. Sekiguchi. 1999. Interannual variation of bivalve populations on temperate tidal flats. Fisheries Science, 65: 817-829.

107 Miyawaki, D., and H. Sekiguchi. 2000. Long-term observations on larval recruitment processes of bivalve assemblages on temperate tidal flats. Fisheries Science, 55: 1-16.

Mojica, R., and W. G. Nelson. 1993. Environmental effects of a hard clam (Mercenaria mercenaria) aquaculture site in the Indian River Lagoon, Florida. Aquaculture, 113:313- 329.

Munroe, D. M., D. Bright and S. McKinley. 2004. Separation of recently settled Manila clams (Tapes philippinarum A. Adams and Reeve, 1850) from three sediment types using sucrose density solution. Journal of Shellfish Research, 23:89-92.

Muus, K. 1973. Settling, growth and mortality of young bivalves in the 0resund. Ophelia, 12: 79-116.

Nanbu, R. E. Yokoyama, T. Mizuno, and H. Sekiguchi. 2005. Spatio-temporal variations in density of different life stages of a brackish water clam in the Kiso estuaries, central Japan. Journal of Shellfish Research, 24: 1067-1078.

Noda, T. 2004. Large-scale variability in recruitment of the barnacle Semibalanus cariosus: its cause and effects on the population density and predator. Marine Ecology Progress Series, 278: 241-252.

Nosho, T. Y., and K. K. Chew. 1972. The setting and growth of the Manila clam, Venerupis japonica (Deshayes), in Hood Canal, Washington. Proceedings of the National Shellfisheries Association, 62: 50-58.

Nowell, A. R. M., and P. A. Jumars. 1984. Flow environments of aquatic benthos. Annual Review of Ecological Systems, 15: 303-328.

Olafsson, E. B., C. H. Peterson, and W. G. Ambrose. 1994. Does recruitment limitation structure populations and communities of macro-invertebrates in marine soft-sediments: the relative significance of pre- and post-settlement processes. Oceanography and Marine Biology: An Annual Review, 32: 65-109.

Olivier, F, R. Tremblay, E. Bourget, and D. Rittschof. 2000. Barnacle settlement: field experiments on the influence of larval supply, tidal level, biofilm quality and age on Balanus amphitrite cyprids. Marine Ecology Progress Series, 199: 185-204.

Pawlik, J. R. 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanography and Marine Biology Annual Review, 30: 273-335.

Pearce, C. M., S. M. Gallager, J. C. Manuel, D. A. Manning, R.K. O'Dor, and E. Bouget. 1998. Effect of thermoclines and turbulence on larval settlement and spat recruitment of giant scallop, Placopecten magellanicus, in 9.5 m deep laboratory mesocosms. Marine Ecology Progress Series, 165: 145-215.

Pech, D., P. L. Ardisson, and E. Bourget. 2002. Settlement of a tropical marine epibenthic assemblage on artificial panels: Influence of substratum heterogeneity and complexity scales. Estuarine, Coastal and Shelf Sciences, 55: 743-750.

108 Pemet, F., R. Tremblay and E. Bourget. 2003. Settlement success, spatial pattern and behavior of mussel larvae Mytilus spp. in experimental 'downwelling' systems of varying velocity and turbulence. Marine Ecology Progress Series, 260: 125-140.

Pomerat, C. M., and E. R. Reiner. 1942. The influence of surface angle and of light on the attachment of barnacles and other sedentary organisms. Biological Bulletin, 82: 14-25.

Quayle, D. B. 1951. Structure and biology of the larvae and spat of Venerupis pullustra (Montagu). Transactions of the Royal Society of Edinburgh, 62: 255-297.

Rittschof, D., E. S. Branscomb and J. D. Costlow. 1984. Settlement and behavior in relation to flow and surface in larval barnacles, Balanus amphitrite Darwin. Journal of Experimental Marine Biology and Ecology. 82: 131-146.

Rittschof, D., R. B. Forward, G. Cannon, J. M. Welch, M. McClary, E. R. Holm, A. S. Clare, S. Conova, L. M. McKelvey, P. Bryan, and C. L. Van Dover. 1998. Cues and context: Larval responses to physical and chemical cues. Biofouling, 12: 31-44.

Roughgarden, J., S. Gaines, and H. Possingham. 1988. Recruitment dynamics in complex life cycles. Science, 241: 1460-1466.

Sakai, A., and Sekiguchi, H. 1992. Identification of planktonic late-stage larval and settled bivalves in a tidal flat. Bulletin of the Japanese Society Fisheries and Oceanography, 56: 410-425.

Snelgrove, P. V. R 1999. Getting to the bottom of marine biodiversity: Sedimentary habitats. Bioscience, 49: 129-138.

Snelgrove, P. V. R, C. A. Butman, and J. P. Grassle. 1993. Hydrodynamic enhancement of larval settlement in the bivalve Mulinia lareralis (Say) and the polychaete Capitella sp. I in microdepositional environments. Journal of Experimental Marine Biology and Ecology., 168:71-109.

Snelgrove, P. V. R, J. P. Grassle, and C. A. Butman. 1998. Sediment choice by settling larvae of the bivalve, Spisula solidissima (Dillwyn), in flow and still water. Journal of Experimental Marine Biology and Ecology, 23: 171-190.

Spencer, B. E., 2002. Molluscan shellfish farming. Blackwell Publishing LTD. Oxford, England. 272 pp.

Spencer, B. E. D. B. Edwards, and P. F. Millican. 1992. Protecting Manila clam (Tapes philippinarum) beds with plastic netting. Aquaculture, 105: 251-268.

Spencer, B.E., M. J. Kaiser, and D. B. Edwards. 1996. The effect of Manila clam cultivation on an intertidal benthic community: the early cultivation phase. Aquaculture Research, 27: 261-276.

109 Stoner, D.S., 1990. Recruitment of a tropical ascidian: relative importance of pre-settlement versus post-settlement processes. Ecology, 71: 1682-1690.

Thorson, G. 1950. Reproductive and larval ecology of marine bottom invertebrates. Biological Reviews, 25: 1-45.

Thrush, S. F., J. E. Hewitt, R. D. Pridmore, and V. J. Cummings. 1996. Adult/juvenile interactions of infaunal bivalves: contrasting outcomes in different habitats. Marine Ecology Progress Series, 132: 83-92.

Toba, D. R., D. S. Thompson, K. K. Chew, G. J. Anderson, and M. B. Miller. 1992. Guide to Manila Clam Culture in Washington. Washington Sea Grant Publications 92-001. University of Washington. 80 pp.

Toonen, R. J. and J. R. Pawlik. 2001. Settlement of the gregarious tube worm Hydroides dianthus (Polychaeta : Serpulidae). I. Gregarious and nongregarious settlement. Marine Ecology Progress Series, 224: 103-114.

Underwood, A. J., and P. G. Fairweather. 1989. Supply-side ecology and benthic marine assemblages. Trends in Ecology and Evolution, 4: 16-20.

Underwood, A., and K. Keough. 2001. Supply-side ecology: The nature and consequences of variations in recruitment of intertidal organisms. In: M. Bertness, S. Gaines, and M. Hay (Eds.) Marine Community Ecology. Sinnauer Associates Inc. Sunderland, Massachusetts, pp. 183-200.

Utting, S. D., and B. E. Spencer. 1991. The hatchery culture of bivalve mollusc larvae and juveniles. Lab. Leafl. No. 68., MAFF Fish. Res. Lowenstoft. 31pp.

Williams, J. G. 1978. The influence of adults on the settlement, growth and survival of spat in the commercially important clams, Tapes japonica (Deshayes). PhD. Thesis, University of Washington.

Williams, J. G. 1980. The influence of adults on the settlement of spat of the clam, Tapes japonica. Journal of Marine Research, 38:729-741.

Woodin, S. A. 1976. Adult-larval interactions in dense infaunal assemblages: Patterns of abundance. Journal of Marine Research, 34:25-41.

Woodin, S.A., S.M. Lindsay and D.S. Wethey. 1995. Process-specific recruitment cues in marine sedimentary systems. Biological Bulletin, 189: 49-58.

Woodin, S.A., R.L. Marinelli and S.M. Lindsay. 1998. Process-specific cues for recruitment in sedimentary environments: Geochemical signals? Journal of Marine Research, 56: 535- 558.

Young, C. M. 1990. Larval ecology of marine invertebrates - A sesquicentennial history. Ophelia, 32: 1-48.

110 CHAPTER 5: Settlement of larvae in experimental flumes'

Introduction

Settlement patterns of invertebrate larvae are a basis of marine community dynamics

(Gaines and Roughgarden, 1985; Hadfield, 1998). The cues involved in larval settlement are poorly understood and have become the focus of many studies (reviewed by: Butman, 1987;

Pawlik, 1992; Qian, 1999). Many factors, both biological and physical, have been shown to influence settlement in a variety of species including cues from conspecifics (Knight-Jones,

1953; Pawlik and Butman, 1993; Toonen and Pawlik, 1994; Turner et al, 1994), prey

(Williamson, et al., 2000; Hadfield and Paul, 2001), sediment chemistry (Butman et al., 1988;

Engstrom and Marinelli; 2005), physical relief of the bottom (Wethey, 1986; Gregoire et al.,

1996; Kohler, et al, 1999) and turbulence structure of the water column (Crisp, 1955; Ableson and Denny, 1997; Boxshall, 2000; Pernet, et al., 2003; Fuchs et al., 2004).

Flow structure was highlighted by Ableson and Denny (1997) as influencing settlement

in a number of ways. The authors note that flow can be a settlement cue unto itself, it can help to mediate other settlement cues in the water by distributing them, and it can help place larvae in physical contact with a surface. High resolution measurements made within the benthic boundary layer above smooth and rough bottoms have shown that even small changes in roughness height (1.5 mm) can invoke distinct differences in small scale turbulence structure

(Shafi and Antonia, 1997; Poggi et al., 2003; Hendriks et al., 2006). Others have shown

differences in larval settlement in relation to roughness elements that interrupt and alter flow

(Baggerman, 1953; Eckman, 1979; Gallagher et al., 1983; Snelgrove et al., 1993).

* A version of this chapter has been submitted for publication as: Munroe, D. M., and R. S. McKinley. Settlement of Manila clam (Tapes philippinarum, Adams and Reeve, 1850) larvae to netted and non-netted sediments in a flume. Journal of Shellfish Research, submitted: May 2006.

Ill Intertidal clam farming involves placement of large nets over the substrate to protect clams from predation; these nets have the potential to alter tidal flow near the sediment surface and the benthic boundary layer, in turn possibly affecting recruitment patterns of shellfish larvae

(Heath et al., 1992; Beal and Kraus, 2002). Aquaculture production of the Manila clam

(Venerupisphilippinarum) has grown rapidly from 315,000 metric tonnes in 1990 to 1,694,000 metric tonnes in 2000, while the capture fishery decreased from 84,000 metric tonnes to 57,000 metric tonnes (FAO, 2004). Clam farming is globally widespread and growing rapidly. Much of the industry practices the use of nets for protection of highly valued seed clams laid out on beaches (Spencer et al., 1992; Toba et al., 1992; Spencer, 2002), however, little is known of the contribution of wild settlement in addition to seeded clams on farmed beaches making the question of potential influence of nets on larval settlement important and timely.

In this experiment, I investigated the influence of bottom roughness elements; clam netting and sediments; on retention of V. philippinarum larvae in flume flows. Although direct

observation of settlement patterns in natural settings can also yield useful information

(Williams, 1980; Ishii et al., 2001) use of a flume allows for greater control over variables of

interest (Muschenheim et al., 1986). In the flume I was able to generate a laminar (based on

calculated Reynolds numbers, summarised in the materials and methods section) and well

characterised flow environment as well as eliminating the potential influence of adults or predators. I predicted that retention of larvae would be higher in flume treatments with no

netting applied to the bottom based on the pattern observed in the field (described in the

previous chapter). This prediction was tested in replicated flume trials with netting and

sediment bottom treatments.

112 Materials and Methods

Four fibreglass flumes were used in this study, all measured 0.47 m wide, 0.25 m deep

(water depth at 0.20 m) and 4.9 m long with a wall of drinking straws (5mm diameter x 200mm length) at the inflow end to entrain water flow and a standpipe at the outflow (Figure 5-1). For each trial, each flume contained a different bottom type based on two factors (net and sediment) with two levels each (present and not present) fully crossed; the resulting bottom types were none (control), sediment only, net only, and sediment and net. Sediment used was pea-sized gravel (15 mm diameter) combined in a 50:50 ratio with filter sand (2 mm diameter) to mimic the heterogeneity of field sediments. All gravel and sand was washed thoroughly with dechlorinated freshwater to ensure that it was clean and free of fine sediments. The nets used in the trial were cotton netting (mesh opening size 2 cm x 2 cm) used for predator protection on clam farms, cut to fit the bottom of the flume and sewn around the edge with lead line to sink the edges of the net (this is common farming practice). The net type used is only one of a variety employed on beaches in British Columbia.

0.3 m 3.0 m Outflow 0.2 m AV. o

T Straws 4.9 m

Figure 5-1: Top view of flume dimensions. Flow is from left to right. Acoustic Doppler measurements were made at the position marked by the X. Bottom treatments were applied between the straws and the outflow.

113 Two separate batches of V. philippinarum larvae were used in the trials. Larvae were reared at a commercial hatchery facility (Taylor Shellfish Farms, Kona, , USA) for 10 days before being sent to the experimental facility at the Centre for Shellfish Research at

Malaspina University in Nanaimo, British Columbia, Canada. Upon arrival, each batch was split into three groups and placed into aerated static larval rearing tanks, fed at a rate of 15,000 algae cells per mL (50:50 combination of Isochrysis galbana (Tahitian strain) and Chaetoceros muellerii) twice daily and water was changed every 2.5 days. Splitting of the batch allowed each group to be delayed slightly to facilitate a 4 day lag between trial runs (the time necessary to "re-set" the flumes). A diagram of the batch splitting, delay period and resulting trial that the group was used in is shown in Figure 5-2. The larvae were observed daily until the presence of pediveligers was confirmed indicating competence to metamorphose. Once the larvae were competent they were siphoned from the larval tanks, length measurements were made and they were then counted and randomly assigned to a flume for the trial (see Table 5-1 for summary of

larval lengths used in trials).

Figure 5-2: Larval batch spawning and splitting dates and resulting groups used in each trial.

114 Table 5-1: Lengths of Venerupis philippinarum larvae (um ± SD) used for each trial and source batch; n = 20 for each measure. Trial Batch Number Larval Length (pm)

1 1 196(±14)

2 1 211(±13)

3 1 226(±12)

4 2 198(±15)

5 2 216 (±14)

6 2 224 (±13)

Each flume was set up with bottom treatment randomly assigned 48 hours prior to running the trial with larvae. The flumes were filled completely with filtered sea water and a small amount of algae (C. muellerii) was added to each. The flumes were left for 48 hours to establish biofilms on the surfaces as biofilms have been shown to be important in settlement of many invertebrate larvae (Scheltema, 1974; Hadfield and Paul, 2001).

During each trial, larvae were introduced into the inflow end of each flume on the treatment side of the straws across the width of the flume at 3 cm depth. Water flow was driven by submersible pumps in an 40 L tub located beneath the outflow of the flume and flow was held steady at 0.3 L/sec (free-stream velocity of 0.45 cm/sec, depth averaged fluid velocity of

0.35 cm/sec). A 105 pm screen was used at the outflow end of the flume to catch any animals exiting the flume. Approximately 100,000 larvae were introduced into each flume for each trial

(equal to 44,500/m2: twice the maximum settlement density observed in the field for this species: (Williams, 1980; Ishii et al., 2001)). The screens at the downstream end were exchanged every 15 minutes and washed into a beaker. Larval concentration in the beaker was

115 counted using a Sedgewick Rafter (1 mL) counting slide and the number of animals was calculated from the concentration and volume in the beaker. A separate trial was also conducted using polystyrene microshperes (manufactured by Alfa Aesar, Parkridge Massachusetts, USA,

75.0 um diameter) as larval controls (Butman et al., 1988; Ertman and Jumars, 1988). The microspheres were used and counted in the same manner as for the larvae listed above.

During each trial all four flumes were used with a different bottom type in each. The trial was repeated six times, each time the troughs were emptied, the bottom treatments were removed and all components (including sediments) were cleaned. On each replicate trial, bottom treatment was randomly re-assigned to flumes. Flow in each of the flumes was profiled using a Sontek Acoustic Doppler Velocimeter (Sontek/YSI Inc., San Diego, California, USA) mounted above each flume. Measurements were made at steps of 1 cm from the bottom up to a depth of 10 cm. At each height above the bottom, 15 measurements were taken at 10 Hz over a period of 120 seconds. Each flume was characterised in this manner and one randomly selected

flume was characterised under all four of the bottom treatments. The calculated Reynolds number (Re) for the length of the flume (distance from the leading edge of the treatment section to the measurement point) was Re=l2,000 - 15,000. The Reynolds number for the channel

(using the flume width as the length value) was Re=l,880-2,350. The Reynolds number for open channel flow using the depth averaged fluid velocity and the hydraulic radius as the

characteristic length is Ref = 302 indicating that the flow in the flumes were laminar (Khalili et

al, 2001). The boundary layer Reynolds number, as calculated using the free stream velocity

and the BL thickness was Reb = 252 indicating that the boundary layer flow is also laminar

(Khalili et al., 2001).

116 Results

I compared the percentage of larvae leaving the system during the entire trial (number leaving/number input x 100) using ANOVA with trial as a block and netting and sediment as factors. The data were tested for normality using the Shapiro-Wilk test, and homogeneity of variance was tested using Levene's test statistic. The results of the ANOVA are shown in Table

5-2. Trial was significant in the percentage of larvae that left the system (p=0.001) while neither netting nor sediment (p=0.603, p=0.391 respectively) were significant. This can be seen in

Figure 5-2 where the percentage of larvae leaving the system varies greatly between trials; however, in any given trial there is no consistent difference between treatments.

Table 5- 2: Summary statistics from ANOVA test for percentage of Venerupis philippinarum larvae leaving the system during the trial. Source df SS F P

Trial 5 11770 38.76 0.001

Net 1 18.7 0.31 0.603 Sediment 1 111.5 0.80 0.391

Net x Sediment 1 201.4 1.45 0.256

Error 10 138.9

Figure 5-3 also shows the results of the trial using polystyrene spheres. On average,

57% (±2.2 S.D.) of the spheres input left the system over the course of the trial (75 minutes total). The majority of the spheres that left the system (approximately 97% ±1.2% 95% confidence interval) did so within the first 45 minutes of the trial (Figure 5-4). This was in contrast to the larvae which showed approximately 73% (±5% 95% confidence interval) of leavers exiting the flume in the first 45 minutes (Figure 5-5 - note lower proportion of darkest bands for trial 7 using beads). This discrepancy indicates the larvae were leaving the flume in a

117 manner inconsistent with inert particles. A portion of the larvae were "leaving later" than would

be predicted by particle motion in the flows created.

100 • Control S3 Net • Net & Sediment • Sediment

3 4 Polystyrene Trial Number Spheres

Figure 5-3: Percentage of total Venerupis philippinarum larvae input at time zero that left the system by the end of the trial (75 minutes). Error bars represent ± standard deviation (n=5 for each bar). Percentage of polystyrene spheres leaving the system is shown on the right side of the chart.

Figure 5-4: Proportion of all polystyrene spheres exiting the system shown by time collected. Error bars represent ± standard deviation.

118 100% 90% nm llll 80% I 70% • 75 min. 60% E) 60 min. 50% • 45 min. 40% • 30 min. • 15 min. 30%

20%

10%

0%

Treatment and Trial Number

Figure 5-5: Proportion of Venerupis philippinarum larvae (or beads in case of trial 7) exiting the flume over time. White band indicates proportion leaving in the first 15 minutes; the dotted band indicates the proportion leaving in the second 15 minutes and so on. Trial number and treatment listed along the x- axis. ** Trial 7 was conducted with polystyrene beads; all other trials shown were conducted with larvae.

Based on this "leaving later" behaviour, I tested the relative proportion of larvae leaving in the last 30 minutes of the trial (larvae leaving in last 30 minutes/larvae leaving the system in entire trial) using ANOVA with trial as a block and netting and sediment as factors. The data were tested for normality using the Shapiro-Wilk test, and homogeneity of variance was tested using Levene's test statistic. Results are shown in Table 5-3. In this case none of the factors were significant in influencing the proportion of larvae leaving the system later in the trial.

119 Table 5-3: Summary statistics from ANOVA test for proportion of Venerupis philippinarum larvae leaving in the last 30 minutes of the trial. Source df SS F P

Trial 5 1078.4 3.26 0.11

Net 1 0.0126 0.00 0.99

Sediment 1 137.4 1.30 0.281 Net x Sediment 1 194.4 1.83 0.205

Error 10 1059.6

Discussion

Larvae retained within the flumes varied from one trial to the next. Because it was necessary to clean the flumes, re-set the bottom treatments and allow biofilms to establish between trials, there was a delay of about 2.5 days between trials. Although efforts were made to slow development of larvae for trials to be run later (reduced feeding for brief periods and reduced water temperature) as the larvae aged, they became less likely to exit the flume. In flume trials with larvae of the bivalve Mulinia lateralis, Snelgorve et al. (1993) also saw reduced swimming ability with larval age that they note may increase "hydrodynamic trapping" of older larvae. In flume experiments with Mya arenaria larva, Snelgrove et al. (1999) saw a large influence of "run" in their trials demonstrating decreased selectivity at older larval age. In work with barnacle settlement, Rittschof et al. (1984) noted that younger larvae were more discriminating in settlement habitat than older cyprids. Despite this, I was still able to test the effects of the treatments by using trial as a blocking factor. Neither sediment nor netting appeared to have an impact on the number of larvae retained in the flumes. This is contrary to the original hypothesis, however evidence from field surveys described in Chapter 4 indicates that in years when overall recruitment is low, netting has limited influence on small-scale recruitment patterns.

120 Water velocity (free stream = 0.45 cm/sec) used here was low compared to average flows on intertidal V. philippinarum farm plots in British Columbia (4-8 cm/sec averaged over

24 hours on spring tide, Appendix 4). Oscillatory intertidal flows are difficult to match and poorly estimated with unidirectional single speed flows in a flume (Petersen and Hastings, 2001) and field flows have numerous additional sources of turbulence (Hendriks et al., 2006). A novel approach to this problem was presented by Asmus et al. (1995) who placed stationary flume channels in the shallow intertidal and allowed naturally generated tidal flow to drive the currents within it. Lower unidirectional flows were used for this experiment because it has been predicted that larvae may settle on slack tides (Gross et al., 1992) therefore I used lower flows, replicating those closer to slack tide. In addition, lower flows allowed for longer exploration time for the larvae within the flume. It is possible that differences in the larval retention may be observed if trials were carried out under conditions of higher flow that might demonstrate greater interaction between the netting and the benthic boundary layer. As flow rate increases, the influence of roughness elements on the bottom would also likely increase.

It has been suggested by other authors (Kohler, 1999; Pawlik and Butman, 1993) that laminar benthic boundary layer flows may lead to decreased encounter rate of larvae with the sediment surface because turbulence is a mechanism that helps to place larvae in contact with the bottom (Crimaldi et al., 2002). Since each trial was run for a duration of five times the total time required for a particle to travel in bulk flow to the outflow of the flume, and some larvae did not pass through the flume in this time, I suggest that the larvae were able to reach and remain in contact with the bottom. Further, the Rouse number (Ro = fall velocity/(von

Kamann's constant x shear velocity) can be used to indicate the probability of a larvae to control

its position in the water column by swimming. For Ro > 0.75 the larvae can control their vertical position by swimming, while at Ro< 0.75 turbulence dominates the larval position

(Gross et al, 1992). Here, I observed sinking rates of dead larvae of 0.17 cm/sec and the shear

121 velocity was 0.16 cm/sec therefore the Rouse number for trials was 2.69 indicating that the larvae were able to control their vertical position through swimming. In addition, recent evidence from observations with oyster larvae has shown that bivalve larvae may have control over vertical position in the water column in a greater range of velocities than previously predicted (Finelli and Wethey, 2003).

The timing of exit from the flumes differed between larvae and polystyrene beads showing that the larvae were not acting as inert particles. This has been observed in other studies involving larval settlement patterns in flow. Butman et al. (1988) demonstrated that bivalve larvae {Mercenaria mercenaria) did not distribute in the same manner as inert particles in settlement tests. Gregoire et al. (1996) compared the patterns of settlement of inert particles and dead larvae to those seen for wild caught spat and found differences. In flume tests with settlement of abalone (Haliotis rufescens), Boxshall (2000) used dead larvae and empty shells as larval mimics and found live larvae settled in a different pattern than larval mimics. Conversely,

Harvey et al. (1995) reported that larval settlement patterns in the field and distribution of inert particles under controlled conditions showed no difference and that passive process alone could explain the settlement of bivalve larvae onto filamentous structures.

The polystyrene beads showed that most particles moving through the flume will exit within 45 minutes, therefore by examining the patterns of larvae exiting after 45 minutes I can more effectively focus on the portion of the animals that are remaining in the flume for a period before exiting the flume (represented by the black and dark grey bands in Figure 5-5). Results of this comparison showed no significant effect of any of the factors on the proportion of larvae leaving the system in the last 30 minutes. This result may differ when flow rates are increased

leading to greater effects of the roughness elements. In field trials described in Chapter 4, it was observed that in years when larval settlement rates are high, netting appears to depress settlement of V. philippinarum larvae to intertidal areas covered with netting.

122 Conclusions

At the flow rates tested in the flumes used here, I was unable to detect differences in retention of V. philippinarum larvae in relation to either sediment or netting applied to the bottom of the flumes. Possibly, V. philippinarum is a poor candidate for these types of trials because the competent period for this species can be prolonged if conditions are unfavourable and retention in the flume is not necessarily an indication of intention to remain until settlement.

However, this species is part of an important fishery and an even more important culture industry worldwide, therefore understanding the effects of farm practices like netting, on larval settlement and recruitment are crucial in ensuring a sustainable and efficient industry in the future.

123 References

Abelson, A., and M. Denny. 1997. Settlement of marine organisms in flow. Annual Review of Ecology and Systematics, 28: 317-339.

Asmus, H., R. M. Asmus, and G. B. Zubillaga. 1995. Do mussel beds intensify the phosphorus exchange between sediment and tidal waters? Ophelia, 41: 37-55.

Baggerman, B. 1953. Spatfall and transport of Cardium edule. Archives Neerlandaises do Zoologie, 10: 315-342.

Beal, B.F., and G. M. Kraus. 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.

Boxshall, A. J. 2000. The importance of flow and settlement cues to larvae of the abalone, Haliotis rufescens Swainson. Journal of Experimental Marine Biology and Ecology, 254: 143-167.

Butman, C. A. 1987. Larval settlement of soft-sediment invertebrates: the spatial scales of pattern explained by active habitat selection and the emerging role of hydrodynamical processes. Oceanography and Marine Biology: An Annual Review, 25: 113-165.

Butman, C. A., J. P. Grassle, and C. M. Webb. 1988. Substrate choices made by marine larvae settling in still water and in a flume flow. Nature, 333: 771-773.

Crimaldi, J. P., J. K. Thompson, J. H. Rosman, R. J. Lowe, and J. R. Koseff. 2002. Hydrodynamics of larval settlement: The influence of turbulent stress events at potential recruitment sites. Limnology and Oceanography, 47:1137-1151.

Crisp, D. J. 1955. The behaviour of barnacle cyprids in relation to water movement over a surface. Journal of Experimental Biology, 32: 569-590.

Eckman, J. E. 1979. Small-scale patterns and processes in a soft-substratum, intertidal community. Journal of Marine Research, 37: 437-457.

Engstrom, S. J., and R. L. Marinelli. 2005. Recruitment responses of benthic infauna to manipulated sediment geochemical properties in natural flows. Journal of Marine Research, 63: 407-436.

Ertman, S. C, and P. A. Jumars. 1988. Effects of bivalve siphonal currents on the settlement of inert particles and larvae. Journal of Marine Research, 46: 797-813.

FAO Fisheries Department. 2004. The state of the world fisheries and aquaculture. FAO Publishing, Rome Italy, 153 pp.

Finelli, C. M., and D. S. Wethey. 2003. Behaviour of oyster (Crassostrea virginica) larvae in boundary layer flows. Marine Biology, 143: 703-711.

124 Fuchs, H. L., L. S. Mullineaux, and A. R. Solow. 2004. Sinking behavior of gastropod larvae (Ilyanassa obsoleta) in turbulence. Limnol. Oceanogr., 49: 1937-1948.

Gaines, S., and J. Roughgarden. 1985. Larval settlement rate - A leading determinant of structure in an ecological community of the marine intertidal zone. Proceedings of the National Academy of Sciences of the United States of America, 82: 3707-3711.

Gallagher, E. D., P. A. Jumars, and D. D. Trueblood. 1983. Facilitation of soft-bottom benthic succession by tube builders. Ecology, 64: 1200-1216.

Gregoire, Y., E. Bourget, and J. L. Verrette. 1996. Deposition of mimics of planktonic invertebrate larvae on simple and complex substrata in flume flows. Marine Ecology Progress Series, 135:89-100.

Gross, T.F., F. E. Werner, and J. E. Eckman. 1992. Numerical modeling of larval settlement in turbulent bottom boundary layers. Journal of Marine Research, 50: 611-642.

Hadfield, M. G. 1998. The D. P. Wilson lecture. Research on settlement and metamorphosis of marine invertebrate larvae: past, present and future. Biofouling, 12: 9-29.

Hadfield, M. G., and V. J Paul. 2001. Natural chemical cues for settlement and metamorphosis of marine-invertebrate larvae. In: Marine Chemical Ecology. Eds. J.B McClintock and B. J.Baker, pp.431-461. CRC Press, Florida.

Harvey, M., E. Bourget, and R. G. Ingram. 1995. Experimental evidence of passive accumulation of marine bivalve larvae on filamentous epibenthic structures. Limnology and Oceanography, 40: 94-104.

Heath, W. A., T. Broadley, I. Sutherland, and L. Clayton. 1992. A biological assessment of culturing the manila clam in British Columbia. Bulletin of the Aquaculture Association of Canada, 92: 33- 37.

Hendriks, I. E., L. A. van Duren, and P. M. J. Herman. 2006. Turbulence levels in a flume compared to the field:Implication s for larval settlement studies. Journal of Sea Research, 55: 15-29.

Ishii, R, H. Sekiguchi, Y. Nakahara, and Y. Jinnai. 2001. Larval recruitment of the Manila clam Ruditapes philippinarum in Ariake Sound, southern Japan. Fisheries Science, 67: 579-591.

Khalili, A., M. Huetell, and W. Merzkirch. 2001. Fine-scale flow measurements in the benthic boundary layer. In: The Benthic Boundary Layer. Eds. B. P. Bourdreau and B. B. J0rgensen. pp. 44-77. Oxford University Press, New York.

Knight-Jones, E. W. 1953. Laboratory experiments on the gregariousness during setting in Balanus balaoides and other barnacles. Journal of Experimental Biology, 30: 584-599.

125 Kohler, J., P. D. Hansen, and M. Wahl. 1999. Colonization patterns at the substratum-water interface: how does surface microtopography influence recruitment patterns of sessile organisms? Biofouling, 14: 237-248.

Muschenheim, D. K., J. Grant, and E. L. Mills. 1986. Flumes for benthic ecologists: theory, construction and practice. Marine Ecology Progress Series, 28: 185-196.

Pawlik, J. 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanography and Marine Biology Annual Review, 30: 273-335.

Pawlik, J. R., and C. A. Butman. 1993. Settlement of a marine tube worm as a function of current velocity: interacting effects of hydrdynamics and behaviour. Limnology and Oceangraphy, 38: 1730-1740.

Pernet, F., R. Tremblay, and E. Bourget. 2003. Settlement success, spatial pattern and behavior of mussel larvae Mytilus spp. in experimental 'downwelling' systems of varying velocity and turbulence. Marine Ecology Progress Series, 260: 125-140.

Petersen, J. E., and A. Hastings. 2001. Dimensional Approaches to Scaling Experimental Ecosystems: Designing Mousetraps to Catch Elephants. The American Naturalist, 157: 324-333.

Poggi, D., A. Porporato, and L. Ridolfi. 2003. Analysis of the small-scale structure of turbulence on smooth and rough walls. Physics of Fluids, 15: 35-46.

Qian, P. Y. 1999. Larval settlement of polychaetes. Hydrobiologia, 402: 239-253.

Rittschof, D., E. S. Branscomb, and J. D. Costlow. 1984. Settlement and behavior in relation to flow and surface in larval barnacles, Balanus amphitrite Darwin. J.Exp. Mar. Biol. Ecol. 82: 131-146.

Scheltema, R. S. 1974. Biological interactions determining larval settlement of marine invertebrates. Thalassia Jugoslavia, 10:263-296.

Shafi, H. S., and R. A. Antonia. 1997. Small-scale characteristics of a turbulent boundary layer over a rough wall. Journal of Fluid Mechanics, 342: 263-293.

Snelgrove, P. V. R, C. A. Butman, and J. P. Grassle. 1993. Hydrodynamic enhancement of larval settlement in the bivalve Mulinia lareralis (Say) and the polychaete Capitella sp. I in microdepositional environments. Journal of Experimental Marine Biology and Ecology, 168:71-109.

Snelgrove, P. V. R, J. Grant, and C. A. Pilditch. 1999. Habitat selection and adult-larvae interactions in settling larvae of soft-shell clam Mya arenaria. Marine Ecology Progress Series, 182: 149-159.

Spencer, B. E., 2002. Molluscan shellfish farming. Blackwell Publishing LTD. Oxford, England. 272 pp.

126 Spencer, B. E., D. B. Edwards, and P. F. Millican. 1992. Protecting Manila clam (Tapes philippinarum) beds with plastic netting. Aquaculture, 105: 251-268.

Toba, D. R., D. S. Thompson, K. K. Chew, G. J. Anderson, and M. B. Miller. 1992. Guide to Manila Clam Culture in Washington. Washington Sea Grant Publications 92-001. University of Washington. 80 pp.

Toonen, R. J., and J. R. Pawlik. 1994. Foundations of gregariousness. Nature, 370: 511-512.

Turner, E. J., R. K. Zimmer-Faust, M. A. Palmer, M. Luchenbach, and N. D. Pentcheff. 1994. Settlement of oyster Crassostrea virginica larvae: effects of water flow and a water- soluble chemical cue. Limnology and Oceanography, 39: 1579-1593.

Wethey, D.S. 1986. Ranking of settlement cues by barnacle larvae: influence of surface contour. Bulletin of Marine Science, 39: 393-400.

Williams, J. G. 1980. The influence of adults on the settlement of spat of the clam, Tapes japonica. Journal of Marine Research, 38:729-741.

Williamson, J. E., R. DeNys, N. Kumar, D. G. Carson, and P. D. Steinberg. 2000. Induction of metamorphosis in the sea urchin Holopneustespurpurascens by a metabolite complex from the algal host Deliseapulchra. Biological Bulletin, 198: 332-345.

127 CHAPTER 6: Conclusions and General Discussion

Density separation of newly settled bivalves from sediments is simple and effective for field sampling. I was able to prove the accuracy of this method in various sediment types allowing us the awareness that results of field sampling are an accurate reflection of the magnitude of early settlement. Beyond that, these methods are easily applicable for other researchers or even shellfish growers interested in tracking larval settlement on their beaches.

Results of field measurements of sediment properties indicated that the netting is not altering the sediment grain size distribution or carbon levels substantially on the study sites.

This does not mean however, that differences do not exist as I measured only a small sub-set of possible parameters. It is likely that the influence of netting on sediment properties is site specific and depends largely on local hydrography. I did not measure biofilms or sediment geochemical properties that could alter the sediment environment. The motion of the netting on the sediment surface may create areas of frequent disturbance that might host different populations of biofilms or change sediment chemistry. Further investigation of this possibility could illuminate driving forces in succession of biofilms.

An unexpected result of field measurements was the "buffering" of low tide sediment/air interface temperature by netting. These results were not explored in depth but may be of significance to the metabolic rates and stress levels of animals beneath the netting particularly at temperature extremes. This may prove to be a worthwhile question to explore further in future research as it may pertain to optimal growth of farmed animals within those plots.

I observed large annual and spatial variation in settlement of larval clams. Plots with netting and dense populations of adult bivalves had depressed levels of early recruits in 2004 when settlement was high. An interesting trend appeared at higher populations of adult clams.

As adult population increased, early recruits initially decreased then levelled off or possibly

128 increased. Larger populations of filter feeders may actually facilitate settlement of larvae by disruption of near bed flows with siphon currents. Questions surrounding the influence of filter feeding benthos on larval delivery and settlement patterns are interesting and as yet, inconclusive. Why smaller populations tend to decrease larval settlement while larger populations do not should be studied further to gain insight in this area.

The presence of adult filter feeders at study plots made isolating the influence of netting alone impossible. A controlled experiment where adults and predators were removed was run in laboratory flumes to try to isolate the effect of netting on settlement of larvae. The experiment showed no change in retention of larvae within flumes as a result of netting. This might be because the animals are poorly suited to this type of trial because they do not cement permanently and take a long time searching for suitable settlement sites. In addition, the water velocity used in the trial may have been too low to generate a turbulence structure that would influence settlement. Repeated trials at a variety of water velocities could be useful in determining if this is the case.

Although laboratory experiments are desirable for the control over variables and the accuracy of the observations that can be made when dealing with invertebrate larvae, field-based research is also important. As was the case here, field experimentations are often confounded with uncontrolled factors; however, laboratory-based experiments often lack the kind of complexity found in natural systems. It is therefore important to partner field and laboratory research in an attempt to understand the patterns of larval settlement in both a controlled and relevant manner. This thesis described a combination of laboratory and field experimentation and highlites some of the discrepancies that exist. Nonetheless, these types of experiments are more relevant and informative than either field or laboratory work can be when used exclusively.

129 Early recruitment patterns measured here did not appear to be random, nor did they follow the pattern predicted by deposition of silt and passive particles. This indicates an active role in habitat selection by these early settlers that should be investigated further. The majority of the contributions to the field of larval settlement involve observations on attached settlers on hard substrates or conspicous species like polychaetes. This thesis described field settlement patterns of a mobile species in soft-sediments and is an important addition to this field of literature. Further studies, like this one, will allow paradigms and hypothesis that have been established and tested for attached and conspicous species in the field and laboratory to be applied and tested on this less-examined group.

The trend in settlement patterns observed at field sites could be used by farmers to increase wild recruitment to culture plots. Timing harvest of adults and temporary removal of nets to coincide with larval settlement could lead to higher recruitment, particularly in years when larval settlement is high. The relationship between early recruits and the recruitment into adult populations is unknown however. Research focussed on determining survival and migration of early recruits is crucial to connecting larval recruitment with adult populations.

Without this connection, patterns of early recruitment cannot be used to understand population dynamics.

130 Appendix 1: Summary table of juvenile bivalve dispersal research

Drift Shell length Location Season studied Reference Lab/Field Species observed mechanism Ahn et al., Flax Pond, New York, both Mercenaria mercenaria 0.2 mm - 0.65 mm crawling September 1989 1993 USA Spring - Amyot and Lac de l'Achigan Quebec, 52 -67 mm active processes summer 1988, Downing, field Elliptio complanata Canada 1998 1989,1990 Ensis directus, Cerastoderma edule, Macoma0.7- 4 mm M.balthica, June - Armonies, byssus Wadden Sea, Netherlands field balthica, Mytilus edulis, Venerupis pullustra,0.5-3. 5 mm C. edule, 1-5 September 1991 1992 Mya arenaria mm E. directus 0.5-4mm C. edule & M. Cerastoderma edule, Macoma balthica, Ensisbalthica, l-18mm£. Summer 1990- Armonies, byssus, climbing Wadden Sea, Netherlands both americanus, Mytilus edulis, Venerupis americanus, l-2mmM. 1992 1994(a) pullustra, Mya arenaria edulis, V. pullustra & M. arenaria Armonies, June - August field Macoma balthica, Cerastoderma edule > 0.5 mm movement noted Wadden Sea, Netherlands 1994(b) 1992 May - August Cerastoderma edule, Macoma balthica, Ensis active and Armonies, 0.125 -2 mm Wadden Sea, Netherlands 1993 April- field americanus, Mytilus edulis, Venerupis passive processes 1996 pullustra, Mya arenaria August 1994 Baggerman, Cardium edule, Mya arenaria, Mytilus edulis, field 0.4- 11.0 mm bedload transport Wadden Sea, Netherlands Summer 1950 1953 Macoma balthica, Petricola pholadiformis July - August Baker and Anadara transversa, Geukensia demissa, pelagic drift Chesapeake Bay, Virginia, 1990 and May - field 0.25 - 0.5 mm Mann, 1997 Tellina agilis (possibly byssus) USA September 1991-1993 April - July foot protrusion Menai Strait, North Wales Bayne, 1964 both Mytilus edulis 0.25 -3 mm 1963 3.7-18.8 mm P. maximus, Beaumont and lab Pecten maximus, Aequipecten opercularis 6.4-13.0 mm A. byssus Barnes, 1992 opercularis Beukema, field Macoma balthica 0.5 - 5 mm byssus Wadden Sea, Netherlands 1993 Beukema and to 10 mm field, both Macoma balthica byssus, mucous Wadden Sea, Netherlands winter de Vlas, 1989 2-9 mm lab Boozer and January - July field Musculium partumeium, Corbiculafluminea 1.2-6.2 mm crawling Savannah River, USA Mirkes, 1979 1976 Drift Reference Lab/Field Species observed Shell length Location Season studied mechanism oriented Brafield and field Macoma balthica adults crawling, short Whitstable, U.K. Newell, 1961 distances Caceres- crawling, mucous Martinez et al., both Mytilus galloprovincalis 0.25-2 mm Ria do Vigo, Spain 1991 - 1993 drift 1994 October 1983 Commito et Assateague Island, field Gemma gemma (brooding) 0.2 - 2.2 mm active drift and January Virginia, USA al., 1995 1984 Cummings et lab Macomona lilliana 1-2 mm byssus New Zealand al., 1993 Macomona lilliana, Arthritica bifurca, Cummings et Auatrovenus stuchburyi, Mactra ovata, Manakau Harbour, New October & field < 3 mm movement noted al., 1995 Mytilus sp., hartvigiana, Zealand December 1991 australis, Hiatula siliquens, Zenatia acinaces Emerson and Eastern Passage, Nova June 1988- field Mya arenaria 8-15 mm bedload transport Grant, 1991 Scotia Canada April 1989 Hiddink et al., Groninger Wad, The December 1999 both Macoma balthica 0.5 - 7 mm byssus 2002 Netherlands -March 2001 May to August Highsmith, Friday Harbour, Transemlla trantilla (brooding) > 0.5 mm floating 1982 July to lab Washington, USA 1985 August 1984 Lane et al., lab Mytilus edulis to 2 mm byssus 1985 Musculus sp, Lasaea sp, Transemlla trantilla Martel and field - brooders, Mytilus sp, Hiatella arctica, unk. 0.5 - 0.95 mm byssus Bamfield, BC Canada Summer 1989 Chia, 1991 Clam - non-brooders 1.4-2.1 mm C. edule, Montaudouin, lab Cerastoderma edule, Ruditapes philippinarum0.4-5.1 mm R. byssus, valves 1997 philippinarum Frenchman Bay, Maine, August 1924 Mytilus edulis 0.35 - 0.94 mm air bubble Nelson, 1928 field USA and 1927 Mt. Desert Narrows, Mytilus edulis 0.25-2 mm byssus 1985 - 1991 Newell, 1994 field Maine, USA Norkko et al., Manakau Harbour, New field Macomona lilliana, Austravenus stuchburyi to 4 mm active processes February 1997 2001 Zealand Olivier et al Abra alba, Cultelluspellucidus, Mysella field byssus, valves English Channel June 1992 1996 bidentata, Tellina fabula Drift Reference Lab/Field Species observed Shell length Location Season studied mechanism Prezant and Chalermwat, lab Corbicula fluminea 7- 14 mm byssus, mucous Mississippi, USA March 1984 1984 Roegner et al., lab Mya arenaria 0.24 - 0.29 mm bedload transport 1995 crawling, Trondheimsfj orden, Rygg, 1970 lab Cerastoderma edule, Cerastoderma glaucum young juveniles 1967-1968 climbing Norway Union Beach, New Jersey, Sellmer, 1967 field Gemma gemma (brooding) 0.33-0.51 mm passive processes 1955-1958 USA Nucula tenuis, Mytilus edulis, Modiolus sp., Musculus marmoratus, Heteranomia squamula, Turtonia minuta, Montacuta ferruginosa, Montacuta substriata, Cardium Sigurdsson et both echinatum, Venerupis pullastra, Mactra byssus al, 1976 corallina, Spisula sp., Tellina tenuis, Gari fervensis, Abra alba, Donax vittatus,Cultellus pellucidus, Ensis sp.,Hiatella arctica, Hiatella gallicana, , Corbula gibba byssus, foot Winter/spring lab Macoma balthica 4-14 mm Wadden Sea, Netherlands Sorlin, 1988 protrusion 1982 Sugawara et lab Ruditapes philippinarum byssus Japan al., 1953 Malpeque Bay, P.E.I, Gemma gemma (brooding) 0.35 mm passive processes Sullivan, 1948 field Canada Turner et al, Manakau Harbour, New field Macomona lilliana, Austrovenus stuchburyi to 4 mm passive processes February 1994 1997 Zealand Wang and XU, Ronhai County, Fujian, lab Sinonovacula constricta 0.55 - 0.90 mm byssus, climbing 1997 China water expulsion, Ensis directus, Tagelus divisus, Solemya Williams and kicking with Eastern North Carolina, field velum, Solen viridis, Dondax variabilis, 1.7-46.0 mm 1957 - 1966 Porter, 1971 foot, bedload USA Petricola pholadiformis, Spisula raveneli transport Yankson, 1986 lab Cerastoderma edule, Cerastoderma glaucum 8 -10.3 mm byssus, climbing

Table Al-1: Summary of research on juvenile bivalve dispersal. References Appendix 1

Ahn, I., G. Lopez and R. Malouf. 1993. Effects of the gem clam Gemma gemma on early post- settlment emigration, growth and survival of the hard clam Mercenaria mercenaria. Marine Ecology Progress Series, 99: 61-70.

Amyot, J., and J. A. Downing. 1998. Locomotion in Elliptio complanata (Mollusca: Unionidae): a reproductive function? , 39: 351-358.

Armonies, W. 1992. Migratory rhythms of drifting juvenile molluscs in tidal waters of the Wadden Sea. Marine Ecology Progress Series, 83: 197-206.

Armonies, W. 1994(a). Drifting meio - and macrobenthic invertebrates on tidal flats in Konigshafen: a review. Helgolander Meeresuntersuchungen, 48: 299-320.

Armonies, W. 1994(b). Turnover of postlarval bivalves in sediments of tidal flats in Konigshafen (German Wadden Sea). Helgolander Meeresuntersuchungen, 48: 291-297.

Armonies, W. 1996. Changes in distribution patterns of 0-group bivalves in the Wadden Sea: byssus-drifting releases juveniles from the constraints of hydrography. Journal of Sea Research, 35: 323-334.

Baggerman, B. 1953. Spatfall and transport of Cardium edule L. Archives Neerlandaises de Zoologie, 10:315-342.

Baker, P., and R. Mann. 1997. The postlarval phase of bivalve molluscs: a review of functional ecology and new records of postlarval drifting of Chesapeake Bay bivalves. Bulletin of Marine Science 61: 409-430.

Bayne, B.L. 1964. Primary and secondary settlement in Mytilus edulis L. (Mollusca). Journal of Animal Ecology, 33: 513-523.

Beaumont, A. R., and D. A. Barnes. 1992. Aspects of veliger larval growth and byssus drifting of the spat of Pecten maximus and Aequipecten (Chlamys) opercularis. ICES Journal of Marine Science, 49: 417-423.

Beukema, J. J. 1993. Successive changes in distribution patterns as an adaptive strategy in the bivalve Macoma balthica (L) in the Wadden Sea. Helgolander Meersuntersuchungen, 47: 287-304.

Beukema, J. J., and J. de Vlas. 1989. Tidal-current transport of thread-drifting postlarval juveniles of the bivalve Macoma balthica from the Wadden Sea to the North Sea. Marine Ecology Progress Series, 52: 193-200.

Boozer, A. C, and P. E. Mirkes. 1979. Observations on the fingernail clam, Musculium partumeium (Pisidiidae), and its association with the introduced Asiatic clam, Corbicula fluminea. The Nautilus 93: 73-83.

134 Brafield, A. E., and G. E. Newell. 1961. The behaviour of Macoma balthica (L.). Journal of the Marine Biological Association of U.K., 41: 81-87.

Caceres-Martinez, J., J. A. F. Robledo, A. Figueras. 1994. Settlement and post-larvae behaviour of Mytilus galloprovincialis: Field and laboratory experiments. Marine Ecology Progress Series, 112: 107-117.

Commito, J. A., C. A. Currier, L. R. Kane, K. A. Reinsel, and I. M. Ulm. 1995. Dispersal dynamics of the bivalve Gemma gemma in a patchy environment. Ecological Monographs, 65:1-20.

Cummings, V. J., R. D. Pridmore and S. F. Thrush. 1993. Emergence and floating behaviours of post-settlement juveniles of Maomona liliana (Bivalvia: Tellinacea). Marine Behaviour Physiology, 24: 25-32.

Cummings, V. J., R. D. Pridmore, S. F. Thrush, and J. E. Hewitt. 1995. Post-settlement movement by intertidal benthic macro invertebrates: Do common New Zealand species drift in the water column? New Zealand Journal of Marine and Freshwater Research, 29: 59-67.

Emerson, C. W., and J. Grant. 1991. The control of soft-shell clam (Mya arenaria) recruitment on intertidal sandflats by bedload sediment transport. Limnology and Oceanography, 36: 1288-1300.

Hiddink, J. G., R. P. Kock, and W. J. Wolff. 2002. Active pelagic migrations of the bivalve Macoma balthica are dangerous. Marine Biology, 140: 1149-1156.

Highsmith, R. C. 1985. Floating and algal rafting as potential dispersal mechanisms in brooding invertebrates. Marine Ecology Progress Series, 25: 169-179.

Lane, D. J. W., A. R. Beamont, and J. R. Hunter. 1985. Byssus drifting and the drifting threads of the young post-larval mussel Mytilus edulis. Marine Biology, 84: 301-308.

Martel, A., and F. Chia. 1991. Drifting and dispersal of small bivalves and gastropods with direct development. Journal of Experimental Marine Biology and Ecology, 150: 131-147.

Montaudouin, X de. 1997. Potential of bivalves' secondary settlement differs with species: a comparison between cockle (Cerastoderma edule) and clam (Ruditapes philippinarum) juvenile resuspension. Marine Biology, 128: 639-648.

Nelson, T. C. 1928. Pelagic dissoconchs of the common mussel, Mytilus edulis, with observations on the behaviour of the larvae of allied genera. Biological Bulletin, 55: 180- 192.

Newell, C. R. 1994. Estuary-scale dispersal of post-larval mussels, Mytilus edulis, among eelgrass (Zostera marina) meadows and subsequent recruitment to planted live and mussel shell cultch. Journal of Shellfish Research, 13:281.

135 Norkko, A., V. J. Cummings, S. F. Thrush, and J. E. Hewitt and T. Hume. 2001. Local dispersal of juvenile bivalves: implications for sandflat ecology. Marine Ecology Progress Series, 212: 131-144.

Olivier, F., C. Vallet, J. Dauvin, and C. Retiere. 1996. Drifting in post-larvae and juveniles in an Abra alba (Wood) community of the eastern part of the bay of Seine (English Channell). Journal of Experimental Marine Biology and Ecology, 199: 89-109.

Prezant, R. S., and K. Chalermwat. 1984. Floatation of the bivalve Corbicula fluminea as a means of dispersal. Science, 225: 1491-1493.

Roegner, C, C. Andre, M. Lindegarth, J. Eckman, and J. Grant. 1995. Transport of recently settled soft-shell clams (Mya arenaria L.) in laboratory flume flow. Journal of Experimental Marine Biology and Ecology, 187: 13-26.

Rygg, B. 1970. Studies on Cerastoderma edule (L.) and Cerastoderma glaucum (Poiret). Sarsia, 43: 65-80.

Sellmer, G. P. 1967. Functional morphology and ecological life history of the gem clam, Gemma gemma (Eulamellibranchia: Veneridae). Malacologia, 5: 137-223.

Sigurdsson, J. B., C. W. Titman, and P. A. Davies. 1976. The dispersal of young post-larval bivalve molluscs by byssus threads. Nature, 262: 386-387.

Sorlin, T. 1988. Floating behaviour in the tellinid bivalve Macoma balthica (L.). Oecologia, 77: 273-277.

Sugawara, K., and M. Tamaga. 1953. Floatation by drifting threads in Ruditapesphilippinarum in laboratory. Bulletin of the Japanese Society of Fisheries and Oceanography, 18: 81-82. (in Japanese).

Sullivan, C. M. 1948. Bivalve larvae of Malpeque Bay, P.E.I. Fisheries Research Board of Canada Bulletin, 77. 59pp.

Turner, S. J., J. Grant, R. D. Pridmore, J. E. Hewitt, M. R. Wilkinson, T. M. Hume, and D. J Morrisey. 1997. Bedload and water-column transport and colonization processes by post- settlement benthic macro fauna: Does infaunal density matter? Journal of Experimental Marine Biology and Ecology 216: 51-75.

Wang, W. X., and Z. Z. Xu. 1997. Larval swimming and postlarval drifting behaviour in the infaunal bivalve Sinonovacula constricta. Marine Ecology Progress Series, 148: 71-81.

Williams, A. B., and H. J. Porter. 1971. A ten-year study of meroplankton in North Carolina estuaries: occurrence of postmetamorphal bivalves. Chesapeake Science, 12: 26-32.

Yankson, K. 1986. Observations of byssus systems in the spat of Cerastoderma glaucum and C. edule. Journal of the Marine Biological Association of U.K., 66: 277-292.

136 Appendix 2: Comparison of methods for the determination of carbon in intertidal sediments.

Introduction

Classification of habitats is essential to understanding behaviours and life histories of the benthos that live in the sediment. Determination of organic and inorganic carbon concentrations in sediments is an important aspect of this classification and has been used as a measurement tool and classification scheme for many years. Despite years of use as a descriptor of sediment, separation of organic and inorganic carbon fractions remains challenging and the methods employed are varied. Frequently used methods differ in the amount of time to carry them out, their expense and accuracy.

Three of the most common methods include:

• Acid leaching of the sample to dissolve the inorganic carbonates followed by dry

combustion at 500°C - 600°C to determine the residual organic carbon fraction (referred to

here as "acid-burn" ) (Gibbs, 1977).

• Loss on ignition at 500°C - 600°C to oxidise the organic matter to carbon dioxide and ash.

Then a second loss on ignition at 900°C - 1000°C to oxidise the inorganic fraction, or

carbonate, into carbon dioxide (referred to here as "LOI" ) (Luczak et al., 1997).

• Determination of inorganic carbon by coulometry (Huffman, 1977) and elemental analysis

by flash combustion to determine total carbon. Then subtraction of inorganic from total

carbon to give organic carbon (referred to here as "CHN") (Verardo et al., 1990).

Other methods exist, however the three above are those most commonly used. The literature contains many examples of conflicting evidence supporting and criticizing the use of both acid-burn and LOI (Weliky et al., 1983; Tung & Tanner, 2003; and Bisutti et al., 2004).

137 The LOI method is inexpensive, easy to carry out and widely used, however it has limitations. Luczak et al. (1997) and Heiri et al. (2001) noted that use of this method involves great variability in temperature, duration of combustion and amount of sample used. These discrepancies in methodology produce results that make comparison between studies difficult.

Many authors ( e.g. Welicky et al. 1983, Yamamuro & Kayanne, 1995; 1983; Bisutti et al.,

2004) point out that the combustion temperatures of organic and inorganic forms of carbon overlap and therefore one can not achieve full separation of the two forms using ignition methods. Clay particles can also lead to large inaccuracies when using this method; the loss of associated structural water upon combustion can lead to overestimation of carbon (Barille-Boyer et al., 2003).

The acid-burn method is slightly more complicated compared to LOI, yet is still an inexpensive and simple method. Nevertheless, it also has shortcomings that have been noted in the literature (Byers et al., 1978). Froelich (1980) and Weliky et al. (1983) explain that upon acidification, acid-soluble organic carbon compounds will be dissolved along with carbonate dissolution producing errors. Acid pre-treatment can also alter the ratios of elements that may be of interest such at hydrogen, nitrogen and oxygen (Lohse et al., 2000; Ryba & Burgess, 2002) leading to problems with subsequent analysis for those compounds. The acidification treatment is followed by three repetitions of rinsing, centrifuging then decanting to remove the acid. With a greater number of steps comes increased human error and possible sample loss on decanting

(Hedges and Stern, 1984).

It is generally agreed that the CHN elemental analysis is an accurate method for analysis of total carbon (Verardo et al., 1990; Leong & Tanner, 1999). The most obvious drawback of this procedure is the expense of the equipment involved in the analysis. It has also been noted that this method may lead to inaccurate results when samples with high carbonate content are being analysed (Weliky et al., 1983; Bisutti et al, 2004). This error comes from the subtraction

138 of two large values (total carbon and inorganic carbon) to determine a small value (organic carbon).

For a researcher seeking an inexpensive method for analysing carbon in sediment samples it is difficult to know which of LOI or acid-burn is more accurate or if either is adequate. Further, no comparison has been made among these three methods to determine performance when analysing intertidal or gravel/shell/sand substrates. And finally, when working with whole intertidal samples that include small rocks and shell fragments, it is unclear whether these samples must be milled to get an accurate value for carbon. In this experiment, I address these uncertainties by comparing the performance of LOI and acid-burn for measuring both organic and inorganic carbon from milled and unmilled intertidal sediment samples.

Materials and Methods

Sample Collection and Preparation:

Intertidal sediment was collected from a clam beach near Ladysmith British Columbia,

Canada (N 48°59'18.8", W 123°45'33.2") on 19 January 2005 during an evening low tide event.

Sediment from the mid-tidal range was excavated from a grid 20 cm by 20 cm to a depth of approximately 2 cm. The sediment consisted primarily of mixed sand, gravel and broken shell.

The excavated sediment was placed in two large plastic sealable bags (Ziplock brand), labelled and returned to the lab.

The sample was kept in refrigeration for two days before being dried in a drying oven

(Precision oven by Thermo Electron Corp.). The sediment was spread out on a metal pan and dried in the oven at 60°C for 48 hours. Once dried, the sediment was fully homogenised by hand in a large mortar and pestle. The homogenised, dry sediment (roughly 80 g) was split into two portions. Representative separation was achieved by taking 1 scoop and placing it in the first portion, the second scoop into the second portion, the third scoop into the first portion

139 again, the fourth scoop into the second portion and so on until the sample was completely split.

From the first portion, 10, 2 g subsamples were taken. Each of these subsamples was randomly assigned to either acid-burn or LOI (described in detail below) for analysis (5, 2g samples were analysed using each method).

The second portion of the sample was milled to <63pm in a Rocklabs Swing Mill

(Model # Rocklabs CH2). The milled sample was stirred to ensure complete mixing then was subsampled into 15 2g subsamples that were again randomly assigned to acid-burn, LOI, or

CHN (again 5 subsamples were analysed with each method). The analysis of sediment via acid- burn and LOI was repeated with these milled samples to examine the effect of milling on the value of carbon and the precision of measurement.

Acid-Burn:

Each of the 2g subsamples from both the milled and unmilled portions (10 subsamples total) were analysed as follows. Each subsample was re-dried in the drying oven at 60°C for 24 hours, left in a glass desiccator to cool to room temperature then weighed on an analytical balance (Denver Instruments APX-200) to obtain Wtj.

Each subsample was then placed in a 50 mL glass beaker in a fume hood. To each beaker, 10% HC1 was added dropwise. As the carbonate present reacted with the acid, effervescence was apparent. The acid must be added slowly and be dilute (no stronger than

10%) for this step to be effective (Froelich, 1980). Drops of acid continued to be added until no more effervescence could be observed; this acidification process took approximately 8 hours to complete.

Once acidified, the subsamples and acid were washed with distilled water into 50mL centrifuge tubes and centrifuged for 10 minutes at 3500 rpm at room temperature in a Thermo

IEC Centra CL3 refrigerated centrifuge. The supernatant was decanted, then more distilled water was added (roughly 40mL) to wash the acid from the subsample. The subsample and distilled water was again centrifuged for 10 minutes. This rinsing process was repeated three times then the subsamples were washed with distilled water into pre-muffled foil pans for drying. They were dried at 60°C for 24 hours, and then weighed again to obtain Wt2.

The difference between Wtj.AB and Wt2-AB gave the mass of carbonate lost in acidification (see equation 1 below).

Equation 1: Wtj.AB - Wt2-AB = Wtcaco3-AB

After drying and weighing, the subsamples were ready for determination of organic carbon by ashing. The dry subsamples were placed in ceramic crucibles. Each crucible was pre-muffled at 600°C for 2 hours before adding the subsample to ensure there was no residual carbon on the crucible. The crucible and subsample were placed in a muffle furnace (Barnstead

Thermolyne 114300) at 550°C for 4 hours, then into a glass desiccator to cool to room temperature. After cooling, the final weight of the subsample was obtained (Wtf-AB).

The difference between Wt2-AB and Wtf-AB gave the mass of organic carbon lost during muffling (see equation 2 below).

Equation 2: Wt2-AB - Wtf.AB = Wtorg.c-AB

LOI:

Each of the 2g subsamples from both milled and unmilled portions (10 subsamples total) were analysed as follows. Each subsample was re-dried in the drying oven at 60°C for 24 hours, left in a glass desiccator to cool to room temperature then weighed on an analytical balance

(Denver Instruments APX-200) to obtain Wtj-Loi-

Each subsample was put into a labelled crucible that had been pre-muffled at 600°C for 2 hours to remove residual carbon. The crucibles and subsamples were muffled in a Barnstead

Thermolyne 114300 furnace at 550°C for 4 hours. After muffling the crucibles were left to cool

in a glass desiccator until they reached room temperature. Once cooled, the subsamples were re-

141 weighed to obtain \Vt2.L01. The difference between Wtj.Loi and Wt2-Loi gave the mass of organic carbon lost during muffling at 550°C (see equation 3 below).

Equation 3: Wtj.Loi - Wt2-LOi = Wtorg. C-LOI

The crucibles and subsamples were put back into the furnace and muffled again at 950°C for 2 hours. They were again cooled in a glass desiccator to room temperature and final weights

taken (Wtf.LOi).

The difference between \Vt2-L01and Wtf.LOi gave the mass of carbonate lost during muffling at 950°C (see equation 4 below).

Equation 4: Wt2-Loi - Wtf.Loi = WtCaco3-LOi

CHN:

The CHN analysis required only a small amount of sample for analysis; therefore each

2g subsample was further subsampled for each step in the CHN analysis. It is impossible to carry out this method with unmilled samples because of the small amount of sample is used, therefore only milled sediments were analysed by CHN.

First, approximately 30 mg of milled sediment was weighed out on a Mettler H20 analytical balance. The samples were weighed into glass weighing vials then placed in sample tubes. The air in the lines of the coulometer system was purged for at least 1 minute to eliminate contamination by atmospheric CO2. The sample was injected with 20% HCL with a CM5130

Acidification Module and the C02 gas evolved from the sample was carried to and titrated by a

CM5014 Coulometer, UIC Inc. This device has been shown to be accurate and reliable for measurement of CO2 by Huffman (1977). Once the titration endpoint was reached, the coulometer reading showed the amount of inorganic carbon in the sample.

Measurement of total carbon was achieved by flash combustion and elemental analysis using a Carlo Erba NA-1500 Analyzer following the methods outlined in Verardo et al., 1990.

Approximately 20 mg of milled sediment was measured into each tin cup (pressed cups, 8x5

142 mm) using a spatula and weighed on a Mettler Toledo MT5 Microbalance for elemental analysis. From this step a value for total carbon was obtained.

To calculate a value for organic carbon (OCCHN), the inorganic carbon value (ICCHN) from coulometric analysis was subtracted from the total carbon (TCCHN) value from elemental analysis (see equation 5 below).

Equation 5: TCCHN - ICCHN= OCCHN

Results

Each of the three methods outlined in the introduction were used to analyze the same homogeneous sediment sample for organic and inorganic carbon. Results of those tests are used to compare LOI and acid-burn with CHN (CHN is assumed here to accurately measure carbon levels) for the accuracy of analysis of carbon from intertidal gravel/sand/shell samples.

Resulting organic carbon values (expressed in % by weight) from the three methods are presented in figure A2-1 (mean value from each test and sample type with 95% confidence interval) and summarized in table A2-1. Resulting inorganic carbon values are presented in figure A2-2 (mean value from each test and sample type with 95% confidence interval) and summarized in table A2-1.

Analysis of variance determined that in both the organic and inorganic trials the means were not equal (F=84.9 and F=l 14.2 respectively). Levene's test for equality of error variances also determined that variances were not equal for organic and inorganic measurements (F=4.036 and F=6.541 respectively), therefore the assumption of equal variances was violated and non• parametric tests were used for multiple comparisons.

143 Organic Carbon For Each Test Type

o a,b,c ro 3 O o

2 rocn i O o 1

in 0 5 5 Acid Burn Milled CHN LOI Acid-Burn LOl-Mlled

Test Used

Figure A2-1: Mean values of organic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison).

Because means were not equal, multiple comparison of means was carried out using a

Tamhane non-parametric comparison to determine which means differed. For the values of organic carbon, the acid-burn mean value did not differ significantly from any of the other means, likely due to the large variance in the data. The means for acid-burn-milled and LOI- milled were not significantly different (p=1.00) but differed from CHN and LOI (p<0.0001, p<0.013 respectively). Significant differences are shown in figure A2-1, means with the same letter next to it are not significantly different from each other.

144 Inorganic Carbon For Each Test Type 301

c 20- o n i_ ro O o 'c co 10- O) i o b _c O in o> 5 5 5 5 Acid Burn Mlled CHN LOI Acid-Burn LOI-Mlled

Test Used

Figure A2-2: Mean values of inorganic carbon obtained from each test performed with 95% confidence intervals. N for each test is listed along the x-axis. Letters next to the data points indicate means that are not significantly different (based on non-parametric multiple comparison).

Table A2-1: Means and standard errors for each test for carbon analysis method and each value measured. Organic Carbon Inorganic Carbon Test N Mean SE N Mean SE Acid-Burn 4 3.094 0.334 5 19.355 1.252 Acid-Bum-Milled 5 5.014 0.097 5 14.299 0.564 CHN 5 1.036 0.049 5 1.449 0.039 LOI 5 3.239 0.258 5 4.895 0.753 LOI-Milled 5 5.067 0.096 5 5.031 0.087

For the inorganic carbon means, acid-burn and acid-burn-milled showed no significant difference (p=0.112) but both means differed significantly from the rest of the means. The mean inorganic carbon value for CHN and LOI-milled were significantly different (p<0.0001) but both showed no significant difference when compared to the mean for LOI (p=0.097; p= 1.000 respectively). Significant differences are shown in figure A2-2 as means with different letter labels.

145 Variances were determined to be unequal, therefore I compared variances using multiple comparison of the variances according to Levy (1975). Results of the multiple comparison of variances for the organic carbon values showed that the error variance for all tests except CHN were not significantly different, while the variance of CHN was not significantly different from the acid-burn-milled and LOI-milled variance (table A2-2). Figure A2-3 shows the results of the multiple comparison for organic carbon values, the underline connects variances that were not significantly different.

Table A2-2: Significance values for multiple comparisons of means for each comparison of test type for organic carbon values. Acid-Burn Acid-Burn-Milled CHN LOI LOI-Milled Acid-Burn 0.074* 0.076* 1.000* 0.068* Acid-Burn-Milled 0.074* 0.000 0.012 1.000 CHN 0.076* 0.000 0.008 0.000 LOI 1.000* 0.012 0.008 0.011 LOI-Milled 0.068* 1.000 0.000 0.011 *SE=0.2702 for these comparisons SE=0.2548 for the remaining comparisons

a acid-burn a LOI a acid-burn-mill a LOI-Mil a CHN

Figure A2-3: Result of multiple comparison of error variances for organic carbon values. Variances connected with underline represent variances that were not significantly different.

For the inorganic carbon values, the error variance for acid-burn, LOI and acid-burn- milled showed no significant difference. The variance for LOI-milled and CHN also showed no

significant difference. Table A2-3 and figure A2-4 shows the results of the multiple comparison

for inorganic carbon values, the underline connects variances that were not significantly

different.

146 Table A2-3: Significance values for multiple comparisons of means for each comparison of test type for

Acid-Burn Acid-Burn-Milled CHN LOI LOI-Milled : Acid-Burn 3 s«9 0.112 0.001 0.000 0.003 Acid-Bum-Milled 0.112 0.000 0.000 0.001 CHN 0.001 0.000 0.097 0.000 LOI 0.000 0.000 0.097 1.000 LOI-Milled 0.003 0.001 0.000 1.000 SE=0.9924 for all comparisons

q acid-burn a LOI a acid-burn-mill a LOI-Mil Q CHN

Figure A2-4: Result of multiple comparison of error variances for inorganic carbon values. Variances connected with underline represent variances that were not significantly different.

The LOI and acid-burn methods presented essentially the same mean level of organic carbon for the sample analysed and had comparable variance. Both methods use muffling to determine the amount of organic carbon present so this result shows that pre-treatment with acid does not significantly affect the level of organic carbon measured. LOI and acid-burn both overestimated the amount of organic carbon present by nearly three times compared to CHN.

The amount of organic carbon appeared to increase after milling by the same amount in both the

LOI-milled and acid-burn-milled tests. This is possibly due to contamination on handling during milling, or more likely caused by increased surface area allowing for more of the sample to be oxidised on muffling.

The acid-burn method showed significantly higher values for inorganic carbon measurements compared to both CHN and LOI, however both LOI and acid-burn methods overestimated the amount of inorganic carbon present in the samples compared to CHN. LOI overestimated the level of inorganic carbon by approximately three times compared to CHN, while acid-burn overestimated by nearly 12 times. Milling greatly decreased the error variance for LOI measurement of inorganic carbon in the sample.

147 Although comparably inexpensive to run, acid-burn demanded a greater time investment compared to LOI. Furthermore, the steps involved in acidifying, washing and decanting the sample were tedious and I found that this resulted in a greater potential for error such as loss on decanting. LOI is a simple and straightforward method to carry out. It involves less handling of the sample and fewer transfers from one container to another thus decreasing the opportunity for false sample loss. One drawback of LOI is that there are no widely accepted temperatures and length of time for each step (Heiri et al., 2001).

Discussion

When comparison of samples from several sites is the goal of analysis, both LOI and acid-burn would provide sufficient data for relative comparison. In comparison studies the overestimation of carbon levels should remain constant across samples and therefore not impact the end comparison of one group of samples to another. However, in cases where quantitative analysis is being carried out and the specific levels of carbon are of interest, both of these methods would lead to false overestimation and possible incorrect conclusions. The values estimated for inorganic carbon by acid-burn are of greatest concern as they largely overestimated the levels in this study when compared to the more accurate (and costly) CHN analysis.

This study makes it possible to assess the most appropriate method for analysis of intertidal sediments and provides an estimation of the errors inherent in each method. In this case, LOI was both easier to carry out and more accurate when measuring inorganic carbon. In the future, investigators with a particular interest in inorganic carbon from intertidal samples should avoid the use of acid-burn and interpret previous data collected in this manner appropriately. Past research using acid-burn to determine carbon levels for intertidal sediments are likely to have estimations of organic carbon that are comparable to research using LOI,

148 however the estimations for inorganic carbon are likely a far greater overestimation than if LOI had been used to measure carbon.

Conclusions

The overall performance of the two methods (LOI and acid-burn) was comparable when measuring organic carbon and preformed best on the unmilled sample in that case. However, when measuring inorganic carbon, LOI performed better than acid-burn. In cases when elemental analysis is not affordable, intertidal samples consisting of gravel/shell/sand substrates should be analysed using LOI on homogenised samples. Milling can be done to increase precision; however that additional step does not change the accuracy of inorganic carbon measurement. It is cautioned that milling may produce overestimations of organic carbon.

I advise that investigators analysing levels of organic and inorganic carbon in intertidal sediments initially compare reference sample LOI determinations with levels determined by elemental analysis to quantify the level of overestimation that LOI yields. In cases where comparisons are being made and the actual carbon values are not the goal, this overestimation becomes less of a concern.

149 References Appendix 2

Barille-Boyer, A., L. Barille, H. Masse, D. Razet, and M. Heral, 2003. Correction for particulate organic matter as estimated by loss on ignition in estuarine ecosystems. Estuarine Coastal and Shelf Sciences, 58: 147-153.

Bisutti, I., I. Hilke, and M. Raessler, 2004. Determination of total organic carbon - an overview of current methods. Trends in Analytical Chemistry, 23: 716-726.

Byers, S. C, E. L. Mills, and P. L. Stewart, 1978. A comparison of methods of determining organic carbon in marine sediments, with suggestions for a standard method. Hydrobiologia, 58: 43-47.

Froelich, P. N, 1980. Analysis of organic carbon in marine sediments. Limnology and Oceanography, 25: 564-572.

Gibbs, R. J. 1977, Effects of combustion temperature and time, and of the oxidation agent used in organic carbon and nitrogen analysis of marine sediments and dissolved organic material. Journal of Sedimentary Petrology, 47:547-550.

Hedges, J.L, and J.H. Stern, 1984. Carbon and nitrogen determinations of carbonate-containing solids. Limnology and Oceanography, 29: 657-663.

Heiri, O., A. F. Lotter, and G. Lemcke, 2001. Loss on ignition as a method for estimation organic and carbonate content in sediments: reproducibility and comparability of results. Journal of Paleolimnology, 25: 101-110.

Huffman, E. W. D, 1977. Performance of a new automatic carbon dioxide coulometer. Microchemistry Journal, 22: 567-573.

Leong, L. S., and P. A. Tanner, 1999. Comparison of methods for determination of organic carbon in marine sediment. Bulletin, 38: 875-879.

Levy, K. J, 1975. An empirical comparison of several multiple range tests for variances. Journal of the American Statistical Association, 70: 180-183.

Lohse, L., R. T. Kloosterhuis, H. C. de Stiger, W. Helder, W. V. Raaphorst, and T. C. E. Van Weering, 2000. Carbonate removal by acidification causes loss of nitrogenous compounds in continental margin sediments. Marine Chemistry, 69:193-201.

Luczak, C, M. Janquin, and A. Kupka, 1997. Simple standard procedure for the routine determination of organic matter in marine sediment. Hydrobiologia, 345: 87-94.

Ryba, S. A., and R. M. Burgess, 2002. Effects of sample preparation on the measurement of organic carbon, hydrogen, nitrogen, sulphur and oxygen concentrations in marine sediments. Chemosphere, 48: 139-147.

Tung, J. W. T., and P. A. Tanner, 2003. Instrumental determination of organic carbon in marine sediments. Marine Chemistry, 80: 161-170.

150 Verardo, D. J., P. N. Froelich, and A. Mclntyre, 1990. Determination of organic carbon and nitrogen in marine sediments using the Carlo Erba NA-1500 Analyzer. Deep Sea Research, 37: 157-165.

Weliky, K., E. Suess, C. A. Ungerer, P. J. Muller, and K. Fisher, 1983. Problems with the accurate carbon measurements in marine sediments and particulate matter in seawater: A new approach. Limnology and Oceanography, 28: 1252-1259.

Yamamuro, M., and H. Kayanne, 1995. Rapid direct determination of organic carbon and nitrogen in carbonate-bearing sediments with a Yanaco MT-5 CHN analyzer. Limnology and Oceanography, 40: 1001-1005.

151 Appendix 3: Larval settlement data from 2002

In addition to larval sampling in 2003 and 2004 reported in Chapter 4, samples were taken from Beach 1 only in 2002 and counted for recently settled Venerupis philippinarum. The methods of sampling and counting were the same as reported previously in Chapter 4. The following is a brief summary of the results of the 2002 counts.

Counts of V. philippinarum early recruits were made at the netted and non-netted plot at

Beach 1 over 4 sampling events in 2002. The dates of sampling were August 8th, August 19th,

September 5th and October 10th. Twenty-four cores were taken at each plot on each sampling date, except October when only twelve were taken. These cores were frozen and later counted for the number of newly settled clam larvae. The results of these counts are shown in Figure

A3-1. The first sampling date showed very few settlers, followed by a larger set on the three

subsequent sampling dates. I tested the mean number of early recruits per m2 at netted and non- netted plots using ANOVA for each sample date separately. The first and third sample date

(August 8th and September 5th) showed no significant difference between early recruit density at netted versus non-netted plots (p=0.25, n=48; p=0.173, n=49 respectively). The second and

fourth sample dates (August 19th and October 10th) did show a significant difference between netted and non-netted early recruit density (p=0.018, n=44; p=0.023, n=24 respectively).

Length measurements were also made on the clams counted from the samples taken in

2002. Average lengths for each sample date for netted and non-netted plots are shown in Figure

A3-2. Over all sample dates combined, average early recruit length for the netted plot was 290 pm (± 78 pm standard deviation) and for the non-netted plot was 299 um (±81 pm S.D.). Early recruit length showed little difference between the netted and non-netted plots. Analysis of variance was performed on length measurements for each sample date separately to compare mean length of clams at netted versus non-netted plots. There was no significant difference

152 between shell lengths at netted and non-netted plots (Date 1: p=0.67, n=20; Date 2: p=0.34, n=91; Date 3: p=0.29, n=l 10; Date 4: p-0.14, n=56).

7000 Early Recruit Density Beach 1 2002

6000

-®—NoNet 5000 A ••— Net

| 4000 CO C

J: 3000

co 2000

1000

Aug. 8 Aug. 19 Sept. 5 Oct. 10 Sample Date

Figure A3-1: Density of settled Venerupis philippinarum larvae per m2 in 2002 at Beach 1 site. Netted plot is shown with the black hatched line, non-netted plot is shown in the grey solid line. Error bars represent the 95% confidence interval. For each point, n=24 except the October data where n=12.

Length frequency of the larvae between the second and third sample dates (August 19th -

September 5th) allow us to examine growth as I did in Chapter 4. Data from both plots shows a peak at length 250 um on the second sampling date, that peak shifts to 380 pm on the next

sampling date (Figure A3-3). Based on the difference in shell length and time elapsed, the

growth rate was approximately 7.6 um/day. This estimate is based on the assumption that the

group measured on date 2 is the same cohort that was measured on the third date. This does not

account for immigration/emigration and mortality losses and therefore is a generalised estimate

of growth rate.

153 Early Recruit Length Beach 1 2002 • Net 450 • NoNet 400 350

E 300 3 250 cn c Sfef 200 0) 150 -\ CO 100 50 0 Aug. 8 Aug. 19 Sept. 5 Oct. 10 Sample Date

Figure A3-2: Lengths of Venerupis philippinarum clams sampled in 2002. Netted plots shown with hatched bars and non-netted plots shown with grey bars. Error bars represent 95% confidence interval.

154 Length Frequency Beach 1 Net 2002

200 250 300 350 400 450 500 550 600 Shell Length (um)

Figure A3-3: Length frequency graphs for Venerupis philippinarum early recruits collected in 2002 Beach 1. Solid black line shows the length frequency for August 19th, the open dotted line shows September 5th. The top panel shows data from the neted plot, the bottom panel shows the non-netted plot. Appendix 4: Field site velocity measurements

Clod cards were used to estimate the water velocity at each field site. Each clod card was made following the methods outlined in Appendix 5. A spike 3 cm in length attached to the bottom of each plaster clod allowed it to be anchored in the sediment (Figure A4-1). Each clod was dried in a drying oven and weighed before deployment in the field. After deployment the clods were again dried and re-weighed to determine mass lost during deployment. The mass lost can be converted to an estimate of velocity using controlled calibration in the laboratory. I calibrated the clods using both laminar and turbulent flow regimes (see Appendix 5 for the calibration).

Six clods were deployed at each plot within each beach for a period of 24 hours. An initial deployment was carried out on August 24th 2004 however an overnight storm occurred and those data were not used. A second deployment on August 30th 2006 was used to generate the data shown in Figure A4-2. This figure shows the estimated velocities based on the turbulent calibration relationship. Velocity estimates ranged from three to eight cm/s. Beach 4 showed the slowest velocity and Beach 2 showed the highest. These measurements were made on a spring tide and therefore are likely to be a slight overestimation of average intertidal velocities for these locations.

156 Figure A4-1: Positioning of the clod card in the sediment. A small plastic spike attached to the bottom of the clod allows it to be inserted into the sediment to keep it in place.

10 Velocity predicted by Clod Cards Based on turbulence calibration 9

8

7

« 6 E 3 5

No Yes No Yes No

Beach 1 Beach 2 Beach 3 Figure A4-2: Velocity measurements from field sites as estimated by clod card dissolution. Beach 1 shown in white bars, Beach 2 in light grey bars, Beach 3 in dark grey bars and Beach 4 in black bars. Bars with hatch marks represent netted plots, bars without hatching represents non-netted plots. Error bars show the 95% confidence interval, n=6 for each bar. Relationship for turbulent calibration is shown in Appendix 5.

157 Appendix 5: Consideration of turbulence in calibration of plaster blocks used for flow measurement.

Introduction

Measurement of rate of water flow is important to the study of marine ecological systems, particularly those involving settlement of invertebrate larvae (Butman, 1990). A simple and inexpensive method for assessing relative rate of water movement was first described by Muus (1968). The rate of dissolution of plaster balls in seawater is dependant on the rate of water flow past the plaster surface. Muus placed plaster balls of known dry mass into flowing water where a portion of the ball would dissolve. The balls were then removed from the water, dried and weighed again to determine the amount of dissolution. This technique has been used by many investigators to determine water motion in the field (see Porter et al. (2000) for references listed therein).

The preparation of plaster blocks, or clod cards as they were called by Doty (1971), involves casting plaster of paris (gypsum) in ice cube trays (Doty, 1971). Although a radially symmetrical shape is preferable for dissolution trials (Denny, 1988), the efficiency and ease that is allowed by using ice cube trays makes this a desirable cast. The success of this method is dependant on accurate laboratory calibration prior to placement of cards into the field.

Calibration involves measurement of dissolution of the clods under known flow rates at the same temperature and salinity as those that will be encountered in the field. Rate of dissolution increases with temperature (Denny, 1988), therefore, temperature must be matched in the laboratory calibrations to what will be seen the field.

A version of this appendix was published in: Munroe, D., and S. McKinley. (2005) Consideration of turbulence in calibration of plaster blocks used for flow measurement. Aquaculture Canada 2004 Proceedings of Contributed Papers. AAC Spec. Publ. No. 9. C.I. Henrdry Editor.

158 One problem with this method that is rarely adequately accounted for by investigators is the influence of steady flow versus turbulent flow (Porter et al., 2000). In many calibrations, flow is steady and smooth, while flows in the field are likely to be more turbulent or mixed.

Turbulent flow leads to a greater exchange between a surface and the overlying water (Denny,

1988) and thus greater dissolution of the clod card. Without consideration of this effect velocity of water would be overestimated when steady flow calibrations are used to evaluate turbulent field flows (Porter, et al., 2000).

In this experiment, both turbulent and steady flow calibrations were carried out at overall flow rates ranging from 0 cm/sec to 4 cm/sec to determine the difference in dissolution rate created by the difference in flow structure.

Materials and Methods

Clod cards were prepared following the methods used by Thompson and Glenn (1994).

Plaster of Paris dry mix (produced by DAP Inc. 2002) was mixed 2 parts plaster to 1 part clean cold water. The liquid was transferred into ice cube trays with a baster then the side of the tray was tapped to remove air bubbles. Plastic cocktail swords were inserted; handle down, into each cast to be used later for attachment in the field and for labelling. The plastic swords were used to avoid metal that may corrode and break apart in the field and thus affect the final weight of the clod card. The clod cards were allowed to harden for at least 30 minutes before removal from the tray. After removal from the tray they were dried in a drying oven for 68 hours at 30

°C.

Prior to initiation of the experiment, each dry block was pre-weighed and labelled with a unique number. Each block was 4.4 cm by 3.6 cm at the base and 3.0 cm by 1.9 cm at the top and had a height of 2.6 cm. The final dimensions of the clod cards are shown in figure A5-1.

The weight of each card was 25.39 g (standard error = 0.1 Og). Flume tanks were set up with bulk flow of 1, 2, 3, or 4 cm/s. Two tanks were used, each was 250 cm long, one was 40.5 cm wide while the other was 35 cm wide and both were filled to a depth of 10.5 cm. Water supplied to each flume was from a re-circulating system that contains roughly 7600L of filtered salt water, therefore saturation as the blocks dissolved was not a concern. In each flume, one end contained turbulence while the other flowed smoothly. Laminar flows were achieved by placing a large honeycomb shaped manifold (holes 0.5 cm diameter, 15 cm long) within the flow to constrain it. Both turbulent and laminar flows were confirmed by using dye to visualize the streaklines (Vogel, 1996). At each bulk flow rate 4 blocks were placed in each type of flow for

24 hours. Still water (0 cm/s) calibrations were carried out with 1 block suspended in a 20 L tank with no inflow or outflow for 24 hours. Still water calibrations were repeated 4 times with water replacement each time. All trials were run at a temperature of 13°C. After 24 hours in the water the blocks were retrieved and again placed in a drying oven for 68 hours at 30 °C. After drying, each block was weighed a second time to determine mass lost over 24 hours.

4.4 cm

Figure A5-1: Dimensions of clod cards used.

160 Results

The dissolution of the blocks showed a linear relationship between percent mass lost and water velocity for both laminar and turbulent flows (figure A5-2). The slope of the graph of percentage mass lost and water velocity was 1.65 for laminar flows (R2=0.83) and 2.74 for turbulent flows (R2=0.71). The dissolution rate of blocks in turbulent flow was significantly higher than in laminar flow at the same bulk flow rate (P<0.0001). The slope of the turbulent flow relationship is greater than that of the laminar flow and therefore the difference in percent mass lost between laminar and turbulent flows is enhanced at higher flow rates. The variability of the data points around the linear relationship is greater for turbulent flow than for laminar flow (lower R2).

37

m

R2 = 0.708 5 • "'" m • .o

A R2 = 0.8291

• turbulent

A laminar

1.5 2 2.5 4.5 Velocity (cm/sec)

Figure A5-2: Graph of percentage mass lost from blocks over 24 hour period at flows from 0-4 cm/sec. Turbulent data points shown with black squares and solid trendline (R2=0.7085), laminar datapoints shown with grey triangles and dashed trendline (R2=0.8291).

161 Discussion

Dissolution of plaster blocks has been used extensively as a method for field estimation for flow rates (Peticrew and Kalff, 1991; Komatsu and Kawai, 1992). Calibration of the blocks prior to placement in the field is crucial to the success of the method. Calibration has been primarily carried out under smooth flow conditions and the observed relationships between dissolution and flow are good in most studies (Porter et al., 2000). However, one factor that is often overlooked is the type of flow being investigated and laminar calibrations are commonly applied to turbulent flows in the field (Komatsu and Kawai, 1992; Porter et al, 2000).

In this experiment, I found that dissolution of the blocks increased with increasing water flow in a linear relationship, as expected. I also found that the rate of increase was higher when the flow was turbulent versus when it was smooth. The difference in dissolution rates between

laminar and turbulent flows must be considered when calibrations are carried out. In cases where calibrations are carried out in a laminar environment and field measurements are made in a turbulent environment, the water velocity will be overestimated due to this increase in dissolution. The degree of overestimation is increased at higher bulk flow rates. Another result of this experiment was that in turbulent flows there was greater variability in the relationship between flow and dissolution. This is also important to note when carrying out calibrations that will be used to measure turbulent flows in the field.

Overall, this method of flow measurement is effective and easy to use. If the intended outcome is comparison of sites with similar turbidity, then an appropriate calibration will lead to reliable results. However, if sites to be examined show differences in the level of turbulence, then the effect of the turbulence on the dissolution must be considered.

162 References Appendix 5

Butman, C. A. 1990. Sediment-trap experiments on the importance of hydrodynamical processes in distributing settling invertebrate larvae in near-bottom waters. Journal of Experimental Marine Biology and Ecology, 134: 37-88.

Denny, M. W. 1988. Biology and the Mechanics of the wave-swept environment. Princeton University Press, Princeton, New Jersey. 329 pp.

Doty, M. S. 1971. Measurement of water movement in reference to benthic algal growth. Botanica Marina, 14: 32-35.

Komatsu, T., and H. Kawai. 1992. Measurement of time-averaged intensity of water motion with plaster balls. Journal of Oceanography, 48:353-365.

Muus, B. 1968. A field method for "exposure" by means of plaster balls. A preliminary account. Sarsia, 34: 61-68.

Petticrew, E. L., and J. Kalff. 1991. Calibration of a gypsum source for freshwater flow measurements. Canadian Journal of Fisheries and Aquatic Sciences, 48: 1244-1249.

Porter, E. T., L. P. Sanford, and S. E. Suttles. 2000. Gypsum dissolution is not a universal integrator of 'water motion'. Limnology and Oceanography, 45: 145-158.

Thompson, T. L., and E. P. Glenn. 1994. Plaster standards to measure water motion. Limnology and Oceanography, 39: 1768-1779.

Vogel, S. 1996. Life in moving fluids. Princeton University Press, New Jersey. 467 p.

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