Lobster Vitellogenesis: Uniformity or Heterogeneity in Egg Storage Proteins PI: Joseph G. Kunkel Biology Department University of Massachusetts Amherst MA 01003-5810 TEL: (413) 545-0468 FAX: (413) 545-3243 E-MAIL: joe@bio.umass.edu

Jason Link & lobster, fall '98 on AL9811.

The developing oocytes of almost all animals receive storage molecules from the bloodstream as provisions for the embryo during embryogenesis and early larval stages.  For most animals the principal storage protein is vitellogenin, homologous across the animal kingdom (Hagedorn and Kunkel, 1979; Kunkel and Nordin, 1986); however in many organisms a second, third and even fourth protein is also stored in substantial quantities.  Calmodulin, normally thought of as a regulatory protein, makes up 10% of insect egg protein (Zhang & Kunkel, 1991; Zhang and Kunkel, 1994; Iyengar and Kunkel. 1995) as well as frog yolk protein (Cicirelli and Smith, 1986).  Lipophorin, a diglyceride carrier protein found throughout the Arthropoda, makes up 5-20% of some insect yolk proteins (Kunkel and Pan, 1976; Dompenciel, Ph.D.).  In Gypsy moths the first and last eggs to be ovulated have dramatically different storage protein profiles and the proteins are used in different proportions during embryonic and first instar phases of life (Dompenciel, Yin, Leonard and Kunkel, 1992) in addition we have demonstrated that the female gypsy moth accumulates large amounts of the serum storage hexamerin, arylphorin in the instar prior to metamorphosis (Karpels, Leonard and Kunkel, 1990).  The role of hexamerins in the reproductive process is of growing interest (Telfer and Kunkel, 1991).  Lobster reproduction has been studied with the aid of antisera to vitellogenin (Quackenbush, 1994). Recently a pseudo-hemocyanin, which is a hexamerin, has been identified in Homarus americanus which is synthesized in the ovary and is likely to be associated with molting and reproduction (Burmester, 1999).   Vitellin and Lipovitellin are also known to be spared from major digestion during several vertebrate (Hartling and Kunkel, 1999) and invertebrate embryonic periods (Dompenciel, Ph.D.) and, interestingly, are reserved mainly for early larval development prior to larvae gaining access to extrinsic food.  These examples suggest that serum and egg storage proteins may be a rich source of information about lobster development and health.

I suggest studying the heterogeneity of lobster storage proteins in the ovaries and egg masses of vitellogenic and berried female lobsters.  What are the major storage proteins in lobster eggs?  What is the percentage of each as a component of the total yolk protein.  Is there a functional heterogeneity of egg storage proteins in the lobster, seasonally or spatially within an individual female?  The analytical approach to measuring them could be developed in an initial year.  If it were found that there are multiple yolk proteins, we could proceed to purify them and develop antisera to the individual proteins.  We could then evaluate natural populations for the heterogeneity of the yolk protein storage in eggs from berried females.  I would also be interested in determining the timing of each protein’s utilization in the embryonic stages from individual lab housed berried females and early larval development of hatchery reared early stages if feasible.   We would then proceed to apply what we learn to field collected samples of serum and eggs.  Indeed there is some suggestive evidence that storage protein disease is already observed in lobsters.  Non-blue (i.e. red) eggs are considered to be infertile.  The normal blue color could be an indication that hemocyanin (a hexamerin) makes up a substantial proportion of normal eggs.  Alternately there may be another blue protein that makes up a fraction of normal egg storage protein.

The project will use the general biochemical tools of cell and molecular biology.  Serial serum samples can be obtained using non-intrusive bleeding through the intersegmental membranes of the lobster.  Eggs from berried females can also be obtained non-intrusively.   Initially the serum of male and females and egg proteins of females would be analyzed by SDS-PAGE electrophoresis and fractionated by combinations of gel permeation and ion exchange chromatography.  These techniques have been successful in the past in separating the major classes of serum and egg proteins of arthropods.  These purified proteins would be used to immunize rabbits to produce monospecific antisera against each protein.  The intention would be to build up a battery of several antisera that would allow the proteins in individual eggs and serum samples from individual females to be profiled.  We would quantify the heterogeneity of the egg population from individual females via quantitative-immunoelectrophoresis (QIEP) and enzyme-linked-immunoassay (ELISA).

We will describe the serum and egg protein titers through the lobster molting and reproductive cycles in association with morphometric data taken on each animal.    This approach may allow us to establish the reproductive health of individual females as well as particular populations by sampling serum and eggs from berried lobsters in the field.  The profile of serum and egg proteins will be diagnostic of their stage of development and perhaps their health.  We hope to apply the approach to particular problems such as obviously off-colored eggs and marginal cases which mught be more subjective.  In addition we could apply our profiling to different distinct populations such as on-shore and off-shore populations which could be distinct in their status.

Undergraduate and graduate student participation in this research will be implicit to the success of  the project.  Dr Kunkel teaches the Cell and Molecular Biology Lab, Bio 297C during the spring semester each year.  The students in this course will be invited to participate in an honors section that will use the lobster storage protein story as a research theme.  In addition, a masters-level graduate student will be enlisted to participate in the protein purification and antiserum production phase of the project.  The naive students will be perfect subjects to test whether the methods of sampling, sample preparation and analysis we develop can be taught to a minimally technical audience.

We will need cooperation from NMFS personnel to obtain access to lobsters of various types, males, females, immature, mature, berried and unberried.  Initial field testing would be carried out on NMFS Fall/Spring Bottom Surveys.