| Using Serum properties as an indicator of stage of
maturation of the American lobster, Homarus americanus.
CMER RESEARCH TOPICS - 2000 #12 2nd Semiannual Report PI: Joseph G. Kunkel
|
Diane Cowan & lobster at The Lobster Conservancy, Friendship Long Island Lobster Pound, Nov 17, 2000 |
Further Progress in Lobster Hemolymph Chemistry
The four proteins we have targeted to be able to purify and follow are:
(1) Vitellogenin (Vg)/Lipovitellin (Lv). (2) Hemocyanin (HbCy).
(3) pseudo-HbCy. (4) Lipophorin (Lp).
We have suceeded in isolating substantially pure HbCy and
Lv, Fig. 1., as seen in this SDS-PAGE pair of gels produced by graduate student
Syed Hussan. This degree of purity is sufficient for characterizing the protein and
making an antiserum in rabbits. The HbCy was purified from serum of an intermolt
male lobster collected by Joe Kunkel on the Spring 2001 Albatross IV Bottom Survey
cruise. It should be low in pseudoHbCy and have none of the female
specific storage proteins. The HbCy does have a small impurity of
a higher molecular weight which may corespond to pseudo-HbCy. We
will be pursuing this possibility to obtain the pseudo HbCy protein in a pure state
also. The Lv was purified from eggs collected from mature ovaries of a female
lobster sampled by Joe Kunkel on a Spring 2001 Albatross IV Bottom Survey cruise.
Typical of Lvs it consists of several peptides in the 80-120 kD size range.
The antiserum made against this protein should suffice to allow titering
of Vitellogenin, green-serum-Lv as well as (green) egg-Lv.
The sera
of lobsters are variably colored light blue to dark blue, light orange
to dark orange, light green to dark green. Some lobstermen report black
lobster serum which may be a combination of orange and green or orange
and blue serum proteins -or- an indication of phenolase activity which
may be associated with wound or reponse to infection. Often, when
freshly drawn lobster hemolymph is kept chilled and centrifuged quickly,
the hemocytes are removed and a clear colorless serum results. Biology
undergraduate student Jennifer Schnorbus took colorless serum (Fig 2 thin line)
and by oxygenating it in a dialysis sack bathed in airated buffer, turned the
colorless serum dark blue (Fig 2 thick line). The difference between the two
spectra (Fig 2 stippled line) represents the oxygenated HbCy spectrum which
has a maximum transmission in the blue region of the visible spectrum. This
dark blue color represents the hemocyanin (HbCy) which was usually dexoygenated
in lobsters freshly bled after being caught in
a trawl. Our objective is to see if we can used the oxygenated blue level
as a measure of HbCy concentration in the serum. If so, a simple color
-based test for HbCy could be developed. To accomplish this we must
first have an antibody to HbCy which we can use to definitively quantify
it based on a purified standard and then relate anti-HbCy measured titer
to the achievable blue color using oxygenation.
The Online Database for lobster work reported in the previous report has been generalised to cover any project that peocedes in the lab. That allows each worker in the lab to have a separate password that allows him and a supervisior and/or coworker to access the important data and analysis files of their projects:
Teaching the Cell and Molecular Biology Laboratory, Biology 297C, has
given me the opportunity to introduce undergraduates to the applications of
molecular biology techniques to practical biological problems. I have introduced
lobster population biology as a practical problem that needs work. DNA extraction from
lobster tissue collected on Spring 2001 NE Groundfish Survey, Leg III was carried out as
a lab exercise in our Spring 2001 Biol 297C Lab. In addition a summer undergraduate SPUR
REU student, Jailyn Toro-Rodriquez (U. Puerto Rico Mayaguez Undergraduate) developed the
DNA technology further during two months of the Summer 2001. This work was supported by a
$500 transfer to my Overhead Account from the NE Alliance SPUR Program. A UMass
undergraduate senior Biology Major, Ted Hartenstein,
was supported by the UMass Biology Woods Hole Fellowship to travel with Joe Kunkel to
the Mass. Lobster Hatchery in Vinyardhaven to collect serum and DNA samples and also to
The Lobster Conservency facility on Friendship Long Island to explore the use of scuba
diving for recovering PIT tagged lobsters.
I have started a collaboration with Dr. Diane Cowan to extend our interest
in the normal serum patters of lobsters to the possible stress related changes in storage
proteins that might result in greater susceptability to shell disease. There are workers
who believe that shell disease represents a new set or combination of microorganisms that
are now attacking lobsters. I suspect that several stresses on current lobster
populations could result in changing their priorities from storage protein production to
more immediate survival strateggies. They might respond to stress by decreasing Vg and
pseudo-HbCy production since these proteins are energetically expensive to produce and
are not necessary for immediate survival. While these changes in resource management might
help them survive in the current instar, decreasing Vg production might lead to
involution of the currently developing oocytes. Decreasing pseudo-HbCy could lead to
production of a weaker new cuticle which might make the next instar more vulnerable to existing
microorganisms. We propose to study the frequency of orange serum (healthy vitellogenesis),
green serum (ovarian involution) in populations that are high in shell disease (Elizabethan
Islands, MA) vs areas that are low in shell disease (Friendship, ME). Toward this end I
talked with George Cadwalader a retired lobster fisherman from Woods Hole who remains
interested in lobster biology. He has volunteered his time checking his 125 lobster
traps in support of our serum protein studies. Diane Cowan will arrange for a
Friendship ME lobster fisherman to work with us in Maine. This study will also
attempt to relate the level of HSP-70, a commonly studied integrator gene for stress, to
the levels of shell disease and storage protein disease in the two populations.
Bruce Estrella of The Massachusetts Division of Marine Fisheries has also
assured us that we are welcome to use the Vinyardhaven Lobster Hatchery facility
for any focal research that might require holding lobsters during an
experimental treatment (such as stress).
Personnel
Dr. Diane Cowan. President of The Lobster Conservency collaborated in providing lobsters that could be bled at regular intervals.
Jeff Xu applied his skills at database management improving an online database of Lobster serum collection information.
Syed Hussan. Research Assistant. Jan 2001-August 2001. Syed applied agarose gel electrophoresis, SDS-PAGE electrophoresis, TEAE-cellulose chromatography and Gel Permeation chromatography in his collaboration in purifying the lobster green-egg-lipovitellin and lobster hemocyanin.
Ted Hartenstein is a UMass Senior Biology Major. Ted volunteered his diving certification abilities to participate in our lobster project. We obtained funding ($400) from the Woods Hole Fellowship to fund his travel and expenses to participate in the bleeding of lobsters at the Vinyard Haven Massachusetts Lobster Hatchery and to The Lobster Conservency in Maine to plan diving activities associated with retrieving PIT tagged lobsters from the lobster pound.
Jailyn Toro-Rodriquez (U. Puerto Rico Mayaguez Undergraduate) was a SPUR REU undergraduate during the Summer 2001. She developed a protocol that will allow us to take a DNA sample based on blood cell DNA binding to glass fiber discs. A $500 allocation of supply money was provided through the Northeast Alliance REU Summer Program (SPUR) to support the work.