Adventures in low salinity

December 5, 2013

ABC recently completed a study comparing 13 different constructs of triploids with their diploid counterparts.  There were a couple of bottom lines.  First, location, location, location.  The relative value of triploids is highly dependent on where they are grown.  At the lowest salinity (the Horn Point Lab in the Choptank River), there seems to be no value.  Second, triploids made with our tetraploid line float all boats, including wild oysters, except in low salinity.  What is it about low salinity that ‘retards’ triploids?

Through our regular breeding activities, we have clearly shown the need for having multiple lines to deal with various salinity (and hence, disease) ranges.  Lines bred in lower salinity areas (leases kindly provided by the Bevan’s Oyster Company, and more recently, Cowart Seafood) have shown adaptation to this environment and perform better than their mesohaline counterparts.  The Lola line in particular is highly productive in low salinity.  Interestingly, the Lola line is also quite good in other areas, as long as the disease pressure is not high – it would be a risky business for an oyster farm to use Lola where disease might appear, despite Lola’s edge.Screen Shot 2013-12-17 at 2.19.34 PM

We hypothesize that pushing breeding to the lower edges of the salinity range will actually improve the overall physiological performance of our lines over and above the breeding we could attain in the middle of the Bay alone.

Therefore, we are about to embark on a new chapter of breeding activity, a confluence of three events that are coinciding.  The first event is the data that we obtained from the triploid project that screams, “lower extremes of salinity in the Bay need special consideration.”

The second event is the advent of ABC’s transition to family breeding.  In short, in the future, we are going to make breeding progress by testing numerous families, instead of lines.  In the York River, we are about to install an Australian long-line system (right under the window of the Dean and Director’s office) because we see this system as the perfect option for family deployments.  Each basket can accommodate around 75-80 oysters at market size and growing conditions among baskets are much more uniform than bags.  It also looks cool, although this system is probably not right for Bay aquaculture writ large.

The third event is the recent addition of a new faculty member at University of Maryland’s Center for Environmental Science at Horn Point.  Louis Plough has a pedigree in oyster research beginning with his Doctorate at the University of Southern California in Dr. Dennis Hedgecock’s lab.  He started at Horn Point this year with expertise in population genetics of marine animals, quantitative genetics, and experimental breeding of shellfish.  ABC has had a long standing collaboration (i.e., as long as we have existed) with the Horn Point lab through Dr. Don (Mutt) Meritt; the addition of Louis to the breeding portfolio of the Bay enriches this collaboration all the more.  And coincidentally, his arrival coincides with our realization that low salinity is another ‘disease’ that should be addressed with our family breeding.  We have entered a collaborative arrangement with Horn Point, through Mutt and Louis, to build a second Australian long line system in the Choptank River.

This new endeavor into low salinity is somewhat unprecedented in oyster breeding.  Few places in the world where oysters are farmed face issues of low salinity – the Chesapeake is unusual in that regard.  Therefore, other breeding programs in the world never delve into the netherworld of salinities lower than, perhaps, 25 ppt.  We are going into the ‘salinity basement’ of 8-10 ppt.  We will see what these adventures in low salinity reap in terms of newer, more robust, lines of farmed oysters.

 

 

 

The OAT experience: Graduation 5.0

November 12, 2013

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About six years ago, a representative for a generous (anonymous) donor in the Chesapeake Bay area visited ABC to see what contributions we were making to the Bay’s welfare.  About that time, the vital industry we have now was incipient, and I made the case, on behalf of ABC, that we were trying to enable oyster aquaculture.  It was a propitious time for this visit because we had just come off of two years of severe budget cuts that threatened to reduce our technical staff.  The donors offered support that, ultimately, helped us remain whole and continue our work unabated.

The donors offered another line of support as well, support for a new program we had been conceptualizing for a number of years, called the Oyster Aquaculture Training program.  The origin of the idea was somewhat selfishly rooted.  During the early years, as the industry grew, I was losing experienced people to new and growing companies.  The way to stop the bleeding so to speak was to provide a continuous supply of newly minted aquaculture technicians, leaving my professionals to, well, remain at ABC.  However, the rub was that to provide a truly meaningful experience and provide thorough training, we would need a participant to reside for a significant period of time.  I originally thought this would be about a year.  We pitched the idea to the donors and they liked it; they agreed to fund the first year – two trainees for a year.  After further consideration, however, we decided that hosting four trainees for half a year was probably twice as effective.  A half year would encompass early spawning season through early Fall when we deploy seed.  And so, OAT was born with the first ‘year class’ in 2009.

This year, we graduated our fifth class, graduation 5.0.  In all, 22 OATs have been sown.  Most are employed in aquaculture, ranging from positions at Cherrystone to Rappahannock River Oysters to Shores and Ruark to Ward Oyster Company to Coast Oyster Company (Quilcene, WA).  While it was initially our intention to stock, so to speak, the roles of local industry, we found that the applicants each year have become increasingly far ranging.  Next year, there are signs that there may be some international applicants.

It might be time to think about the intention of the OAT program:  should it be wide open or restricted to trying to provide a trained workforce locally?  Frankly, I am torn.  I am torn because of the unanticipated effect that the OAT program has had on the ABC staff.  The hallmark of the OAT training is immersion, hands-on, person-to-person tutorial, deep understanding of the principles of oyster culture.  Immersion, hands-on, person-to-person means that trainees interact with all of the ABC employees, all the time.  In effect, then, everyone at ABC is a teacher, colleague, and sometimes mentor.  This exercise in sharing our activities with willing learners has had a profound effect on the morale of ABCites – they love it.  Now, each year, as the applicant pool begins to trickle in at the beginning of the calendar year, there is a palpable anticipation.  Who is applying?  What is their background?  What kind of training do they need and what specialization would they like to learn about?  Where are they from?

The arrival of OAT trainees is Christmas in April.  What is in these packages under the wrapping of their application?  How good of a worker are they?  Do they have the right stuff?

Let’s be clear.  This is a quid pro quo program.  While we provide training, we also get seasonal assistance in the busiest time of the year.  As a result, we no longer hire summer technicians.  And the funding is also sufficiently ge
nerous to enable us to place OAT trainees in what we call externships – stints, from a couple of days to a week, at willing oyster culture businesses.  These businesses are our silent partners in the OAT program, and highly influential in shaping the perspective of impressionable trainees.  A couple of years, we were able to send OATs as far as Washington State (and actually, one remained and works there now).

So back to the question, should we restrict the bounds of the OAT program to serve the provincial needs of Virginia?  Of the Chesapeake?  Of the East Coast?  Of the United States?  Maybe you have some thoughts on the matter.  In the meantime, we are enjoying a program that turned out to be more than the sum of its parts.

The case for a research hatchery

Recently, VIMS forwarded their annual capital request to Richmond, which included a general description of a new shellfish hatchery.  In short, ABC has grown out of the facilities it has now.  What do we have now?  The building that houses the diploid breeding program in the Gloucester Point boat basin at VIMS was built nearly four decades ago and was originally designed as a production facility for planting large numbers of seed oysters in Chesapeake Bay.  It is crumbling.  It is medieval.

What do we need?  Oyster breeding requires production of numerous small groups of genetically distinct crosses.  (See June 1, 2013 blog, “Spawning Season at Gloucester Point”).  This contrasts to how a commercial hatchery operates – or for that matter, the original purpose of our current hatchery building.  Commercial hatcheries deal in very large groups of only a few different types.  Oyster breeding just requires a lot of space.

We need space that is designed for the meticulous care of up to 200 larval tanks simultaneously; we need space for expansion of algal cultures during our busiest times; we need both batch and continuous algal systems; we need dedicated space for setting larvae – as many containers for seed as we have larvae, in the example above, 200; we need state of the art water filtration capabilities to insulate us from the ever changing nature of Chesapeake Bay water; we need temperature control of water, both for tempering incoming water and capturing heat from the outflow via heat exchangers so that we can start our season earlier; and, we need conditioned air (i.e., control of air temperature in certain rooms) to keep larval cultures optimal, not to mention people, who now work in completely un-tempered summer heat.  One of the ridiculous examples of the lack of this latter capability has been the need to make a run to the ice machine in August to chill water around the larval tanks when air temperature surpasses the maximum.  The needs listed above – in no way complete – are just to optimize our current program.

What about future needs?  Unanticipated research issues?  What about serving as a crucible of future hatchery advancements, such as, high density larval culture, nutritional studies, advanced micro-nursery systems, or research on important life stages of larvae, such as, metamorphosis?  Our current facilities are so jam-packed with current programmatic needs, we are utterly incapable of asking questions like these.  For example, a few years ago, Stephanie Reiner, then a Master’s student with ABC, did research on high density larval culture.  We had to set the project up in a commercial hatchery nearby for lack of space and appropriate infrastructure at Gloucester Point.

What is completely ironic is that neighboring states – Maryland, North Carolina, New Jersey – have state-of-the-art facilities but their commercial aquaculture lags far behind what is going on in Virginia.

ABC is currently seeking support from anyone and everyone who sees the value that ABC has, and can in the future, provide the Commonwealth and specifically the dynamic, still expanding industry.  With State financial support, help in planning the hatchery from industry advisors, and a shrewd breeding strategy, the investment in a new R&D hatchery can pay off in major ways, in ways that the investment in ABC in 1997 has to date.  Support your local hatchery initiative.  The Bay will thank you.

The REU Contribution

Does it matter that some of the tetraploids the hatcheries are using to make triploids are mosaics?  Does it affect the nature of the larvae?

Are there alternative means of inducing tetraploidy from triploid eggs besides the fairly toxic cytochalasin B?  Is it better or worse than the standard operating procedure now in use?

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These are some of the research questions that have real implications for the Virginia industry, especially hatcheries, that undergraduate scholars have addressed in a National Science Foundation sponsored Research Experiences for Undergraduates.  Each year about a dozen promising young researchers come into the program at VIMS and hook-up with a mentor in anything from fish systematics to benthic ecology to – yes, I am happy to add – oyster aquaculture, specifically aquaculture genetics.  While ABC’s only other REU student was in 2002, we have had the pleasure of hosting students in 2012 and 2013.

Last year, Joseph “Joey” Matt (Wake Forest University) took on a project stemming from a question we got from AJ Erskine about mosaic tetraploids (a mosaic has lost some of its chromosomes, a subject of AJ’s thesis at VIMS).  Specifically, AJ questioned the idea that mosaic tetraploids were being used for spawning triploid production batches and asked whether it might be more apt to distribute “pure” tetraploids.  (While this is an excellent question, the fact of the matter is that most tetraploids are mosaic, and sorting them out would be a logistical nightmare.)  So, does it matter?

Joey spent the summer doing crosses using mosaic tetraploids (labeled M – often selected for their severity) and also using pure tetraploids (labeled 4) to make triploids.  Triploid crosses were made all ways – M (♂) x 2n; 2n (♂) x M, 4 (♂) x 2n, 2n (♂) x 4, along with diploid controls.  These crosses were replicated over and over, early larvae were analyzed by flow cytometry to look for abnormalities, and survival recorded.  Three pairs of M(♂) x 2n and 4(♂) x 2n triploids were set and grown up to examine this summer.  The bottom line of this 2012 research project was that we could detect nothing unusual about the larvae obtained from crosses that involved mosaic tetraploids.  (The sperm from every male – M or 4 – were always ‘perfectly’ di-haploid.)

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This year, Brittany Peachey (Grove City College) joined us for the REU summer.  Brittany accomplished an experiment that may never be repeated again in the annals of polyploidy.  Specifically, Brittany examined the effectiveness of using 6DMAP as an alternative to CB for chemical induction of tetraploidy in eastern oysters.  The reason for the unique aspects of this work is that it corresponded precisely with ABC’s overall push to develop new tetraploid lines from some of our best diploid lines.  This is a process that takes four generations: diploid -> chemical triploid -> chemical tetraploid -> mated tetraploids as brood stock.

As ABC just completed a study of 13 different varieties of triploids, we had an unprecedented supply of three year-old triploids for Brittany’s work, and the basic experiment was replicated nearly 20 times.  The bottom line for Brittany’s project was that 6DMAP is very effective in making chemical tetraploids, it is also less dangerous to the user, and, it seems, survival is better.  A paradigm has been changed.  We will switch to 6DMAP.

Both Joey and Brittany were rising seniors as REU students.  Both are writing up their results for publication – the results were that tight.  Joey re-joined us for the summer to help operate our hatchery at KAC.  He will stay next year in a new position we are calling the “tetraploid technician” to help keep track of our increasingly intense breeding program with tetraploids.

Brittany will complete her senior year at Grove City, and then – who knows?

Spawning season at KAC

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As I carefully wash a thousand larvae down into the corner of a mesh screen, sized to catch tetraploid eyed larvae, I think about how ridiculous this must look to anyone who has cultured larvae before.  A thousand?  Really?  A thousand spills on the floor of a commercial hatchery when someone sneezes.  Welcome to the game of tetraploids, where no number of larvae is too small.

The arena for the game of tetraploids is located in Topping at the Kauffman Aquaculture Center (KAC), conveniently down the road from Merroir at RRO, where, if the numbers of larvae get too small, I can drown my disappointment in Oyster Stout.  The role of KAC in ABC operations has morphed considerably over the years, but has always been crucial.  Built during the “era of ariakensis” exploration, it is no longer a quarantine facility per se.  It really has two principle functions for us now: i)conditioning brood stock for spawning and then holding them back once ripe, and ii) the arena for the game of tetraploids – our hatchery away from the hatchery.  As such, we operate it seasonally.  The latter activity includes not only propagation of the next generation of tetraploids destined for industry distribution, but also R&D on tetraploids, including the generation of experimental families and, for the last two seasons, a site for experiments for NSF REU students working with me on tetraploid related issues.

For example, last year Joseph Matt looked into the fate of di-haploid sperm from mosaic tetraploid versus non-mosaic (i.e., “pure” tetraploids).  This is a timely question insofar as the majority of tetraploids used for making triploids are mosaics (a combination of triploid and tetraploid cells).  (It seems to be a feature in C. virginica tetraploids.)  We found no differences in early larvae attributable to mosaics.  This year, Brittany Peachey will look into more effective ways of making tetraploids from triploids using an alternative means of induction.

The game of tetraploids is one of those long duration games requiring patience.  To get to the tetraploid state, you must first pass from diploid to triploid, and then from triploid to tetraploid.  The latter step is not fun, requiring one to find triploid females that can be used in the very special cross that obtains to tetraploidy.  In this step, I have carefully coaxed as few as a couple of dozen larvae into the corner of a screen for setting.

The good news (sort of) is that once you have your first generation of tetraploids, you can cross them to each other – 4n x 4n à 4n.  The bad news (sort of) is that even this cross has “issues.”  Once in a while, a 4n x 4n cross will be really good, which means average for any other kind of larval culture.  But most of the time, the loss of larvae leaves you with a few thousand that reach setting size.  Average hatching rate for a 4n x 4n cross, for example, is less than 20%.  The one I did today was 6%.

KAC is an excellent place to do this work, however, partly because it is a hatchery away from the cacophony of the Gloucester Point hatchery, partly because it has a modicum of air conditioning (something absent at Gloucester Point), and partly because I get to choose the music – literally and figuratively.  I guess you could say it’s my hobby hatchery – the oyster equivalent of a busman’s holiday.

Spawning Season at Gloucester Point

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Individual families are kept in their own mini-downwellers before moving out to the nursery when they reach 1-2mm.

Our spawning season at ABC gets off to a later start than for commercial hatcheries.  But when we do, the scope of work is intense for about three months, until the slate of crosses is complete for the year.  For a while, I used to think of the hatchery as the center of operations for our breeding work, as in, it defined us.  But that notion has changed.

The hatchery is only one of our tools in the breeding process.  Sure, the crosses start here, but really they only stick around for 5 weeks before they are out in the nursery and, then, only in the nursery for about the same amount of time before they are in the field.  Ten weeks out of about 90 (the total length of time from spawn to selection) for any given cohort (a cohort is a group of spawns that were done at the same time).  Of course, what defines a breeding program is the number spawns in a cohort as well as the number of cohorts overall.

For example, first up this season was the founding population of families for our new family breeding approach.  It consisted of making 130 families (a mating between one male and one female) about three weeks ago, and then a re-spawn to fill in the families that were going less well than we wanted – another 44 crosses.  In the end we ended up with 115 individual cultures that made it to setting.  Our first cohort, then, was 115 low salinity families.  These families will be deployed to two low salinity sites in the early Fall.

Next up, our line spawns.  We continue to produce our bread and butter lines – DBY, XB, Lola, and hANA – even while we are starting our new families.  For lines, a cohort will consist of three lines from each of our test sites, York, Lynnhaven, and Kinsale.  Three lines from York will be one cohort, three lines from Lynnhaven – another, etc.

Finally, while the larvae from the lines are still growing, we do the final set of high salinity families – another 130 or so of them.  At the peak of the season, we might have about 150 individual, albeit small scale, spawns running simultaneously.  During “drops” (water changes) 6 microscopes will be in use examining the larvae.

All these crosses make for a flurry of activity in the hatchery.  The starting date is dictated by the arrival of our OAT (Oyster Aquaculture Training program) participants in about mid-April so we have sufficient help for the glut of spawns.  The end date is dictated by the deterioration of water quality for growing larvae in mid-July.  Overall, about three months of hatchery activity determines the scope of work for field tests in the following year.

Next up – Spawning Season at KAC.

Industry on a different scale

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I am writing this from my Spartan hotel accommodation in Tongyoung city in the southern part of South Korea.  Early morning is a good time to write because my biological clock is exactly opposite the actual time here.  The Tongyoung area, along with sister city Geoje, is the epicenter for Korean oyster production. I am on what amounts to another foray in my personal mission to plaster the planet with polyploid oysters.

If you want to understand the extent of oyster culture in a global sense, then I recommend a trip to Asia, where the big three producers – China, Japan, Korea – together culture 95% of the oysters, Crassostrea gigas, on Earth (although in China, recent findings suggests that a good proportion of Chinese production comes from C. hongkongensis).  China produces about 85% of the world’s oysters, or around 3,800,000 metric tonnes (mt = 1,000 kilograms or 2,200 pounds), and the “laggards” – Japan and Korea – about 250,000 mt each.  Virtually all of this production is what we would call “spat-on-shell,” with the shell hung from long-lines whose buoys fill many of the bays around here (see video).

In Korea, the major product from culture is oyster meat with a production of about 40,000 mt.  Koreans like their oysters small so they are marketed at an average meat weight of 12g, which translates to about 3.3B oysters per year.  Some are grown larger, of course, and go to half shell trade mostly in Japan and China.

Although only 10% of production in Korea comes from hatcheries, 10% of a very large number is pretty big itself – in the neighborhood of 50B eyed larvae per year.  The scale of the operations are industrial-size and measurements are expressed in unfamiliar units.  “How many eggs do you stock in your 120 tonne (= 120,000L or 32,000 gal.) tank.”  Answer – “70 grams.”  “How many eggs is that?”  Answer:  “We don’t know.”  Or “How many eyed larvae do you put in your setting tank?”  Answer: “One kilogram.”  “How many larvae is that?”  Answer: “About 120M.”

In that the major product is meat, there is increasing interest in growing triploids.  In addition to the likely increase in meat yield from a triploid compared to a diploid, triploids in Korea portend an interesting twist in marketability.  Korea has occasional outbreaks of norovirus that occur primarily in the winter, while rare in the summer.  In summer, diploid oysters are not in prime condition, so some see triploids as a way to more productively fill the summer niche.  Marketability during the spawning season is the theme song of triploid (spawnless) oysters.

Korea is but another case of the hatchery world awakening to the possibility of adding value to the oyster product.  In the Chesapeake, added value from breeding is now axiomatic.  In Korea, and Asia in general – again where 95% of the oysters come from – adding value through breeding, triploidy, or both is rudimentary principally because the vast majority of seed still comes from natural spatfall.  But hatchery production is picking up especially to get those specialty products, and triploids are leading that charge, as they did in France.  To paraphrase Everett Dirkson in his infamous “real money“ quote, a billion triploids here and a billions triploids there, pretty soon you’re talking about real added value.

Video: The below video was taken on Saturday, May 25 looking out at one of the many Bays where oyster culture is practiced in the Tonyoung region, southeast Korea. Click the link below to view the video.

Panorama oyster culture

Maintenance

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Changing oil, winterizing, cleaning – not the most glamorous of activities, but arguably among the most important for sustaining operations.  In a sense, “maintenance” is the origin of this blog – maintenance of communications, that is.

ABC has had an interesting history.  In retrospect, it seems that the Virginia Legislature was prescient in passing the initiative that launched ABC in 1997.  To some extent, perhaps they were, but the road to “today” has been winding and, at times, opportunistic.  For example, ABC has been variously involved in restoration, molecular genetics, clam breeding, breeding disease resistance, non-native species, polyploidy, and remote setting.  Some of these things were on the path to where we are today and some were merely dead ends.  Ultimately, ABC has provided something of value to the emerging and growing industry and our aspirations – ABC’s and industry’s – have become common.  As Anu is always pointing out to me, ABC and the oyster industry are both, absolutely concerned with the bottom line.  We grew together to this state.

Given our common goals, it seems surprising to me that we are only now coming to mutual understanding and collaborative outlook on our activities.  Frankly, this is probably my fault.  I thought that our annual Stakeholders meeting, our distribution of brood stock to hatcheries, and an occasional public talk was sufficient communication.  In the early days, it probably was, but not today.  Not when so many businesses have so much of their bottom line tied up in what we are producing.  For example, the Stakeholder’s meeting provided a forum for exposition by ABC to industry but not a forum for the obverse – industry to ABC.

Recently, an Industry Advisory Committee (IAC) for ABC was established to enable two way conversation.  The IAC will probably become one of the seminal developments in ABC’s history, I can tell already.  Not only does it provide a colloquium for exchange of important issues from industry to ABC and vice versa, it provides a new avenue of transparency between us.

And that brings us to maintenance.  The Stakeholders meeting was once a year; IAC is three to four times a year.  But between those months is silence, there is scant opportunity for communicating.  Industry has businesses to run; ABC had crosses to spawn and experiments to deploy.  The Director’s Blog, then, is an attempt to shorten the intervals between communications.  Although it is largely one-way (although there will be opportunity to comment in the blog), the Director’s Blog means to promote transparency, the antidote to misconception and misinterpretation.  Frequently, but irregularly, this blog will contain information on happenings at ABC, on field observations from our research farms, occasion outbursts of data that may be of interest to our constituents, and, maybe, even updates on the Director’s occasional forays to distant places to promote tetraploid technology.  The content kind of depends on how well the maintenance is working.