HomeMy WebLinkAbout20081764 Ver 2_Corps of Engineer Correspondence_20141215Steenhuis, Joanne
From:
Sugg, Mickey T SAW [Mickey.T.Sugg @usace. army. mi1]
Sent:
Monday, December 15, 2014 2:00 PM
To:
Matthews, Kathryn; Fritz Rohde - NOAA Federal; Baker, Jessi E; Dunn, Maria T.; Wilson,
Debra; Dail, Jason; Huggett, Doug; Steenhuis, Joanne; Stuart Turille; Rosov, Brad; Willson,
Kenneth
Cc:
Dana Schmidt; Skip; David Cerino
Subject:
FW: Final Report Attachments NTB Macrofauna (Coquina clam) (UNCLASSIFIED)
Attachments:
CCC Upwell Temps 02262013 10232014.xlsx; Donax variabilis Larval Logs 2014.xlsx;
Donax variabilis Spawn Log 2014.xlsx; Donax variabilis Brochure NTB 2013.pdf; Donax
variabilis CCC Aqua Brd Brief 03242014.pptx; Donax variabilis Monogean Trematode.pptx
Classification: UNCLASSIFIED
Caveats: NONE
Second and last one.
- mickey
Mickey Sugg, Project Manager
U.S. Army Corps of Engineers
69 Darlington Avenue
Wilmington, NC 28403
(910) 251 -4811 (direct line)
(910) 251 -4025 (fax)
"The Wilmington District is committed to providing the highest level of support to the
public. To help us ensure we continue to do so, please complete the Customer Satisfaction
Survey located at: http : / /regulatory.usacesurvey.com /"
- - - -- Original Message---- -
From: Dana Schmidt [mailto:schmidtd(@carteret.edu]
Sent: Saturday, December 13, 2014 9:49 PM
To: Sugg, Mickey T SAW
Cc: David Cerino; Skip
Subject: [EXTERNAL] Final Report Attachments NTB Macrofauna (Coquina clam)
6 files attached
Dana L Schmidt
Aquaculture Facility Assistant
Carteret Community College
301 College Circle, Howard Bldg.
Morehead City, NC 28557
1
Classification: UNCLASSIFIED
Caveats: NONE
Steenhuis, Joanne
From:
Sugg, Mickey T SAW [Mickey.T.Sugg @usace. army. mi1]
Sent:
Monday, December 15, 2014 2:00 PM
To:
Matthews, Kathryn; Fritz Rohde - NOAA Federal; Baker, Jessi E; Dunn, Maria T.; Wilson,
Debra; Dail, Jason; Huggett, Doug; Steenhuis, Joanne; Stuart Turille; Rosov, Brad; Willson,
Kenneth
Cc:
Dana Schmidt; Skip; David Cerino
Subject:
FW: Final Report Attachments NTB Macrofauna (Coquina clam) (UNCLASSIFIED)
Attachments:
CCC Upwell Temps 02262013 10232014.xlsx; Donax variabilis Larval Logs 2014.xlsx;
Donax variabilis Spawn Log 2014.xlsx; Donax variabilis Brochure NTB 2013.pdf; Donax
variabilis CCC Aqua Brd Brief 03242014.pptx; Donax variabilis Monogean Trematode.pptx
Classification: UNCLASSIFIED
Caveats: NONE
Second and last one.
- mickey
Mickey Sugg, Project Manager
U.S. Army Corps of Engineers
69 Darlington Avenue
Wilmington, NC 28403
(910) 251 -4811 (direct line)
(910) 251 -4025 (fax)
"The Wilmington District is committed to providing the highest level of support to the
public. To help us ensure we continue to do so, please complete the Customer Satisfaction
Survey located at: http : / /regulatory.usacesurvey.com /"
- - - -- Original Message---- -
From: Dana Schmidt [mailto:schmidtd(@carteret.edu]
Sent: Saturday, December 13, 2014 9:49 PM
To: Sugg, Mickey T SAW
Cc: David Cerino; Skip
Subject: [EXTERNAL] Final Report Attachments NTB Macrofauna (Coquina clam)
6 files attached
Dana L Schmidt
Aquaculture Facility Assistant
Carteret Community College
301 College Circle, Howard Bldg.
Morehead City, NC 28557
1
Classification: UNCLASSIFIED
Caveats: NONE
Steenhuis, Joanne
From: Sugg, Mickey T SAW [Mickey.T.Sugg @usace. army. mi1]
Sent: Monday, December 15, 2014 1:55 PM
To: Matthews, Kathryn; Fritz Rohde - NOAA Federal; Baker, Jessi E; Dunn, Maria T.; Wilson,
Debra; Dail, Jason; Huggett, Doug; Steenhuis, Joanne; Stuart Turille; Rosov, Brad; Willson,
Kenneth
Cc: Dana Schmidt; David Cerino; Skip
Subject: FW: Final Report NTB Macrofauna (Coquina clam) (UNCLASSIFIED)
Attachments: Donax NTB Macrofauna Project Final Report.docx
Classification: UNCLASSIFIED
Caveats: NONE
Good afternoon-
The enclosed is the final report for the Donax study as required by our May 27, 2011
authorization to North Topsail Beach Shoreline Protection Project. A second e -mail will
follow this one and will include supplemental information that gives a full picture of the
work that was completed by Carteret County Community College.
Now, the hope would be for someone to pick up the last step in studying the methodologies in
beach reseeding.
If you have questions, pls call.
- mickey
Mickey Sugg, Project Manager
U.S. Army Corps of Engineers
69 Darlington Avenue
Wilmington, NC 28403
(910) 251 -4811 (direct line)
(910) 251 -4025 (fax)
"The Wilmington District is committed to providing the highest level of support to the
public. To help us ensure we continue to do so, please complete the Customer Satisfaction
Survey located at: http : / /regulatory.usacesurvey.com /"
- - - -- Original Message---- -
From: Dana Schmidt [mailto:schmidtd(@carteret.edu]
Sent: Saturday, December 13, 2014 9:43 PM
To: Sugg, Mickey T SAW
Cc: David Cerino; Skip
Subject: [EXTERNAL] Final Report NTB Macrofauna (Coquina clam)
Hi Mickey,
Here is the final NTB Macrofauna (Coquina clam) Report and total attachments list (below).
All the videos were uploaded to YouTube. The links are listed in the Executive Summary
(beginning) of the final report. Sorry for the delay. I hope everyone learns something from
it; I know I did!
Best Regards,
Dana
1
Total Appendices Emailed:
* Donax variabilis Spawn Log 2014.xls - spawn data
* Donax variabilis Larval Logs 2014.xls - larvae data
* CCC Upwell Temps 02262013 10232014.xls -Rogue Sound Temperatures (Morehead City, NC)
* Donax variabilis Brochure NTB 2013 - Informative Project Brochure (Beach Handout)
* Donax variabilis Monogean Trematode Unknown- Power Point overview
* Donax variabilis CCC Aquaculture Board Briefing - Power Point overview
Dana L Schmidt
Aquaculture Facility Assistant
Carteret Community College
301 College Circle, Howard Bldg.
Morehead City, NC 28557
Classification: UNCLASSIFIED
Caveats: NONE
N
Improving the Aquaculture Propagation Techniques for
Restocking Nourished Beaches with
Coquina Clams (Donax variabilis)
Principal Investigator — Philip S. Kemp, Jr.
Project Manager— Dana L. Schmidt
Carteret Community College Aquaculture Technology Department
Morehead City, North Carolina 28557
Final Report
November 2014
r _ Don ax variabilis S¢xs juvenile figur mefita talpoi da zoe a
November 2014 NTB Macrofauna Final Report Page 1 of 37
Project Objectives
A. Test the standard clam larviculture methods for production of juvenile Coquina Clams. The
following variations for gamete collection from broodstock (adult clams) will be tried:
a. Environmental manipulation of broodstock to achieve mass spawns.
b. Out -of- season maturation and holding in broodstock conditioning system.
c. Collection of trickle- spawned gametes from broodstock holding systems.
d. Direct strip spawn of mature adults.
B. Develop semi - continuous batch larviculture of comingled trickle spawned gametes.
C. Determine growth rates and time in nursery from post -set juvenile to stocking seed size.
Executive Summary
The primary goal to spawn Donax variabilis and obtain larvae was achieved on multiple
occasions using all proposed methods. The most reliable spawning method utilized in our hatchery was
a two -step temperature manipulation process of naturally ripe broodstock. The clams were retrieved
from their holding facility (15 ° ±1 °C brood room or ambient outdoor upwell system) and purged up to
twenty four hours prior to spawning while gradually increasing their temperature six to eight degrees
Celsius (6 °C to 8 °C). During the purge process excessive feces were reduced while the gamete condition
and monogean trematode level were evaluated; undesirable stock was eliminated. Extremely ripe
individuals did occasionally spawn while the group purged which ended the purge cycle. The purge and
acclimation process was followed by a new bath at a constant hatchery water temperature (winter
19 °C /summer 24 °C). Gamete release enhancement was obtained using hydrogen peroxide at fifty parts
per million (H2O2 at 50 ppm) without detrimental effects on the larvae.
Spawns of one to eighteen million eggs were readily obtained from hydrogen peroxide induced
spawns. Originally, the hydrogen peroxide was unsuccessfully tested as a treatment for the monogean
trematode infestation. Without the use of hydrogen peroxide, "trickle" spawns normally resulted with
ova releases in the thousands versus millions. April through July 2014 provided repeatable viable large
spawn yields by utilizing the combination procedure of "purge conditioning" followed by a tempered
bath with hydrogen peroxide at fifty parts per million. An average spawning broodstock volume of three
hundred milliliters (300 ml; about 100 -150 clams) produced twenty one (21) successful spawn events
during 2014 that had an average yield of 2.6 million zygotes per spawn (55.7 million zygotes total). Strip
spawning proved to be ineffective. Detailed information of broodstock management, purge
conditioning, spawning, and comingling of spawns are described.
Naturally ripe broodstock display a tertiary gamete conditioning pattern in our area (western
Atlantic Ocean, south of Cape Hatteras) uncommon to shellfish: a winter spawn season mid - January
through February, a spring spawn season April through July, and a fall spawn season October through
November. Coquina clams are heterosexual; females have pink gonads (ova lobes) and males have
white gonads (sperm lobes). The sectional lobes of the gonads, another uncommon bivalve
characteristic, allowed the clams to readily ripen a portion of gonad for gamete release. Animals that
had "trickle spawned" (released some, not all of their gametes) were kept for immediate continued
November 2014 NTB Macrofauna Final Report Page 2 of 37
spawns with positive results. Individuals sacrificed throughout the year repeatedly showed a variable
index of gonad ripeness. A onetime gradual temperature increase (60-8 0C) immediately followed by a
constant temperature acclimation period for one day (winter 19 °C, summer 23 °C, ±1 °C) yielded cohorts
ripe for spawning most of the year.
The goal to produce juvenile Donax reached a new milestone during this project. One batch
reached post - larval juvenile stage in thirty -six days. To our knowledge, juvenile coquina clams had never
been produced in a hatchery previously per reports in the scientific literature. The batch was
subsequently lost, however this success was photographically documented. Semi - continuous batch
larviculture was utilized by comingling multiple spawns weekly and reared as one spawn. One
experimental larviculture feeding study was attempted but terminated without results due to protozoan
and mycelial contamination in the algal cultures. A growth rate assessment of wild collected juvenile
clam seed held in the upwell system for one year was attempted. Insufficient data resulted due to
mortalities from heavy sedimentation and "over spat" from natural competitive planktons in the
unfiltered seawater supply such as oyster, sea star, mussel, and macro algae. The primary obstacles
encountered in hatchery operations were: a) protozoan and mycelial contamination of algae, b) mycelial
contamination of conditioned seawater tanks before adding algae or larvae, c) monogean trematode
infestation of collected broodstock clams resulting in d) monogean trematode contamination (and
competition) in larval cultures. A trial culture of another key beach species was conducted; the mole
crab (Emerita talpoida), Approximately 1000 mole crab larvae grown to the megalop larval stage are
described.
The objective to inform the public and involve students was achieved. Student involvement
included engaging aquaculture classes in broodstock collection and demonstrations of algae and
hatchery techniques. The general public was engaged and informed through frequent campus tours for
high school and elementary school groups and other civic groups. The project was reported in the
Carteret County News -Times on April 20, 2014. Three summer camps from NCSU Outreach Education
Program, Raleigh, NC toured the hatchery and collected broodstock while they learned about the beach
environment. Informative brochures were disseminated to interested onlookers on beach collection
trips. Many brochure recipients came to see the hatchery operations.
The additional appendices contain data files of spawning and larval methods, YouTube links for
spawning and larval videos, hourly seawater temperatures of Bogue Sound in Morehead City, NC, and
other informational material presented throughout the project period:
• Donax variabilis Spawn Log 2014.xls — spawn data
• Donax variabilis Larval Logs 2014.xls — larvae data
• CCC Upwell Temps 02262013 10232014.xls — Bogue Sound Temperatures (Morehead City, NC)
• Donax variabilis Brochure NTB 2013 - Informative Project Brochure (Beach Handout)
(continued)
November 2014 NTB Macrofauna Final Report Page 3 of 37
• Donax variabilis spawning videos ( "MacJar" and tray methods)
• Coquina clam "MacJar" Spawn YouTube tray video link: http: / /youtu.be ybxcCpSjiuU
• Coquina clam tray spawn YouTube tray video link: http: / /youtu.be /kHv6tRSj39k
• Donax variabilis veliger videos
• YouTube veliger 2 days old video link: http: / /youtu.be /aeT103cIC00
• YouTube veliger 7 days old video link: http: / /youtu.be WoaIIOXRt0
• YouTube veliger 13 days old video link: http: / /youtu.be�o62UJkeHvxj4
• Donax variabilis pediveliger 34 day old videos
• YouTube moving pediveliger video link: http: / /youtu.be arMvtQltrrg
• YouTube pediveliger video link: h11p1/youtu.be�LXm6RcS9YW3
• Emerita talpoida (Mole Crab) videos
• YouTube crab zoea 2 weeks old video link: http: / /youtu.be�RmKFIs7PdQrw
• YouTube crab zoea 2 and 3 weeks old video link: http: / /youtu.be Y04SOJk4KSk
• YouTube crab zoea 4 weeks old video link: h11p1/youtu.beJFFlov7el q6M
• Donax variabilis Monogean trematode infestation— Power Point overview
• Donax variabilis CCC Aquaculture Board Briefing - Power Point overview
Overview and Summation of Proiect
The project as a whole was a success. Valuable information was gained on the species (Donax
variabilis) that will aid future spawning attempts to produce viable seed for beach nourishment areas.
The tertiary conditioning pattern along with the rapid gonad conditioning proven by multiple spawns
throughout the year provided previously unknown knowledge that will be useful in future spawn trials.
Nutritional requirements of the larvae were our greatest obstacle and will possibly be tested at the
college. After attending a NOAA algal workshop in November 2014, Tetraselmis spp. is the algal food of
choice to supply adequate shellfish nutrition for optimal survival through larval morphological stages.
(Gary Wikfors, NOAA Fisheries Service, NEFSC, Milford, CT, personal communication, November 2014).
Our choices of Skeletonema sp., Dunaliella sp., and Nannochloropsis sp. were potentially not optimal
food for the Coquina clam. Many of the experiences gained in this process lead us to believe that further
research will lead to successful cultivation of this species. In the future the Coquina clam would be a
great species for the community college to spawn as class to learn the shellfish spawning and rearing
process while providing a community service.
Future research areas:
1) Treatments of broodstock to effectively remove monogean trematodes
2) Continue spawning Donax variabilis with the ultimate goal of seed production
3) Feeding studies to determine proper larval diet and feeding regimes
4) A juvenile growth rate of hatchery seed in coarsely filtered (60 micron) seawater systems.
November 2014 NTB Macrofauna Final Report Page 4 of 37
Broodstock Grading
The Coquina clam broodstock displayed a tertiary conditioning pattern, uncommon to shellfish
that was effectively used in our hatchery. Individuals were sacrificed to grade the gonad for gamete
ripeness indicative of the broodstock as a whole. The gonad held the gamete in sectional lobes that
allowed the clam to have variable conditions of ripeness. Individuals used their lobed gonad to "trickle"
small spawns through entire seasons. The gamete lobes observed maintained a pre -spawn C1 condition
using the Donax variabilis Gonadal Index Chart (Manning, 2004); peduncle shaped ova and spermatid.
The fully ripe gamete condition, C2 condition of round ova with large yolks and free swimming sperm,
was rare to observe. Gonad lobes (female and male) ripened as needed, proved by larvae producing
gamete released at controlled spawns throughout all of the seasons. The three spawn seasons we
discovered were winter (January and February), spring (April through July), and fall (October and
November).
Figure : ; Female gonad, gamete lobes, and peduncle ova (Cl, re-ripe stage)
ale gonad, gamete lobes, and clumpy! spermatid (Cl, pre -ripe stake)
November 2014 NTB Macrofauna Final Report Page 5 of 37
sir ; C2 ripe gamete of Coquina clams per Don ax variabilas Gonadal index (Manning 2004);
round eggs with large yolk of female left, and individual free swimming sperm of male right
Parasitic Monogean Trematode in Gonad of Adult Clams
Holding broodstock was problematic due to monogean trem
gonad and muscle tissue. The monogean trematodes came to the fa(
The closed cultures of the indoor broodstock holding system
allowed the parasitizing monogean trematodes to infest all the
stock. When held in the ambient (outdoor temperature) upwell
system, unfiltered seawater flow fed broodstock and flushed
loose monogean trematodes from the stock. However, infested
individuals would release parasites and gamete during a spawn
due to infestation within the gonad and the trematodes would
infect others. The purging cycle was imperative for clean spawns.
Trichodina, a protozoan, was also routinely found during gamete
investigations (within the loose tissue folds where siphons
attached to the main body) but was not problematic to
spawning.
The amount of monogean trematodes was so numerous
and widespread that a standardized method was utilized to
quantify the infestation load of a brood stock set. A set was
purged for fifteen minutes in a MacDonald Fish Hatching Jar
(MacJar) set up with stacked sieves (75µ and 20µ sieves) at the exit flow
trematodes. Sets of broodstock with monogean trematodes in excess of
within fifteen minute were eliminated from spawning for the day.
Sets with less than 100 collected were allowed to purge for longer
periods until trematodes were not found or the stock was
eliminated.
Preliminary treatments were tried without positive results;
hydrogen peroxide, copper sulfate, low salinities (10 to 15ppt),
freshwater, extreme high and low temperatures (8 °C and 45 °C),
and prolonged flushing. Most chemicals tried resulted in closed
clams; safe guarding the parasites from treatment. Unfortunately,
November 2014 NTB Macrofauna Final Report
Female gonad infested
with parasitic monogean trematode
lure ;'tale gonad infested with
parasitic monogean tre atode
cold winter temperatures (8 °C) had no effect on the parasites nor did heated baths (up to 45 °C).
Elimination of infested stock by purging small one hundred milliliter (100 ml) sets was the only method
we found to reduce the monogean trematodes to a manageable level. Infected sets were discarded to
dry ground. Additional broodstock was collected monthly to maintain spawning stock.
November 2014 NTB Macrofauna Final Report Page 7 of 37
an adult monogean trema ode with cysts (left) and a 20 micron siege cater from a set of brood stack (right)
ano aarr tra atode cysts within Eaat muscle tissue (last) and a 1000x image of a cyst (right)
November 2014 NTB Macrofauna Final Report Page 8 of 37
CD a single gamete lobe of a female Coquina clam infested with adult monogean tremat des (left);
the overall gamete lobes of the infested female; no clam eggs remained (right).
Broodstock Handling
Broodstock was held
chilled indoors (15 ° ±1 °C)
and in the outdoor upwell
system. Stock held in our
outdoor upwell system was
supplied with unfiltered
seawater which provided an
abundance of natural algal
food supply from Bogue
Sound. Beaufort Inlet in
Morehead City, NC provides
ample flow and ebb of
ocean waters twice daily to our location on Bogue Sound providing algal supplies similar to the clam's
natural beach environment of the Atlantic Ocean. Their voracious appetite and heightened metabolism,
possibly due to their normal high velocity beach environment, required abundant feeding of diversified
nutritional algae. The stock's holding temperature had little effect as to the clams' ability to readily
produce viable gametes; algal food supply was a limiting factor. The temperatures for Bogue Sound
were collected hourly from February 26, 2013 through October 23, 2014. The weekly and monthly
averages are at the end of this report (page 30).
November 2014 NTB Macrofauna Final Report Page 9 of 37
Broodstock was held indoors at 15 ° ±1 °C in aerated static baths of one micron
(1µm) filtered seawater. Each culture tub was fed one to four liters of live algal blend
daily consisting of equal parts of Isochrysis spp., Dunaliella sp., and Chaetocerus spp.
The culture tub was cleaned every two days and restocked with pre - chilled one micron
seawater, algae, and the rack of clean live broodstock. Full seawater salinity was held.
Figures.11: Broodstock (:300 ml each) i
icroalgae Density Stick (MDS) to
Live algal blend was premixed and pre - chilled
(15 ° ±1 °C) before fed to clams. The algal density was
measured by a microalgae density stick (designed and
s Filtered seawater and algal feed was chillers
prior to use with clam cultures
manufactured by Florida Aqua Farms Inc.) and maintained a density depth of fourteen to six centimeters
(14 -6 cm). The measured depth translated to algal cell density estimations of 3 to 18 million cells per
milliliter (3M to 18M cells /ml) respectively. Thirty liter (30 liter) capacity PVC tubs held up to five
hundred milliliters (500 ml) of stock. The small PVC mortar tubs measured 2'x 1%' x %' available at
hardware stores. The clams were retained on a 1/8" diamond patterned poly mesh rack supported by a
square frame made of %" PVC pipe (with 1/8" drilled submersion holes) secured around the edges with
plastic cable ties.
November 2014 NTB Macrofauna Final Report Page 10 of 37
In 2014 the clams were held in small groups (100 ml to 500 ml) which reduced the clams' mass
infestation of monogean trematodes. The total volume of broodstock held was dramatically reduced the
second season to a maximum of two liters of large clams, most with a length greater than eight
millimeters. There were only a few mortalities as compared to 2013. Thirty liters of clam stock died in
August 2013. Broodstock, growth stock, and extra stock
all became infected with monogean trematodes and died
when the temperatures rose to 30 °C multiple times in
August.
Individual upwell buckets or "miniupwells" (an
outdoor version of MacDonald fish egg hatching jar) held
small groups of stock. Each had an average continuous
flow rate of seven liters per minute (7 Ipm) supplying
oxygen and food and flushing wastes. Sediment mud and
large zooplankton such as crab larvae, sea star larvae,
and oyster spat came with the flow as well. All units were
adjusted daily and cleaned weekly at a minimum.
Figwe 13, Outdoor da ' iniupwells" flea} and Upwell
Backus (rear right) held small sets of broodstock
New broodstock collected anytime proved to be a reliable resource given their ability to ripen
quickly. A period (usually a day) of gradual temperature rise (6 °C to 8 °C) from their immediate holding
temperature to a constant spawn temperature (winter 19 °C, summer 23 °C, ±1 °C) cued the clams to
ripen gonad lobes and release gamete. The husbandry methods employed maintained healthy
broodstock with viable gamete when needed.
Spawning Preparation (Purge Conditioning)
Clams were taken from their holding cultures and thoroughly cleansed beginning with a fresh
water rinse. Next the clams were flushed with one micron ambient seawater for an hour to flush
internal debris and feces. A filtered seawater hose with twenty six liters per minute flow capacity (26
Ipm) flushed clams in a container; a tray or bucket with 20 to 30 liter capacity. Ebbed or pool areas
within the small high velocity environment enticed clams to open their shell to flush internal debris.
Clams readily opened, extending their siphons and feet. Seawater temperatures had to be monitored
during flush cycles to prevent (or collect) spawns of very ripe stock. Prolonged flushing of increasing
water temperatures of 6 °C to 8 °C occasionally induced a spawn without hydrogen peroxide.
November 2014 NTB Macrofauna Final Report Page 11 of 37
r ...1 Before spawning, clams required a cleansing cycle. After a fresh water rinse, clams were flushed multiple ways
(above) with 26 liters per minute flow (26 I ) axe ambient filtered seawater for up to one hour to remove internal clam
debris. High volatility environments enticed clams to open their shell to flush internal debris.
This unique hatchery approach was custom designed to entice the uncommon active
characteristics of the Coquina clam. Turbulent water movement and vibration stimulus enhanced their
shell opening and tactile mobility, verified by full extension of their siphons and active feet. Anecdotal
trials tested the clams' shell opening and siphon extension responses with different volumes of clam.
The opening time was one minute. One hundred milliliters to ten hundred milliliters (100 ml to 1000 ml)
of clam volume were observed for response as the clam volume was increased by one hundred milliliter
(100 ml). At one hundred milliliters (100 ml) the siphons extended partially with very little foot
extension as well; approximately half length. The peak extensions of siphons and foot were at a volume
of seven hundred milliliters (700 ml). Up to one liter of clam stock were placed in the MacJar with
positive results.
After flushing the stock for an hour, the broodstock was purged in a closed recirculating
"MacJar" set up for two to twelve hours prior to spawning with one micron (1µm) filtered seawater. The
initial seawater temperature was the same as their holding culture temperature. Purging allowed the
clams to acclimate to the desired spawn temperature, purge unwanted debris and excessive feces, and
mainly, allowed us to assess the monogean trematode problem within the stock. The exit water of the
MacDonald jar was sieved to test for monogean trematode infection of the brood stock. The gradual
temperature rise (6 °C to 8 °C) from their immediate holding temperature to a constant spawn
temperature engaged the clams to ripen gonad. When purge completed the clams were moved to a tray
or tank treated with hydrogen peroxide at fifty parts
per million (50 ppm H2O2). Purging provided a
healthier environment for larval fertilization and
initial growth.
When ambient seawater temperatures were
above 25 °C, clams were short cycled in a chilled
(15 ° ±1 °C) aerated tub for one week prior to
spawning preparation. When we were ready to
spawn the group, the clams were removed from the
cold water, rinsed with cold fresh water, and purged
for one day in a MacJar. The winter short cycle
November 2014 NTB Macrofauna
followed by a day of gradual temperature rise (6 °C to 8 °C) to a constant spawning temperature was
occasionally used with success in very hot summer months.
The "MacJar" set up utilized a 100 liter culture tank, a MacDonald fish egg hatching jar, a small
recirculating pump, and a piece of tubing. A plastic household utility sink was the 100 liter culture tank.
The MacDonald fish hatching jar (6 liter capacity) was a six inch diameter clear tube vessel eighteen
inches tall (18 "x6" dia.) with an over flow spout at the top and a one inch diameter (1" dia.) center
standpipe. A two foot piece of half inch internal diameter (1/2" I.D.) kink free tubing adapted to a small
variable flow fountain pump (155 -300 gallon per hour flow rate) pushed seawater from the culture tank
down the center stand pipe, past the clams in the bottom, and out the over flow spout of the
MacDonald hatching jar. Sixty liters (60L) of one micron filtered seawater recirculated to purge clams
without hydrogen peroxide or algae.
Stacked sieves filtered the exit water of the "MacJar ". A seventy -five micron sieve atop a twenty
micron sieve collected purged matter to assess the stock. The larger seventy -five micron (75µ) sieve
collected large debris and clam feces. The twenty micron (20µ) sieve collected monogean trematodes,
theirjuveniles, small debris, and clam eggs. A pipette was used to sample the sieve collections and load
a microscope slide for viewing using a compound microscope. The sieve collections were sampled
fifteen minutes after start up and random intervals r 15: The high velocity MacJar set up with stacked
after. The sieves were rinsed clean with fresh water sieges at the exit waters, recirculated 60 liters of filtered
after each sampling. Observational assessments were 23 °c seawater to Surge and acclimate clams for spawning.
.
made of the samples as the following points were considered: the amount of stock purging, the number
of clam eggs, the number of monogean trematodes found, the number of larval or juvenile trematodes
found, and the amount of any other debris collected. The purging continued with an end goal of no
monogean trematodes, most feces and debris purged, and clam activity increased (siphons extended
and active foot use).
When monogean trematodes were found in the sieve, the stock was divided to purge separately
to attempt to eliminate the infested stock. Until better sieve removal methods can be determined or
assessments of juvenile clam parasite treatments and effects can be made, infected larval cultures were
discarded. Our experience showed the monogean trematode reproduced exponentially faster than the
clam larvae grew. Sieve removal of monogean trematodes was ineffective. Elimination of infected stock
prior to spawning by sieve evidence (or lack of) was effective.
Spawning Methods
Basic temperature manipulation shellfish spawning methods were employed with the addition
of hydrogen peroxide for enhanced gamete release usually within three hours. Ripe stock was placed in
a conditioned (winter 19 ° ±1 °C, summer 23 ° ±1 °C) filtered seawater bath which triggered clams to release
gametes in the water column to fertilize. A heater was occasionally used with very cold large volumes of
water. Fifty parts per million of hydrogen peroxide (50 ppm H2O2) was added to the spawning culture to
enhance the spawn without ill effects to the larvae. More than fifty parts per million of hydrogen
peroxide deteriorated the egg wall and created abnormal larvae if fertilization occurred. The fecundity
November 2014 NTB Macrofauna Final Report Page 13 of 37
rate was not quantified due to many individuals, but not all, spawning at once. Also the fact that they
are" trickle spawners ", meaning they do not release all their gamete at one time.
Three different options (below) were successfully used to obtain spawns after flushing and
purge conditioning. The options were used individually and in combinations as the stock continued to
spawn. Broodstock was moved daily to another spawn tank with identical water parameters for up to
ten days to take full advantage of their spawning cycle. Gamete release increased with the second
spawn; thousands to millions of ova released without the use of hydrogen peroxide possibly due to the
longer constant temperature period.
Option 1 The high velocity Mac.lar set up was drained and refilled with sixty to eighty liters (60-
80 liters) of clean conditioned seawater and hydrogen peroxide at fifty parts per million (50 ppm) in a
utility sink. The stock was returned to the MacJar. Sieves collected eggs from the system (20µ sieve for
winter 60µ eggs, 35µ sieve for summer 70µ eggs). When the spawn subsided, the adults were relocated
to another tank with identical water parameters to continue spawning.
: A MacJar set up in a utility sink using 60 liters of filtered seawater before and after a spawn.
Option 2 Stock was moved to an airlifted recirculating conical tank (200 to 300 liters) on a
center rack. This provided a medium velocity environment, plenty of aeration, and added gamete mixing
resembling ocean turbulence. The tank was filled with conditioned seawater and hydrogen peroxide at
fifty parts per million (50 ppm). Larvae were reared in these tanks as well.
November 2014 NTB Macrofauna Final Report Page 14 of 37
lure 1 is A 300 liter conical tank was fitted to recirculate with an airlift and an optional internal rack to retain brood stock
for spawning. . The rack was removed for larval rearing. The airlift added extra oxygen and better~ mixing ability for gamete.
Option 3 Stock was moved to a static bath of conditioned seawater with fifty parts per million
of hydrogen peroxide (50 ppm). Various sized static baths were used. In small trays (30 liter) gamete was
collected from spawning adults. Eggs and sperm were mixed in a controlled environment and stocked to
a clean larval tank with a known stocking density and resulted with a very clean larval culture. This was a
preferred method.
lure 1 : A tray spawn allowed gamete to be removed and restocked to a clean larval culture
The larger aerated baths (200 to 2000 liters) were better for leaving stock overnight to spawn
and minimalized the amount of zygotes that were filtered fed. A very light feeding of Isochrysis sp. and
Choetocerus spp. were added to the bath. A six foot round tank of various depths (200 to 2000 liters)
and a utility sink (40 to 80 liters) were used successfully for spawning. The best results came from stock
started late afternoon and left overnight to spawn, usually in the morning as surmised by the larval size
and stage. The amount of gamete the stock consumed while filter feeding was never quantified.
November 2014 NTB Macrofauna Final Report Page 15 of 37
lure 1 . six foot round tank and a basket of stock (300 l), a six foot round tank with stock (100 l)
scattered on tank bottom, and a utility sink with scattered stack (50 l). All using variable amounts of
23 °1°1 °C filtered seawater with hydrogen peroxide at fifty parts per million (50 ); all positive spawns.
Literature stated that shellfish would spawn within three hours of using hydrogen peroxide.
(Method to Induce Spawning in Shellfish, U.S. Patent #4198929, Morse, 1980). Another uncommon
shellfish characteristic was taking up to twelve hours to release gamete with hydrogen peroxide. The
Coquina clams often spawned around daybreak, so as to instinctively not compete with their natural
environmental counterpart of the beach, the mole crab, Emerita talpoida, who spawned at dusk.
(Zeigler, T.A. and Forward Jr., R. A., Larval Release Rhythm of the Mole Crab Emerita talpoida (Say), Biol.
Bull. 209: 194 -203. December 2005.
Examples of Spawning Culture Vessels, Stock Used, and Capacities
1. The thirty liter trays were used with one hundred milliliters of stock yielding approximately
three million larvae (30Ltray, 100 ml stock, 3M larvae).
2. An eighty liter utility sink was used with one hundred to three hundred milliliters of stock
yielding approximately three million to ten million larvae. (80L sink, 100 -300 ml brood stock,
3M -10M larvae).
3. A two hundred and eighty liter recirculating conical tank was used with one hundred to five
hundred milliliters of brood stock yielding approximately three million to fifteen million
larvae. (280L tank, 100 -500 ml brood stock, 3M -15M larvae).
4. A six foot round tank (thirty six inches maximum depth) with three hundred to a eighteen
hundred liter seawater bath with one hundred milliliters to up to one liter of stock yielding
eight to eighteen million larvae. (1800L tank, 100 -1000 ml brood stock, 8M -18M larvae).
Gamete Handling
The gametes released by Coquina clams were not always visible by the eye. A twenty micron
collection sieve sample was checked using a compound microscope to confirm a negative spawn result.
Routinely, more ova were released than were fertilized. Each ovum had to be fertilized by one sperm
during the spawn cycle to create a viable zygote resulting in a future clam. For example, April 29, 2014,
eleven million ova were released. After two days the actual veliger larva created was four million.
November 2014 NTB Macrofauna Final Report Page 16 of 37
It was imperative to use conditioned seawater for all rinsing, handling, and restocking of
gamete and larval cultures. Conditioned seawater was similar, if not identical, to the original spawn
water; recently aerated 1 micron filtered (1µ) seawater tempered (winter 19 ° ±1 °C, summer 23 ° ±1 °C),
thirty five parts per thousand salinity (35 ppt), and 8.2 pH. Two methods were used to handle gamete
and fertilization: controlled mixing of egg and sperm, and natural fertilization in a large culture tank with
greater than two hundred liters of conditioned seawater. Both worked well in our experience.
Controlled mixing of gamete: the eggs were pipetted directly from
female releases in trays or rinsed from sieve collections from a MacJar set up to
another container to finish hydration, usually into ten liters of mildly aerated
conditioned seawater. Eggs had a seventy micron diameter (70µ dia.) during
spring season and sixty micron diameter (60µ dia.) in winter season.
Fertilization occurred soon after in a controlled environment where polyspermy
could be reduced by rinsing sperm from eggs. A sperm mixture was made with
a male to one hundred milliliters of conditioned seawater; separate mixtures
were made and used for diversity. The sperm activation time started five
minutes after seawater hydration and ended thirty minutes after activation.
Figure.....29: Polysperme egg
Sperm was mixed with hydrated eggs at a rate of one milliliter of sperm mixture to one million
eggs for one hour checking the eggs often for fertilization (polar body on egg) and polyspermy (excessive
sperms attached to each egg); more sperm was added if fertilization rate was low (few polar bodies
observed on eggs and eggs without sperm). This method eliminated feces and debris, and reduced
polyspermy; abnormal development of zygotes, half shell animals and /or rotating ciliated circular
< spots in the central body area that remained in that state indefinitely (Stephano
& Gould 1988, Clotteau & Dube 1993, Encena et al. 1998).
Anecdotal tests were performed with gamete and hydrogen peroxide
at three levels: zero, fifty, and one hundred parts per million (0, 50, and 100
ppm H2O2). At fifty parts per million (50 ppm), hydrogen peroxide had no
effect on larvae or gamete; visibly identical to zero parts per million (0 ppm).
At one hundred parts per million (100 ppm) of hydrogen peroxide, the egg
deteriorated (exterior wall disintegrated and internal yolk split into multiple
pieces), yet some fertilized zygotes that survived created malformed larvae
to polysperm abnormalities (ciliated spinning spheres).
hydrated fertilized eggs
Larviculture Methods
General shellfish husbandry methods were used to rear the spawns obtained. Each larval batch
was drained down every two days. Algal feed (Isochrysis sp. and Choetocerus spp.) was added after
twenty four hours of fertilization for larvae feeding. Larval batches were comingled weekly as they
matured to veliger stage after two days during their first culture restocking ( "drain down ").
November 2014 NTB Macrofauna Final Report Page 17 of 37
The health of the larvae were examined and counted volumetrically to plan for the immediate
restocking and feeding. The culture tank was drained through one or more sieves to retain the larvae,
remove any debris, and rinse away wastes. The sieve collections of larvae were rinsed into a bucket of
known volume, usually fifteen liters, of clean conditioned seawater. After thorough mixing, three one
milliliter larval samples were collected. At this time the larvae was evaluated for general health and
growth progress, measured with an ocular micrometer, photographed, and counted to obtain an
average estimate of the total larvae. The tank was completely cleaned with chlorinated freshwater, de-
chlorinated, and refilled with conditioned seawater similar to their original culture (temperature and
salinity) and filled with adequate algae supplies for another two days of growth. The counted culture
was then added to the fresh culture tank. The conditioned seawater for large cultures were prepared a
day before when temperature and or salinity needed adjustments. Algal supplies were collected the
same day as larval "drain downs ".
Clam larvae remain free swimming in the water column of the culture tank for approximately
three weeks when they begin to use their newly developed foot. At this point they swim less and "set"
at the bottom. During a routine drain down, the large clams with a foot are collected and restocked to a
downwell. The floating container has mesh at the bottom to hold the "setting" clams off the bottom of
the culture tank. Tank culture water was
r : Floating daxwna ell with 100p mesh for clams to "sets
past this point.
This was where our main problem of the
project occurred in the larval process. The larvae were
put in a downwell on their twenty fourth (24) day, but
their algae feeding quality history was poor. The most
larvae had developed a "wooly mouth" from the
mycelial fungus in all of our algal cultures which
seemed to flourish after a roof leak February 8, 2014.
All algal cultures had to be sieved with a twenty
micron (20µ) sieve before feeding, and we lacked a
airlifted into the container at the top
providing dissolved oxygen and algal food and
flushed wastes out the bottom mesh while
retaining the clams. A one hundred micron
(100µ) mesh was the first downwell size. The
clams must also be large enough to be
retained by a 100µ or larger mesh. Clams were
able to set or swim while they transitioned to
the next biological phase. High mortalities
during this transitional phase are common in
shellfish larviculture. Excellent nutritional
quality was required for the larvae to develop
r : Day 19 larvae and vorticelia protozoan
November 2014 NTB Macrofauna Final Report Page 18 of 37
green algae needed for a balanced nutritional diet. Our algal feeding supplies consisted of brown
unicellular and diatom algae; Isochrysis sp., Pavlova sp., Chaetocerus sp., and Thalassiosira sp.. The latter
two species were diatoms. Thalassiosira sp. was generally used for broodstock feeding. Isochrysis sp.
was a small larvae food (first week food) and "background filler" food and had a protozoan problem
(Vorticella sp.).
Figure 25: Day 29 larvae and mycelial ranges in algae
Pavlova sp. was a good algal feed for the time but
it also had mycelial fungus. The Pavlova sp. helped
swimming larvae to "set" within one day of feeding. The
clams were moved to a downwell one day after being fed
Pavlova sp. despite the lack of foot use. The larvae set,
but did not visibly use their foot to move. The missing
algal food was Tetraselmis spp. and /or Dunaliella sp. One
carboy of Dunaliella sp. was produced and half was used
on the larvae's thirty fourth (34th) day routine drain down
and restock. The larvae made a dramatic change
overnight; they were actively moving using their foot to a
full extension. This was a clear example of the
importance of proper nutrition (pictured below).
w .. : Day 33 larvae, visible foot not used
The first chart was used as a quick
r is Day 34 larvae, foot actively used
reference guide for tank stocking at various densities. These charts are for shellfish in general and were
referenced every two days as a check list and guide. The Coquina clams had a slower starting
development rate than other shellfish in the summer and winter months. The second chart was used to
manage larval cultures; their "drain down" sieve sizes, estimated growth rates, restocking densities, and
algal food species and amount recommendations. The Donax larvae set from January 23, 2014 was
included in the chart for comparison and future reference.
Larval Capacity Chart for Tanks Used at CCC
November 2014 NTB Macrofauna Final Report Page 19 of 37
Culture
Tanks
Water Capacity
(liters)
Desired Larval
Restocking Density
Recommended
Recommended
4 /ml
6 /ml
8 /ml
Age
Hatchery Manual
Hatchery Manual
Bucket
17.5 liters
70K
105K
140K
Suggested
15 liters
60K
90K
120K
Larval
Larval
Density
Utility Sink
80 liters
320K
480K
640K
Stage
60 liters
240K
360K
480K
(cells /ml)
50 liters
200K
300K
400K
40 liters
160K
240K
320K
Spawn
egg
50it -70it
8 -10 /ml
20it / 35it
Recirculating Conical
300 liters
1.21VI
1.81VI
2.41VI
280 liters
1.12M
1.71VI
2.21VI
(70�t summer)
250 liters
1M
1.51VI
2M
6' Round Tank
coquina are slow
Tank Depth -33"
2200 liters
8.81VI
13.21VI
17.61VI
TD -30"
2000 liters
8M
12M
16M
TD -27"
1800 liters
7.21VI
10.81VI
14.41VI
TD -23"
1500 liters
6M
9M
12M
TD -18"
1200 liters
4.81VI
7.21VI
9.61VI
TD -15"
1000 liters
4M
6M
8M
TD -12"
800 liters
3.21VI
4.81VI
6.41VI
TD -9"
700 liters
2.81VI
4.21VI
5.61VI
TD -7"
500 liters
2M
3M
4M
TD -6"
400 liters
1.61VI
2.41VI
3.21VI
TD -4.5"
300 liters
1.21VI
1.81VI
2.41VI
TD -3"
200 liters
800K
1.21VI
1.61VI
Chart used to manage larval restocking densities
Hatchery Management Chart for Larval Husbandry
and Donax variabilis winter spawn growth comparison (190 ±1oC)
November 2014 NTB Macrofauna Final Report Page 20 of 37
CCC Oyster
CCC Oyster
Recommended
Recommended
Recommended
Estimated
Actual Winter
Age
Hatchery Manual
Hatchery Manual
(NOAH Milford, CT
Algae Lab; 11/2014)
Spawn 1/23/2014
Suggested
Suggested
Stocking
Catch
Algal
Larval
Larval
Density
Sieve(s)
Density
Donax Size
Stage
Length
(larvae /ml)
Algal Feed
(cells /ml)
(LxwxH)
(microns)
(Species)
Spawn
egg
50it -70it
8 -10 /ml
20it / 35it
60�t winter -
(70�t summer)
1 Day
trocophore
50it -70it
no sieving trocophores! And no
coquina are slow
November 2014 NTB Macrofauna Final Report Page 20 of 37
lure Chart used to manage larval cultures
A Narrative of the Coquina Clam Larval Spawn and Rearing (January 23, 20141
Our best larval rearing achievement to date is a pediveliger of thirty -seven (37) days old,
surpassing our previous record of 15 days in 2013. On January 23, 2014 a total of three million (3
million) larvae were released by three sets of brood stock. One set of five hundred milliliters (500 ml)
was left overnight in the recirculating conical tank with two hundred and eighty liters (280L) of
conditioned seawater, and two sets of two hundred and fifty milliliters (250 ml) of broodstock were left
overnight in a utility sink with eighty liters (80L) of conditioned seawater. Hydrogen peroxide was not
used for any of the spawns; only steady temperature increase to 190±10 and overnight patience. The
spawn waters were checked with a twenty micron (20lt) dip sieve the morning of January 24, 2014. All
November 2014 NTB Macrofauna Final Report Page 21 of 37
8 /ml
food required; prep for D stage
to develop from
zygote to D -stage
D stage
50it -70l t
20it / 35it
Isochrysis spp.
10K
(some took up to 2 days)
Chaetocerus spp.
2Day
veliger
60it -80it
8 /ml
20µ/35µ
Isochrysis spp.
15K
95x70x55
Chaetocerus spp.
4 Day
veliger
90µ -110µ
4 /ml
Isochrysis spp.
20K
100x90x60
Tetraselmis spp.
Chaetocerus spp.
6 Day
veliger
120µ -140µ
4 /ml
20it/ 35µ
Isochrysis spp.
25K
110x85x60
Tetraselmis spp.
Chaetocerus spp.
8 Day
veliger
130µ -150µ
4 /ml
20it/ 35µ
Isochrysis spp.
25K
110x85x60
Tetraselmis spp.
internal foot
Chaetocerus spp.
developing
10
veliger
140µ -160µ
4 /ml
35µ
Isochrysis spp.
30K
110x85x60
Day
Tetraselmis spp.
Chaetocerus spp.
12
veliger
150µ -170µ
4 /ml
35µ
Pavlova asp.
30K
115x90x60
Day
Tetraselmis spp.
Chaetocerus spp.
14
veliger
180µ -200µ
3 /ml
35µ/55µ
Pavlova asp.
40K
120x100x60
Day
Tetraselmis spp.
150x140x60
Chaetocerus spp.
internal foot
visible
16
veliger
220µ -240µ
3 /ml
35µ/55µ
Pavlova asp.
40K
120x100x60
Day
Tetraselmis spp.
160x130x65
Chaetocerus spp.
18
veliger
240µ -260µ
2 /ml
35µ/55µ
Pavlova asp.
40K
110x90x60
Day
Tetraselmis spp.
140x120x80
Chaetocerus spp.
180x155x120
20
veliger
280µ -300µ
2 /ml
35µ/55µ
Tetraselmis spp.
50K
105x85x60
Day
Chaetocerus spp.
175x125x100
Thallassiosira spp
225x175x125
22
pediveliger
300µ -320µ
1 /ml
35µ/55µ
Tetraselmis spp
50K
125x100x70
Day
Chaetocerus spp.
150x125x110
Thallassiosira spp
250x210x120
24
pediveliger
320µ -360µ
1 /ml
105µ
Tetraselmis spp
60K
125x100x80
Da y
105µ catch
75µ/35µ
Chaetocerus spp.
200x160x100
restocked into 1-
100µ downwel l
Thallassiosira spp
230x 190x
26
pediveliger
380µ -400µ
1 /ml
105µ
Tetraselmis spp
80K
120x100x
Da y
1051t catch
75µ/551`1
Chaetocerus spp.
140x120x
restocked into 2_
1001t downwells
Thallassiosira spp
290x250x 150
lure Chart used to manage larval cultures
A Narrative of the Coquina Clam Larval Spawn and Rearing (January 23, 20141
Our best larval rearing achievement to date is a pediveliger of thirty -seven (37) days old,
surpassing our previous record of 15 days in 2013. On January 23, 2014 a total of three million (3
million) larvae were released by three sets of brood stock. One set of five hundred milliliters (500 ml)
was left overnight in the recirculating conical tank with two hundred and eighty liters (280L) of
conditioned seawater, and two sets of two hundred and fifty milliliters (250 ml) of broodstock were left
overnight in a utility sink with eighty liters (80L) of conditioned seawater. Hydrogen peroxide was not
used for any of the spawns; only steady temperature increase to 190±10 and overnight patience. The
spawn waters were checked with a twenty micron (20lt) dip sieve the morning of January 24, 2014. All
November 2014 NTB Macrofauna Final Report Page 21 of 37
tanks showed evidence of spawning (zygotes). The broodstock was relocated to another tank with
similar water parameters to continue spawning.
The three sets of larvae were allowed to mature for at least 24 hours to reach the "D- stage"
before their culture water was sieved for total larval evaluation and restocking. The progression from
fertilized egg to "D- stage" larvae took much longer than normal shellfish progression (an estimated
additional twelve hours) possibly due to the lower temperature (19 °C) of the culture water. The egg size
was sixty microns (60µ); smaller than seventy micron (70µ) spring spawns for the same species.
Figure M Clay 1
Egg Fertilization Cycle
Polar body (exterior burp at 10 o'clock)
Completed first full cellular cleavage
Beginning of cellular cleavage
Figure 32: Day 2, late trot xphore Figure ::: Day 2, "D" stage veliger
November 2014 NTB Macrofauna Final Report Page 22 of 37
lure : Day 3 - vel iger
Every two days each larval culture tank was evaluated, cleaned, and restocked. The tank was
completely drained down using seives to retain the larvae. At this time the larvae were sized, counted,
and observed for general health conditions and development. The tank was thoroughly scrubbed with
chlorinated freshwater, de- chlorinated with sodium thiosulfate, saltwater rinsed, and refilled with
conditioned temperature (19 °C) one micron (1µ) filtered seawater and an adequate algae supply for two
days. As the larvae grew the the stocking density was decreased and the algal concentrations were
increased and varied to supply adaquate nutrition. A mixture of brown algae and diatom algae
(Isochrysis spp. and Chaetocerus spp.) were blended evenly as available to feed. The swimming veliger
stage larvae looked the same every two days except their size increased. Although not used, foot
development was visible at day seven.
sire Day 7 veliger
figure ; Day t, internal foot development
For the first seven days the larvae were stocked at a density of 3 -4 larvae per milliliter of culture
water resulting in a culture tank of 900 liters. By the seventh day the total larvae count was two and a
half million. A 35 micron sieve was used retain the larvae when the culture tank was drained until day 23
because the larvae was varied in size (250 microns to 120 microns in length). A twenty to forty liter
blend of dense (average of 165 million cells per milliliter) live cultured algae was used to feed the larvae
when restocked every two days. Equal amounts of Isochrysis spp. or Pavlova sp. and Thalassiosira spp.
or Chaetocerus spp. were used. Nannochloropsis sp. was not used this year for the larvae. Dunaliaella sp.
was introduced at Day 33 which greatly changed their foot use activity. Dunaliella sp. or Tetraselmis sp.
should be introduced sooner and continued throughout the larval rearing.
November 2014 NTB Macrofauna Final Report Page 23 of 37
At day 23 half of the larvae was retained on a 105 micron seive and moved to a 100 micron
downwell vessel within the general larviculture tank. The remaining larvae were retained on a 55 micron
seive wherethey were returned to the culture water of the tank to swim freely or set at the bottom of
the tank. An estimate of 840,000 larvae remained at a stocking density of 1 larvae per milliliter.
Although the larger larvae had developed a foot, they did not appear to use it. A 900 liter tank minimum
was used to be able to float the downwell vessels within the tank.
lure Vii: Day 23 Pedivefi ers and vefigers
sire 085:. Day 29, 400,000 hrvae sir .... Day 0: , 100,000 pedivefi er
November 2014 NTB Macrofauna Final Report Page 24 of 37
On Day 34 many physiological features had changed overnight. The only culture method change
was the addition of ten liters (10 L) of cultured Dunaliella sp., a green algae, was added to their diet of
Isochrysis spp. and Chaetocerus spp. blend on the previous day. An abrupt visible change of foot
utilization occurred. The pediveligers were actively using their foot for movement. The exit siphon, now
visible, was as an extension of the foot appendage. The developing mantle tissue displayed rib like
structure with constant flowing cilia movement resembling gills. This was not their regular day for a
water exchange and the downwell vessels were free flowing and floating at five o'clock.
r Q; Day 34 (. ) Active foot pediveli ers
s re_41 Day : 6.• Post-set juvenile with characteristic foot extended
r _42 . Day 34, mantle cilia visible
November 2014 NTB Macrofauna Final Report Page 25 of 37
sir _ ; : Day 36, daxwna, eller sank to bottorn & flowed out tans hales
On Day 36 (9 am) the downwell vessels had ominously sunk to the tank bottom. The airlifted
culture water into the downwells was flowing out of the top. Normally, the airlifted culture water exited
the bottom mesh past the larvae with algal food and adequate levels of dissolved oxygen. The entire
twenty two inch diameter (22" dia.) one hundred micron mesh bottom of both downwells had clogged
with debris and dead larvae. The reason for the sudden mass larviculture mortality incident, otherwise
known as a "crash ", was unknown. All of the culture water was sieved during the routine "drain down ",
including the extra saltwater rinses of the tank, to retrieve any live larvae. Twenty thousand (20,000)
larvae, small pediveligers and post -set juveniles, were rescued. A stocking density of one larvae per
milliliter was maintained when the larvae were restocked into an aerated five gallon bucket.
The larviculture data and photographs were reviewed to deduce theories for the devastating
mass mortality incident. The "crash" was probably the result of compounded negative culture issues.
1. A larval nutritional deficiency at the critical morphological stage of pediveliger. A
sufficient algal diversity, such as the addition of Dunaliella sp. or Tetraselmis sp., was
not provided to the larvae through out their rearing resulting in an overall larval
nutritional deficiency at a critical stage.
2. Algae containing unwanted protozoans and debris, such as Vorticella, was
unknowingly added directly to downwells.
3. Adequate amounts dissolved oxygen could not reach the surviving larvae when the
downwells sunk to the tank bottom and normal downwell flow was stopped.
Day 37 - 100% mortality: No live larvae were found during the normal culture water exchange.
November 2014 NTB Macrofauna Final Report Page 26 of 37
Larval Husbandry of the Mole Crab (Emerita talpoida)
Gravid female mole crabs (10) were colleced to test their spawning and potential rearing
conditions by following a journal article. (Zeigler, T.A. and Forward Jr., R. A., Larval Release Rhythm of the Mole
Crab Emerita talpoida (Say), Biol. Bull. 209: 194 -203. December 2005.) On October 7, 2013 the gravid females
were placed in an aerated 20 liter bucket filled with 15 liters of one micron (1µ) filtered seawater of
ambient outdoor temperature (24 °C). The next morning larval mole crabs were found in the same
manor larval clams were found. The larval mole crabs were relocated to another bucket by using a 20
micron seive to collect the larvae. The larvae was fed 2 liters of blended algae (Isochrysis spp. and
Chaetocerus spp.) and artemia. Rotifers were not available at the time. The larvae was evaluated,
drained, and restarted every two days. After four weeks approximately 1,000 larvae had reached the
megalopae stage. Below are their observed life cycle pictures.
_ :Fertilized and unfertilized eggs of a gravid female
November 2014 NTB Macrofauna Final Report Page 27 of 37
s r _4 Day 1 of a hatched mole crab
s r _46 One week old mole crab larvae displaying phototropic behavior
jjg,VK,p,,,,,47: Two week old mole crabs
November 2014 NTB Macrofauna Final Report Page 28 of 37
Figure.....48: three- week -old mole crab atop a 400-micron siege
Figure.....49: Four week old mole crab atop an 800 micron siege with tightly tucked tail.
figure BCD: At four weeks old megalopae had reached 1.2 millimeters.
November 2014 NTB Macrofauna Final Report Page 29 of 37
Larval Chart for January 23, 2014 Spawn (37 day duration)
Spawn Date(s):
January
23, 2014
Standard Larval Calculations required
per Drain Down (every 2days)
Broodstock File: Spawn Log 201
1/22
MacJar /Brood
partial egg
release
Catch
x
Avg.
=
Total
/
Desired
=
Restock
Larval Data
File: DLO1232014
S pm
Brood to
Conical on rack
Volume
Count
Larvae
Density
Volume
1/23
Conical Main Spawn Overnight
(ml)
( # /ml)
( #)
( # /ml)
(ml)
RC= Recirculating Conical
tank
11 am removed
brood
6' =6' Round
Tank with
3airstones & heater
1/24
Added
2 Black
tub spawns of 1/23 also
culture
seawater
1u aged
2 weeks, heated to19C,
aerated
Date
Age
Culture Water
Larvae Size
Health
Drain Down
Catch
Counts per
ml
Total
Restock Density
Temp C/
Feed & Density (20u sieved)
TempC
Salinity
Sieve
Catch
1/2/3
Avg
# /ml
I Vol (ml)
Type
dipstck(cm)
Amt(L)
Days
LxWxH(um)
Life Stage
Volume
Larvae
Salinity
1/22
20
35
60u
egg
20
na
na
20K
0
28OK -RC
na
Tiso / Chaet
na
15L
1/23
0
18
35
60 -70u
blastulas
20dip
na
na
na
na
na
na
28OK -RC
na
na
5pm
0
20
35
trocophore
20dip
na
na
na
na
na
na
28OK -RC
na
na
1/24
RC
19
35
95x70x55
trocs /D's
20
larvae
280K
4
1120K
Tubl
18
35
95x70x56
trocs /D's
20
larvae
5K ml
189
945K
Tub2
18
35
95x70x57
trocs /D's
20
larvae
5K ml
175
875K
2.94M
1
105
trash
Total
10 /ml
290L -RC
19/35
Tiso / Chaet
8
30L
1/25
2
19
35
100x80x60
veliger
1/26
3
19.5
35
100x90x60
105
trash
brn belly
20
veliger
20K ml
128/161/116
135
23M
9 /ml
290L -RC
19/35
Tiso / Chaet
8
30L
1/28
5
18.5
35
110x85x60
brn belly
105
20
veliger
20K ml
142/215/178
178
3.56M
4 /ml
840L- 6'x14'
19/35
Tiso / Chaet
9
40L
1/30
7
19
35
110x85x60
brn belly
35
veliger
20L
110/135/122
122
2.45M
4 /ml
860L -6'
19/35
Tiso / Chaet
9
60L
active
2/1
9
19
35
110x85x60
brn belly
35
veliger
20L
1.1M
4 /ml
860L -6'
19/35
Tiso / Chaet
25.51V
60L
active
cells /ml
2/3
11
19
35
115x90x6O
brn belly
35
veliger
20L
1.8M
4 /ml
290L -RC
19/35
Tiso / Chaet
25.51V
40L
active
cells /ml
2/5
13
19
35
125x100x60
brn belly
35
veliger
15L
82/111/94
96/ml
1.44M
3 /ml
500L -6'
19/35
Tiso / Chaet
30M
15L
150x140x90
active
20
trash
add airstone
cells /ml
2/7
15
19
35
120x100x60
foot
35
veliger
15L
101/79/57
79/ml
1.2M
3 /ml
500L -6'
19/35
Tiso / Chaet
50L
160x130x65
rinse tank bottom into
seive
seived
2/9
17
19
35
180x155x120
pedi's
pedi's
15L
48/45/66
53/ml
795K
2 /ml
600L -6'
19/35
Tiso / Chaet
30M
50L
140x120x80
on
35
n
cells /ml
seived
110x90x60
bottom
veliger
2/11
19
19
35
225x175x125
pedi's
15L
41/36/36
38/ml
570K
2 /ml
290L -RC
20/35
Tiso
7
10L
175x125x100
35
n
Chaet
15
10L
105x85x60
veliger
3H
9
10L
Pavlova
10
10L
2/13
21
19
35
250x210x120
no foot use
55
pedi
3L
221/230/245
232/ml
348K
1 /ml
290L -RC
20/35
Dunaliella
14
10L
150x125x110
full bellies
35
mixed
ChaetC
10
10L
125x100x70
35
veliger
Pavlova
10
10L
mycelial fungus
"wooly mouth"
3H
9
20L
2/15
23
19
35
230x190
same
105
pedi
15L
50/42/48
47/ml
705K
1dw
1000L -6'
19/35
Dunaliella
14
10L
200x160x100
75
pedi
15L
14/5/8
9 /ml
135K
1 /ml
ChaetC
10
15L
_______________125x
100x80________
35__veli�er_
3L ___
21212
2�m1
6K
_______________
Pav lova�__
10___
20L
mycelial fungus
"wooly mouth"
total
846K
3H
9
20L
ChaetB
9
20L
2/17
25
19
35
290x250x150
same
75
pedi
20L
26/29/44
33/ml
660K
2dw
1000L -6'
19/35
3H
9
20L
140x120
veliger
330K
/dw
ChaetB
8
20L
120x100
mycelial fungus
"wooly mouth"
<1 /ml
Pavlova
12
20L
2/19
27
19
35
290x160
same
800
trash
50/42/49
47/ml
705K
2dw
1000L -6'
19/35
3H
9
20L
280x240
75
pedi
15L
352K
/dw
ChaetB
8
20L
260x170
55
veliger
<1 /ml
Pavlova
12
20L
35
trash
2/21
29
19
35
220x210
foot Pic
800
trash
2dw
1000L -6'
19/35
CC
9
30L
140x120
still not usi
75
larvae
15L
28/26/24
26/ml
390K
Tiso
14
30L
mycelial fungus
"wooly
mouth"
195K
/dw
Pavlova
18
30L
2/23
31
19
35
230x210x200
same
800
trash
1000L -6'
19/35
CC
9
30L
300x255
150
pedi
10L
9/10/2011
10 /ml
100K
2dw
Tiso(seived
14
30L
75
mixed
14L
9/21/2016
15/ml
210K
Pavlova
18
30L
mycelial fungus
"wooly
mouth"
310K
2/25
33
19
35
355x300/105dw
foot
105dw
pedi
2L
16/34/20
23/ml
46K
1000L -6'
CC
9
30L
200x160/100dw
"ribs"
100dw
pedi
2L
24/26/11
20 /ml
40K
2dw
19/35
iiso(seived
14
30L
150x130
50% dead
75 tank
pedi
1L
1/1/1
1 /ml
10K
Pavlova
18
30L
mycelial fungus
"wooly
mouth"
96K
Dunaliella
13
20L
2/26
34
330x300
active foot
use! /full
pediveligers /pictures
only- no
drain down
2/27
35
19
35
DWs
on bottom
400
trash
20L (bucket)
Dunaliella
13
4L
Tiso bugs n
150
pedi n
2L
5/17/2009
10 /ml
20K
1 /ml
19/35
CB
9
2L
mycelial
fungus "wooly mouth"
100
dead
3H
14
1L
most dead
75
trash
3/1
37
19
35
100% Mortality
400
trash
200
dead
150
dead
100
trash
November 2014 NTB Macrofauna Final Report Page 30 of 37
CCC Upwell Temps 02262013 10232014.xis -Bogue Sound Temperatures (Morehead City, NC)
November 2014
Values
Year
Month
Week
Monthly
Weekly
Bogue Sound
Ave rage Temperature
Data
2014
January
8.7
Average Temperatures in Celsius
1 -7
1
10.2
2/26/13 through
10/28/14
5 -14
2
9.4
Carteret Community Col lege,
Morehead City, NC
15 -21
3
10.2
22 -31
4
6.2
2014
February
8.5
Year
Month
Week
Monthly
Weekly
1 -7
1
7.5
Start Hobo
8 -14
2
6.9
2013
February
15 -21
3
8.8
26 -28
4
11.7
22 -28
4
10.9
2013
March
11.6
2014
March
11
1 -7
1
10.7
1 -7
1
8.5
8 -14
2
11.8
8 -14
2
10.7
15 -21
3
13.2
15 -21
3
11.2
22 -31
4
11.4
22 -31
4
12.7
2013
April
17.1
2014
April
17.2
1 -7
1
13.0
1 -7
1
15.8
8 -14
2
18.4
8 -14
2
17.9
15 -21
3
19.8
15 -21
3
16
22 -30
4
17.2
22 -30
4
18.5
2013
May
21.5
2014
May
23.2
1 -7
1
18.1
1 -7
1
21.5
8 -14
2
21.1
8 -14
2
23.8
15 -21
3
22.1
82.6
15 -21
3
23.3
22 -31
4
23.6
81.9
22 -31
4
23.9
2013
June
26.5
82.5
2014
June
26.8
1 -7
1
25.5
83.7
1 -7
1
24.6
8 -14
2
26.8
82.5
8 -14
2
26.5
15 -21
3
25.6
80.9
15 -21
3
27.6
22 -30
4
26.8
81.2
22 -30
4
28.2
2013
July
28.1
83.4
2014
July
27.8
1 -7
1
27.7
78.6
1 -7
1
27.7
8 -14
2
28.1
80.5
8 -14
2
28.4
15 -21
3
28.7
78
15 -21
3
27.1
22 -31
4
28.1
82.3
22 -31
4
28
2013
August
27.2
81.2
2014
August
27.3
1 -7
1
27.3
76.9
1 -7
1
26.3
8 -14
2
28.6
73.2
8 -14
2
27.2
15 -21
3
25.9
70.2
15 -21
3
27.9
22 -31
4
26.9
75.1
22 -31
4
27.6
2013
September
25.6
71.2
2014
September
26.3
1 -7
1
28.0
71.3
1 -7
1
29.1
8 -14
2
27.3
65.2
8 -14
2
27.4
15 -21
3
24.9
51.3
15 -21
3
25.5
22 -30
4
22.9
51.8
22 -30
4
23.7
2013
October
21.2
47.3
2014
October
22.4
1 -7
1
23.9
51.3
1 -7
1
23.2
8 -14
2
21.8
52.2
8 -14
2
23.7
15 -21
3
21.8
54.9
15 -21
3
21.3
22 -31
4
18.5
62.9
22 -28
4
18.6
2013
November
15.1
60.4
1 -7
1
18.4
64.2
End Data
8 -14
2
15.0
60.7
15 -21
3
15.1
65.3
22 -30
4
12.8
2013
December
12.5
1 -7
1
12.8
8 -14
2
12.2
15 -21
3
12.0
22 -31
4
12.8
NTB Macrofauna
Final Report
Page 31 of 37
Growth Stock Overview
A growth study was attempted for one year. The initial measurements of July 2013 yielded a
clam size of 3.3 clams per milliliter (3.3 clams /ml) with an average length of 12.3 mm. After one year the
clams yielded a clams size of 1 clam per milliliter (1 clam /ml) with an average length of 15.1 mm. Heavy
sedimentation and over spat of clams by other larval animals (barnacles, oysters, sea stars, etc.) yielded
the trial data a general loss. Unfiltered seawater was used to feed the upwell system where they were
kept. Photographs below show the clam growth lips, macro algae colonization on clams, oyster over
spat, and general discoloration of long term held stock.
Figure 5
Original growth stack (left) and Oyster over spat stack one year later (right)
November 2014 NTB Macrofauna Final Report Page 32 of 37
Sample AVera¢es
Mods Total Vol Subset Vol measured
MUIGSS Files
Length Width Height (4
clams)
(ml)
(ml) (4 clams) clams
/ml
72013
12.3
6.9
4.9
100D
30
10D
3.3
7112013
12.3
6.9
4.9
62
209
3.4
5032013
11.7
6.5
4.6
79
20
5112013
12.6
7.1
5.0
227
20
5212013
12.9
7.2
5.1
287
20
9142013
13.7
7.4
5.2
224
20
102013
13.7
7.6
5.5
30
10D
3.3
10012013
950
50
140
2.8
10242013
13.5
7.4
5.3
1012
63.0
143
2.3
112013
13.7
7.7
5.5
40.0
10D
2.5
122013
14.1
7.9
5.8
20.0
78
3.9
214
13.9
7.0
5.6
80.0
10D
1.3
308
14.1
7.9
5.8
650.0
80.0
0.0
401
14.1
7.9
5.8
50.0
10D
2.0
410
14.2
7.9
5.7
60D
20.0
48
2.4
514
14.9
8.3
6.0
30.0
73
2.4
521
13.0
7.0
4.8
10.0
24
2.4
527
14.7
8.0
6.0
650.0
20.0
48
2.4
529
17.5
9.7
6.9
80.0
10D
1.3
609
14.3
8.1
5.9
40.0
8J
2.0
610
17.5
9.0
6.9
80.0
10D
1.3
612
14.2
8
5.5
70D
70
10D
1.4
617
14.3
8.1
5.8
650.0
60.0
50
0.8
707
14.2
8.2
5.9
500.0
60.0
59
1.0
711
14.5
8.3
5.8
475.0
70.0
10D
1.4
8J6
14.8
8.5
6.6
550.0
100.0
10D
1.0
813 spatted
15.2
8.8
6.2
475.0
250.0
41
0.2
813
15.1
8.5
6.2
475.0
100.0
Teo
1.0
November 2014 NTB Macrofauna Final Report Page 32 of 37
figure 5
Growth set data compiled for one year
New (lower) and one year held stock (upper)
Macro algae grows at the base of clam (center)
figure 5
Donax spawn log 2013
November 2014 NTB Macrofauna Final Report Page 33 of 37
2
SO
32
E
7 7
�C r" T
4 A,
k 0 0 Y.e G�
V
62 R R 2, 2 2 nay.,
6
,F. r
6 4 'ri
flgure,55: Donax spawn log January through March 2014
2 2
November 2014 NTB Macrofauna Final Report Page 34 of 37
'IXA,
M, "I R 14 "Al
?
d, 'A 'h y
November 2014 NTB Macrofauna Final Report Page 34 of 37
haomi"s
Ah;prt.n wttham.he I rnt-n, Mllne,ed eel - natal. c.inperaujuss a.4 s.
AT RisingTempro-ature
RU regular uppyvAl burket FMT - Ectmotso-d Hold TImo
FT pitifing remperaturc,
ML$ cram ordarth 4 , I neiniti
Static
KS-Nwoerr %psnamadl 2 2,4 weeks
Sinka Anvatec-Staij, Whit
PS- isr"joudy spawned 0 weak ";
" - T u, Roper name she, Is
1111111111"(01 tanetip created
PAT-Pxptkbe
exte %s uwee Acdencruo to OfoidW Temperature. and Treat for fluats wath SO pp hcerrogen perorxedir 0 mIJ20 lfhssJ
4202-hydrusten panuadde added to spawn cochrre at 50 pout for 6nducing a tparwtt J5SO ppro deterZrdrits eggs)
air how, 2 uperell Bucket tRC13S pOLIJ
1u has., 40. to Sty"g, L, wo 1, tays rinuad wrclu hosin for 15 nwn.rconoving chair' dkbu
rWre
split Off c"Alection, Stotts as Rolows
200 ml mog', Brient
Iratik, here lot I u For I to 18C no 101 truillfty %ird:
Done
a5ft oil "amm 12 udst
Str6r, bath PW 1u Ap I far - JW1 no Lot Wark "norhan, tub
none
ait 3 set%
re.mp shook,, MY" to 20C atfort bath desegact stabse of isse tub%
firsno
M mi mw'sl amrs
:al Brit, bahr firmynke MINE white buckat #of 9,0 minutes
none
200 m I rnsnk , grerp
Stark trap1h, mlovaho 201)206.' black mo par tub
none
MCI ord 'Arnm slack bontrilmed 2 setsp
Madar, sarth 40LJ20r, to 24('. lipurnia 12 hr's: ?Su & Mat supies collected many 'fluiurs' ordy
holse
1Y.K.1 rol 48mm vlock,
Sranc chair; relocate 2%J20C blank Tiortar tub
none
ad 1 cram
it 202 added IT BW added to Wh up I nal W202 hatio ME tea 3 wbofaaak 2 Art
nbe"
45o rut r8oam qa,,k ijph1I again 2 wl
6,Mttd-9AIJk (W-19 2KA LID& 2 an fat oil blat k I u fis wefe up each 4SO rpt sk. c k, :t I iso/Chaut, 122 I L,
none
200 mi mosi , tivtru,
Manua, With 40re oul iu iSrr,20u fieves Eoffect duke, otflwmecocV4S found an Maciap
1r-,y 10
20C, mdmost > 8rrrMTk
R , r S prru stock Dr rack in conical -overnight 15L Tao,,Chrao rab
1 /21
"'00 ml most , grem
CivarnvgM releaut, at nC r) ain, Mn dip sawe'lourml blaslurrs5� took 29C/drsft, rannoved! adults
49) u'l '8r'm ,'MO
Ovy,,nrailri rW.- .1 20C 9 -, 20. dip bwft f-rid bastiroat: tank 12C)dwsafto r.unred Adults
low
200 crN1 mom x ftnten
Minted learn [151, fu loC, A Itsmirl 11-1, Pop: black rviortarvuh eft fixtho day jamerent pro hArep 2,O(,]
nor"tr
450 cot rRrnrn, pock, (2 W.%)
Amated Both DSI Itt IN dL Tnto/V A 1 5 jam: black prodexub left forthetfiry jamb ant rm leum 2OCI
p W, P.
2W ror most a artwo
torrid Shook; 1I C Peft overnight to Tt 5 am; tended black'. morea, Tob,:. 15J, fu 10( W1 )t tholchgo,
none
28 new daffi% lPKS Cadleviont
Added to 200,c] s Romp tuhupe Variany of sdns� bright pink femairsovsdac
picnic
ASHY "if eZrrrrn -1trut (Ir set%)
terup S Bock I I C rnawmath k to u r. I S mrn aerated bilack men a, tub; 151 to I UC yet 211 11,501C hae t
none
1/atn4
200 not Most ;H Im.
tjai dnft��- %rI 5 Lam Aerated Marc cuoutin tool 151. 1 J IDC', vc� A fins./Cruprd
none
4S,0rr0 ,8rAr, stock f2 sorm)
Put omi�do in upwafl hucket ftrC) farood ine AC down: mcreasing adult flrdvs; put chumdr, tt21hohh
1,000
1/2S,1IA
200 H most Y 8 In rn
Ch'arnight release 191_ at 9 air, mmoved arlfts it, arnfitur anrehrcl black tub Mw.-, no Mate
1000AW
IN cik moct 8Pd1 no
'-nap Snuck: 11C keft ovemetht to it 5 pit,, a-rated iffill lty, sin fi, SOL W IDC w/ I St I sarechaet
no rus
1
200 one "ieW 8mm-
Shcecto VIC off ovehrittif no n I pm„ aerated ruddy siflk.' SOL I u I DC W) 151 Tiso/Chatit
n(use
WAYU
200 net moset Am to
Temp Shock,, 11C; left o-errught to 6 S pm ceruovd utility sink SOL W IW w/ 151 TisaraChaet
more
100 he I nuart bm rn
WAY In BArfi 4 daty,_IK S pm;; aerated utility Npdr„ 501 7u 19C. / jSt TLh,/(',hanj
21$11111
200 Ml most 1A8" ul
Spa.n 000f 2 nigbis hko dosmgi nernoged adults IT) another aerated black tub 20C no algae
-too ral most , Brorn
r
Wa-
- - ------ - S pm"aoratndaflhpy drk: 50k I it 15K, w/ I S�, Tnu,�Crafu
none
2/vm
200, nt I most . Amm
Spawn rrao, 2 naft'Its isdothey OM royrryed adufts to another i&,trphd black tub 20C no aanut
200 Ml Most > opt"
S pro atmanted, urdto, Sink' Sol lu 194, , %n/ Iq LmVchafft
r1oro
2141Q0
Nan Vol most , Arrant
Spawn vs6caight (1 .2 dead) stoussaud, Adulps!.v another etoaled bla,,k tub XK no algane,
Agia"WOWM
NO' ml ITurAl +fimon
Iffil-n f, pro aerated shifty dink, SOL It, 19C w/ 151, 71 ist/Char-t
acme
21'5r 14
NX) ml "frost . San"
Spasm overnitafist LM dead; removed aduris in onusther aersted Mack tub 20C flo, algae
2,00, ml muck , drum
WAM-Auft"AVUE 5 pol; 6Qreked ohUly tathk WL 1,19 14C W1 Ist TaworiC"honm
morn,
2/6111
NIG ml most x 8nno,
WaTM-ILBath -- I dl .4y- AM 5 pro; aersled urf0ity %ink; SCA, to 19C w)" 151, Tfsokh,tdv.
porif
2elfla
;too ml most s Riono
Spaop ovotrualut (� dead) reopoerd adults t up a not fief as rated black t j It 20-C ro a gal
200 ml most, Snem
Mutt Bath I day; IOC S pio, aerated uplKyr skirl W, 14,19C wl 15t Tito,'Chant
none
218VIA
200 rfil mobt `+ found
seem Nuh I clay; 19C 9 pro; aerated un0fty sink ; Set l 19C wl ISL Tistr/Charp
upon
I fifj m6 , Room
"/9:2IA
200 mi most , anur,
Warne Bath I day: M S pffr aerate u0thirvink,' SOL fit 19C w/ 151 TfradChaet
Movie
100 Will 'armo
2!111.1/14
XK) rol mind a tinown
Written Bath I day, I SK, S nor, aerated utiffty, dre; 501, to 191; osir ISL Tusty/Chaal
ft"WW"
7)13/14
100 roll '8'Mm
MmAr I day 5 mut,; aerated uthleV 51'IC SOL Vu S 3L w7 SL ilp,
none
21141,14
100 mf ¢g rarn
Spawn overnight 10 dead) recuoved andulus to anoific, aerided black tub 51L (lbset thel
41",
)L'orsilst
100 un 16MM
Warm tub; 19C n afgae, sucle to aerated bucket; 101, It 19C, 2,St Dun, 2.5 Chatat, stripped Ms added Anne
2/17/14
t00 mi <Ardim
Warm bucker, 19C a Aftere cyck,notatatysi huckad 100 u 20C, q 311. M. Pas
110 M,
2/19/14
100 art �Srffltn
!�QUqnd - Lter n t {,j 0 5 pm; aerated milid, oink, Mt It, 19C 33 hat; Sli 3ri n SO Pau
ftO'4fi
notno,
2120114
100 ML <&MM
Small Spawn aers.rntdA 11,000 ancets nernowhil aduftste another aecalned utffilry sink 501- 1 51 911,51 Priv
2/21114
I'm fat carers
Stock ourtude tougoweRts gaterse, relarasics had viable any unthor; fevc& not healthy
end
3/12)14
Set ml trued
178 taftery main vcaclr� hCoUtehicl 3S% frounuftstarM L114 35 kdyI
Static Bath purge 20L, 2OC135 pId, 1 5 Ban aeratted utilityr sank" 50L In 19f, W/ lot Duriadella
none
3113Y�hit
90 mi miced
Warm with A dvtc 19C vinack to aerated black tub 204,, I'LX, 3S piyr, It Charter allgrav
none
3/14,014
90 PTal mrsted
Cold Shook buck" of vaned nhered 14C in,, that flow then hrvy wnaawxfvr
400re
Slow a cheap Wner. 5 pow aerated utility %Ink, 500 lu 13C to ISC, 35 piu• R Chseek
eml if
31W14
90 and topped
Stock oupside to upairells,
and
November 2014 NTB Macrofauna Final Report Page 35 of 37
figure 5
Dona x spawn log March through August 2014
3xva€ Mae Age
0
Wield #4101`2014
LT
romp
Sa I InII"Y
r0yalu a I c h la:r' &ne
I. °`ra h r�',rF� 4tk 1 "ISwpIQ,;i1
k4 h�fP
B
rl
8
P91
Aokojj4
S H t t
krcrnk Inam fw Neviland dataft
R Al
II �
TJ
posse, Won ftmo ainaArtkur
Mar 2a
Maaslar /PAT (overrarTjh4:)
14 -18
33
270 W
y�
Pb, E TYA onixed site
Mar 25
Conical /AT entarniltl9t
14,18
33
3y.P,t rn4
new and PS, EHT a mixed siRe
Mar X
ConscalPatsvadywrpe
18
33
''WO n4
slao:lk flukkavrisCs..
AtN' U2
Ma dar OPAT (1 @arb
9 S
34
80 rat
new lootleaed Adrf, a,ra .`2ielrou path)..
Apr kit
ruMa:elar PAT (7. hr)
15
'3A..
XV rest
ng MIJ, CKT 2 flukes.
40"
P(,omwa RT nvenight
1 G 2 €1.S
311
280 oral
rarasged dock moved to conjc6 @aclt/no H202 add
Aft CPt
C,Im cast fWrrae
209.5
34
2Ari nil
previous spawns vccx reset In di R erenk rank
hspl os
Matlar6 g & lions,
2.%
34
2901 n„i
some stuck eirrsed well
Apr"I,75
20
34,
290 nr[
Asante dra:ktnnded siamwn
Apr -07
MA(I'Ar /f2TIN i hae4
i7 5-21
M
2tl>I2 rrr1 ',.
saran stock
Apr-C i
i avraacal,JStnadypA1g* e
20
34 .
"stable
pbrwmAdly spawned vfrack. Hns d we4l'
,Afar "08
11filkapShaady, no PIo:st
20
3;A
2,80 Tra4
same sEarck
Agar W
SUnirfPl
18.2x1
3's
80 oil.
p
new stock Ism, Kohl Shtst'ry€ (PKS,eKnoi 4
Aplr,09
RAtlydr5!4mm
20
35...
90ral....
now dock pineKnotlShonn;(PKSdKnWlwu dt
ATSt 1t7
Stattt, K' 02
20
3'S.
AO ml
ammo stock piing knoll tihuom (F#:, KreA[ }
Apsr' IP1
Stat'red •`'im'I'
2t.
35,
SO nvk ea
MU, PS, CHT2 k) new stock. PXS/KnoAIwoW
Aps.r.lfa
SfaticJR'(Atas�rna
18.21.
35
S i ml ea..
n w Aock PK!ijKtyoIhu*od & WOW inn),
Apt, 11
Mrselal VH2rr2
1.8
"IS,
250 real em ,.
oldv ow* A lag ",mez PKS O(ark
Apr, 11
Ma:lar%VH202
19
34 ...
OWN we
naeu stork. PKS a Salkat maih (Sp).
Ap, 22
STathedRT
15 2A
.V.S
3d mi
new 006, PKS N A rryM lengp:H
Apr 29
hP@dal r raYartl...2r 2'.
26
34 ,
S4lrl no
MH 15 062
May 06
Ste'tk H202
2i
23
$00toI
IOU pt EHT2
hiiBk Li'
Static"4£201
f1
4y
S41S3 mh
MU VS t H.T2
May 09
Drnn,dreuarcp112£32'
B 1A
ALL1104,
all flliflkytotlk in StpFapan edlo!°es a.rrM.Cidonlal"
iDniq 1 Liter 0 8rtxyd
%OCN fiepnaJovI iP,pl3+n1:
4id 4pAW1h ati t0
ClkAo0 @t baiyweft t4A $pfd afMQT rrh@SS 5D&Wr` aCtarl@nt
P:May, 16
H202 •'j irOM
32
13'.
:100 ITI1
Sal. i H P 11
.1262.103tvplt
;v °2
'13
H''JJ fin I
fl„ khNfA
M.MaY�lf
H202 mid u a, I.
J'.2
33
300 ml
P%, FH?d
h4art42
H202 -54
:3:
34 ,
i•00 rrel'.
new rn,^b. OR
Pun 13
H202 513 pprra
k3
34 :
%1,S21 In
now e ,1k, SPY
gun 17
li202 -Stt pprn
24
'U
30s) in ,..I
PS UATfk
aern SP
H21-112 50 ppfin
7A
,SA
300",1
PS, FHTJ
,un19
H20 511Porr
24
3A
300rr,,4
A'S,EHTU
2cin 251
H201 °501 ppm
N
34
500 ml
Mfl PS, fkpT2
fur:,22
Q02 Sri pa63rmk
24
34
SW red
s2H PS, EH -12
Meru 27
H102 50f pgar'n
J,4.1a
34 ..
5rrrej to I.
MU PS„ EHT2
Jun -28
11,102 -" psprrn
244%
34 .
500 rnl:,
MU, p5, EHT2
Jun 211
H202 AD ppm
d410
34 .
SCO Inf..
DAU Fv5 CHT2
Jun 3Q
H20i -So plem
2+4 :s Pa
34 :
Sri IA
MLI p5„ EHT2
iul -f2
Mader kAl
irk
34
ASfir nl
MU, NS, EH 14
ir11 -23
Ma €lal IIAI
24
IT
A,3, In t.
MU, PS, EHIN
hil-24
H202 -W ppri'a
2A
311
A50 nq
ML4, PS, N14
i:al' >2A
11"'02 'W pp7'n4
24
34
CIO W
MiJ p5. N74
1:11 -26
1"1202 SR pparrr
¢A
-iA
850 real
MU p,, NTA'..
Aag tat
K'Wy 50 "pn
14
33
You net
MU, PS, EHTA..
Aug 473
QW, co peon
24
33
44at@ fid
OU, PS, &91'4
Atil,12
HM2 �$O topper
23
313
SOD mli
MU K, 0,114
Aap 11
H202 rsr.MU41,
23
33
YV reap
MV, K, FHT4lGtnvarh Stne.k:.p
tiurg, 16
H202 SP ppne
233
33
500 tap
M4 PS, EN114
"d tr amts
3xva€ Mae Age
0
LT
0
r0yalu a I c h la:r' &ne
U
rl
8
1'.n'w i0odi4rymAi Yal tul%�'Ps
r)
5
Q
R Al
II �
TJ
posse, Won ftmo ainaArtkur
fl
qi' 1jp�1i }It,
(7
t
CB
s'
SS
212A Hard 2altk aa11n1 35 to 10t taernatrnties
1a
ti
Ill_
S+flo kAS
fort 4u Adrr;
e�flnrkas
P
A)r
A 5,17 rRriaeaVldn n 844e
combined wT:N
{f�
tStd .�1 to
a pron3tuars irk nipe
ra;wr Tn
1
arl t,l' 8 ^.ormlvinrd mavt'h 0 plvm ¢arik Facknne
12 15 "3 @@
it pink kader'l's
11
30
r0yalu a I c h la:r' &ne
%j "j't1tYttt , .415
rl
8
1'.n'w i0odi4rymAi Yal tul%�'Ps
f'd
5
pxswnr iailed/no air /Attar
R Al
II �
2.
posse, Won ftmo ainaArtkur
qi' 1jp�1i }It,
p,nlrrirod/txyrelallungus
s'
6
212A Hard 2altk aa11n1 35 to 10t taernatrnties
Ill_
II
fort 4u Adrr;
gd 82'19 Rain''Afinhg a3 to 26 . p1K4t
pool foodj"Aral fig@
SS 7 M tnrtal
November 2014 NTB Macrofauna Final Report Page 36 of 37
r is Appreciation letter from C State Outreach
Education Summer Camp Coordinator, Terri Mann.
Three separate groups were brought to Morehead City
from Raleigh for the day to learn about the beach
environment and collect Coquina clam brood stack.
For some children it was their first time to the beach
Cak$H f3 EPflIU6B'bXN i`+�9BdR,9EdQwd[wuu �'VtdMd�l 9UOhyu liunW.ihiu�$dQkuE, iulmrau�ulYggdS�rankNNEYxrvfi&lai IlWEt+`'33.Wdlre.., mu+yy WO
re8
R�I;I- A b., sn asamreu P4ry d,�rs4 .Xsm- x NlPUn3d r Tan ov B�aeXp R b., $f,re 7�aw%s. aae Xxs,m fmFf Crlk" Sed-41 bo,l MI . C5 €1,, Xd c Sa
Pdafmp
MC clam project Kelps restore beach ecosystems
sir _ ; A Carteret News -Times article
about the North Topsail Beach Coquina
clam project and CCC students collecting.
November 2014 NTB Macrofauna Final Report Page 37 of 37
1 IS J 11 1,1,
� Ih,' (
ect
a
✓w 7 9 ,t 4 da
I 4 t h j ,fl
I il .I 7..
f P 1
A,
Up— 111 id
.S.
[an
Ii 1
JlJ
1 i 1
1 I Y .�.
t Ise
4rJ it
�� q
I p
t
t
VA i ''.
Y 5 t
t .
I.
a d l h", 41, 11
1
e
10, 11 If x
-fif K
Ail,
•,+ Ls
fo lid
t rut
_ 1
1 3
�n, tiA ,
Fi Ii II r1 1,i,
1110
111 01, t
"' ""I"', ",'r I
sir _ ; A Carteret News -Times article
about the North Topsail Beach Coquina
clam project and CCC students collecting.
November 2014 NTB Macrofauna Final Report Page 37 of 37