Using Serum properties as an indicator of stage of maturation of the American lobster, Homarus americanus. CMER RESEARCH TOPICS - 2000 #12 1. APPLICANT 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.

2. BACKGROUND

This CMER proposal outlines an approach using a minimally invasive sampling of lobster serum along with standard morphometric data to be used as indicators of maturation and health of lobster populations.  The project will include experimentation and observation with pound lobsters including correlating non-intrusive sampling of lobster weight, morphometrics, external eggs and serum protein development with molting cycle timing and ovarian development.  The non-intrusive data will be used to develop discriminant functions with which to predict molting cycle timing and ovarian development.  This will be followed by field sampling to establish the utility of the techniques.  The long term objective  would be to allow a fisherman or technician to sample large catches or populations of lobsters with a sampling method that would not affect the commercial value of the animals but allow a maturation profile to be established which could predict the maturation state and perhaps health of the catch or population.

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 egg storage protein derives from the serum protein 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.  In addition, a series of serum storage proteins, hexamerins, accumulate in the haemolymph of arthropods, in several cases, in correlation with the molting cycle or reproductive cycle (Telfer & Kunkel, 1991).  Calmodulin, normally thought of as a regulatory protein, makes up 10% of insect non-yolk protein (Zhang & Kunkel, 1991; Zhang and Kunkel, 1994; Iyengar and Kunkel, 1995).  A similar high CaM titer is found  in eggs  of frogs (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.

The well known insect juvenile hormone analogue methyl farnesoate (MF) may be associated with maintaining the status quo of juvenile development in lobsters (Prestwich et al., 1990; Homola & Chang, 1997a,b) and it or its binding protein could be used as an indicator of the timing or quality of an upcoming molt: juvenile-juvenile (status quo) vs juvenile-adult (metamorphic).  While most lobsters do not have measureable MF in their haemolymph (Tsukimura & Borst, 1992), the haemolymph binding protein for MF circulates in the serum (Prestwich et al., 1990) and might increase or decrease in titer, as its insect homologue does, during critical phases in lobster development.  In addition, the titer of the molting hormone, crustecdysone, could be used as an indicator of the imminence of molting in an individual (Snyder and Chang, 1991).  Radio immune assays (RIAs) are available for these compounds which could be applied to an aliquot of lobster serum. In addition, numerous neurohormones are being described, including one which regulates MF production (Liu et al., 1997).  These fine regulation controls via the nervous system are probably unreasonable to follow with current technology and also fall into the category of transitory events which would not allow logical analysis or fit into a scheme of useful items to measure in order to predict an animals physiological state since random animals only rarely exhibit any titer at all.

3. OBJECTIVES OF PROPOSED RESEARCH

I propose to study the hormone binding and serum storage proteins in the serum of males and the serum, ovaries and egg masses of vitellogenic and berried female lobsters.  Major initial effort will be on the major storage molecules.  In my opinion, the regulatory hormones and hormone binding proteins are of considerable academic interest, but the major storage proteins are the main indicators of the physiological health and stage of development of the animal. The hormone regulatory system components are also more ephemeral in that they may appear on short notice and dissapear quickly after having their effect.  However, the major storage proteins have integrated the nutrition and physiology that has gone on for the near distant past and should be a reliable measure of the animals physiological state.  What are the major storage proteins in lobster serum and eggs?  What is the percentage of each as a component of the total yolk protein.  Is there a functional relation between the titers of haemolymph proteins and the storage proteins being deposited in the developing oocytes within the lobster?  Or does the storage protein titer in the haemolymph predict the phase of the molting cycle of either males or females as it does in insects (Karpels et al., 1989)?  Thus one might predict that if lobster eggs are developing internally that Vg titer would be high in the haemolymph.  If molting is imminent the serum storage hexamerins should be high.  If molting has recently occured the serum storage hexamerins should be low.

The analytical approach to measuring hormone binding proteins and the major serum proteins will be developed in an initial year.  I have become an expert in measuring multiple proteins in the serum and eggs of insects using minute samples that would be irrelevant to the health of a commercial lobster.  If it were found that there are multiple serum and yolk proteins that could be informative about the stage of maturation or the molting cycle, we could proceed to purify them and develop antisera to the individual proteins with the objective of having a panel of antisera that could be used to provide a profile of an individual lobster's serum and egg proteins.  The first year would be spent in developing assays based on eggs and haemolymph sampled from NMFS bottom survey trawled or impounded lobsters of known developmental stage.  We would also develop the methodology to measure the maturation state of the ovaries and correlate these with morphometric measurements that have been used previously to judge maturation.

By the second year we would start evaluating natural populations as well as impounded lobsters which can be followed using the minimally invasive serum serial sampling followed by destructive analysis of their maturation state as a last sample.  The development of discriminant functions to predict the maturation state would depend upon good observational data from impounded lobsters which approach what we wish to think as normal development.  We will attempt to obtain data from as varied a set of normal rearing sites as possible to ensure the robustness of our discriminant functions.  A lobster should not be declared abnormal or in an extreme stage just because the base population used to develop the discriminant function is narrow.

4. Methods

The project will use the general biochemical tools of cell and molecular biology.  Serial serum samples will be obtained using non-intrusive bleeding through the intersegmental membranes of the lobster with a fine gauge needle and a tuberculin syringe.  Only microliters of haemolymph are necessary for the immunological tests of serum proteins but larger volumes (1 ml) may be needed to estimate Me-farnesoate titer or crustecdysone titer.  I have experience measuring the ecdysone from 5 µl samples of cockroach haemolymph (Kunkel, 1975).  Eggs from berried females will 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 (Kunkel & Nordin, 1985; Telfer & Kunkel, 1991).  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) on individual eggs.  Serial sampling from empounded females which have berried at known dates will allow us to relate the embryonic changes that may occur to the adults molting cycle.  This could provide another way to estimate the position of the female in the molting cycle.  Since lobsters incubate their eggs for long periods of time (6-9 months) it will be necessary to cooperate with a facility which has the capability of maintaining identified individuals in a close to normal environmental and nutritional state over those time periods.

We will take megapixel digital images of each lobster sampled along with carapace length and gross weight.  An attempt will be made to apply the new morphometric approach of landmark analysis to digital images of a structure that we chose based on initial sampling and discussions with current experts on lobster development (Josef Idoine, Diane Cowan, ...).  The megapixel images will be taken with an extant Kodak DC-120 camera which allows closeup images. These images will be downloaded into a notebook computer (to be purchased).

5. Anticipated Products

A. We will describe the serum and egg protein titers and their ovarian state of maturation through the lobster molting and reproductive cycles in association with the morphometric data taken on each animal.  This information will be of general interest as biological information about the species.  This material will be prepared for publication in a peer reviewed journal as well as being shared via a lobster WWW page,URL: http://marlin.bio.umass.edu/biology/kunkel/fish/lobster/
B. The data collected in (A) may allow us to establish a discriminant function useful in predicting the reproductive state of individual females as well as reproductive profiles of 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.  A NOAA Technical Memorandum will be provided which outlines in detail the approach that we develop.
C. Male and female serum profiles may allow us to predict an individual lobster's stage in the molting cycle.  This information may be of use in computing the commercial value of the catch or a strategy of holding individuals until their value increases.  A NOAA Technical Memorandum will be provided if appropriate.
D. We hope to eventually apply the dicriminant function approach to particular problems such as obviously off-colored eggs and marginal cases which might be more subjective. Following individuals with this analysis may lead to the data upon which a discriminant function for a disease could be developed.  A NOAA Technical Memorandum will be provided if appropriate.
E. 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 reproductive or molting status.
F. Student participation and education.
Undergraduate and graduate student participation in this research will be implicit to the success of the project.  The PI, Dr Kunkel has directly sponsored 8 PhD students in his 30 years at the University of Massachusetts and has cooperated in dozens more via close collaborations with faculty in the Biology, Entomology and the Biochemistry Departments at UMass as well as international guests to his lab.  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 PhD or 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.

6. Cooperation required from NOAA and other units.

We will cooperate with 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 aboard the Albatross IV.  The PI has participated in the past four bottom surveys in conjunction with a CMER sponsored study of cod maturation and is familiar with using their survey design and data to piggy-back a study on top of the normal routine of a bottom survey.  For example the data plotted in the accompanying figure is the lobster poundage collected on the Albatross IV 1999 Fall Bottom Survey.  We could have taken a serum sample and a megapixel image of the lobsters collected in at least one of the legs of this survey by having a research team member aboard as a volunteer scientist.  In addition, if our sampling kit is simple and straight forward enough, we could request sampling by the scientific crew of a leg in which we do not participate.  The NMF Aquarium at Woods Hole has limited capability to maintain a few lobsters which could be used for long term sampling.  The PI has established cooperation with the Aquarium Director, David Radosh, who has provided housing for cod in a previous CMER project.  Cooperation will be also be enlisted from commercial and institutional lobster pounds and fishing vessels.  Associations such as The Lobster Conservancy and the Lobster Hatchery on Martha's Vineyard will be solicited to establish cooperation which may allow this project to use their facilities for long term sampling of cohorts of larvae or tagged adults maintained in their facilities.  Lobster fishermen will be enlisted to participate at a later stage in testing our sampling procedure on particular catches in order to test our methodology.

Equipment Needs

I am requesting a notepad computer to facilitate the field station and shipboard collection of data and images.  An existing megapixel camera is able to download images captured in the flashcard memory of the camera to the notepad computer via a PCMIA flash memory card socket.  The data and images can then be downloaded to our LAN via an PCMIA Ethernet card installed in the notebook.

Storage and movement of survey and image data will require upgrading our disc storage system. We currently have a reserved 4 Gbyte drive on the Biology Department SparcStation.  To upgrade we must replace the current drive with a larger one. The next size up currently is an 18 Gbyte drive. This will provide us with 14 Gbyte more space. Since we will be storing megapixel images of the lobsters sampled, this storage upgrade is not unreasonable. The larger data flow will also be made more feasible by upgrading our connection to the internet to a 100 Mbyte link. George Drake, Director of the NSM Network, has stated that he will instal the wiring for the 100 Mbyte system in the Fall of 2000 connecting us to the campus backbone at 100 Mbyte.  We would be responsible for purchasing a 100 Mbyte hub for our laboratory LAN.

We use a microfuge to separate serum from blood cells prior to flash freezing in the field.  The microfuge we currently use is over 10 years old and also is needed in the Amherst lab.  We request support to purchase a new model which will also allow 100 µl microtube centrifugation which will facilitate reduced serum sample size.  This will allow us to sample from younger lobster stages in a less intrusive fashion.

7. PROPOSED BUDGET

	YR-1	YR-2	2-YR
	00-01	01-02	00-02
JG Kunkel	$0.00	$0.00	$0.00
Other Personnel: 1 grad student ($15.37/hr)	$15,985.00	$15,985.00	$31,970.00
Geo Fringe benefits ($1.923/hr)	$2,000.00	$2,000.00	$4,000.00
2 undergraduate student trainees	$1,000.00	$1,000.00	$2,000.00
Services:    Antibody Production	$500.00	$1,000.00	$1,500.00
Equipment: Laptop Computer.	$2,000.00	$0.00	$2,000.00
18 Gbyte Hard Drive for SparcStation.	$500.00	$0.00	$500.00
100 Mbyte Ethernet hub	$1,500.00	$0.00	$1,500.00
PicoFuge Microcentrifuge	$450.00	$0.00	$450.00
Supplies	$4,000.00	$4,000.00	$8,000.00
Total Direct Cost	$27,935.00	$23,985.00	$51,920.00
TDC-equipment (MTDC)	$23,485.00	$23,985.00	$47,470.00
Overhead (20% MTDC)	$4,697.00	$4,797.00	$9,494.00
Total Cost			$61,414.00

8. SCHEDULE OF EVENTS
An effective project to determine the major storage proteins of lobster serum and eggs will require at least two years of study initially.  The first year will be used to analyze the measurable items and to develop the micro assays for them.   All assays will be able to be applied to all stages using single eggs or 1 ml serum samples.  The exact assays and the stages upon which particular assays will be performed will depend upon our preliminary sampling in the first year.  Reports on progress will be submitted quarterly to CMER and published along with sample data at an extant WWW site, URL:
http://marlin.bio.umass.edu/biology/kunkel/fish/lobster/.
 

Year 1	1st quarter Oct-Dec, 2000	A. Exploratory analysis using Spring 2000 samples.    (1) SDS-PAGE Characterization of serum and egg components.    (2) Measure lobster egg CaM reactivity in dot blot assay.  B. NOAA Albatross IV Fall Bottom Survey:   (1) collect frontal image from normal NMF surveyed lobsters.   (2) collect serum, ovary and egg samples from select surveyed lobsters. C. Purification of major serum and egg proteins. D. 1st Quarterly report published on WWW.
	2nd quarter Jan-Mar, 2001	A. Histology of lobster ovaries. B. Biology 297c Cell & Molecular Bio Lab analysis of lobster samples. C. Analysis of Albatross IV Survey data. D. Continue purification of major serum and egg proteins. E. 2nd Quarterly report published on WWW.
	3rd quarter Apr-June, 2001	A. Begin antiserum production.  B. Development of  morphometric analysis.  C. Preliminary PUFA analyses on various aged fish. D. NOAA Albatross IV Spring Bottom Survey: E. 3rd Quarterly report published on WWW.
	4th quarter July-Sept, 2001	A. Lobster Hatchery and/or Pound Research.   (1) use antiserum to determine Vg utilization in embryo and larva.   (2) correlate serum protein and ovary development within instar.   (3) follow development of berried lobster eggs; freeze samples.  B. Evaluate antiserum/SDS-PAGE in following instar development. C. 4th Quarterly report published on WWW.
Year 2	1st quarter  Oct-Dec, 2001	A. Develop assay kit for non-destructive sampling of lobsters B. NOAA Albatross IV Fall Bottom Survey:   frontal image, weight, serum, ovary sampling of lobsters. C. 1st Quarterly report published on WWW.
	2nd quarter  Jan-Mar, 2002	A. Analysis of Albatross IV Survey data. B. Biol 297c Cell & Molecular Lab analysis of lobster samples. C. 2nd Quarterly report published on WWW.
	3rd quarter Apr-June, 2002	A. NOAA Albatross IV Spring Bottom Survey. B. Evaluate Albatross IV Survey data. C. 3rd Quarterly report published on WWW.
	4th quarter July-Sept, 2002	A. Continue Lobster Hatchery and/or Pound Research. B. Prepare results for publication. C. 4th Quarterly report published on WWW.

 9. CURRICULUM VITAE:  Joseph G. Kunkel

Born: Oceanside, New York, August 17, 1942.	ZZ# XXX-YY-WWWW
Home: 74 Stony Hill Rd.              Amherst MA 01002	University: Biology Department             University of Massachusetts             Amherst MA 01003
Home Phone: (413) 253-3391	Office/Lab Phone: (413) 545-0468 Email: joe@bio.umass.edu URL: http://www.bio.umass.edu/biology/kunkel/

Education:

Columbia College, New York, New York, Zoology A.B. 1964

Case-Western Reserve University, Cleveland, Ohio, Biology Ph.D. 1968

Dissertation: Control of Cockroach Development

Awards and Honors:

Marine Biological Laboratories Corporation Member, elected 1996; NSF Graduate Fellow, Biology, Case Western Reserve University, 1967-68; R.C.A. Scholar in Chemistry, Columbia College, 1961-62; Columbia University Scholar, Columbia College, 1960-64; New York State Regents Scholar, Columbia College, 1960-64; Bnai Brith Scholar, Columbia College, 1960.

Positions and Professional Experience:

Sabbatical, National Vibrating Probe Facility, MBL, Woods Hole, MA	1993-94
Member, Organismal and Evolutionary Biology Program	1996-present
Full Professor, University of Massachusetts at Amherst	1985-present
Adjunct Professor of Entomology, University of Massachusetts.	1985-present
Sabbatical with B. Lanzrein, Zoological Institute, U. of Berne, Switzerland,	1985-86
Member, Cell and Molecular Biology Program	1983-present
Associate Professor of Zoology, University of Massachusetts.	1976-85
Sabbatical with Alan C. Wilson, University of California at Berkeley	1977-78
Assistant Professor of Zoology, University of Massachusetts, Amherst	1970-76
Lecturer in Biology, Yale University	1969-70
NIH Postdoctoral with Gerry R Wyatt, Yale University	1968-70
Postdoctoral in Biometry, Case Western Reserve University Medical School	1968
Graduate Research Assistant, Biology Department, CWRU, Cleveland	1964-68
Research Assistant to Arthur W. Pollister, Columbia University, NY	1963-64
Research Assistant to Francis J. Ryan, Columbia University, NY	1963

Postdoctoral and Research Associates:

Rolf Koenig, Postdoctoral Research Associate, 1985-86.
Elizabeth S. Bowdan, Senior Research Associate, 1986-1993.
Joseph Zydlewski, Predoctoral Research Associate, 1998-2000.

PhD Students: Raymond Duhamel PhD '79, Don Wojchowski PhD '84, Sharon Karpells PhD '88, Yujun Zhang PhD '92, Rachel Dompenciel PhD '93, Anand Iyengar PhD '96, Ruth Hartling PhD '99, Ellen Faszewski PhD '99.

Kunkel JG and JH Nordin. 1985. Yolk Proteins. in Comprehensive Insect Physiology, Biochemistry and Pharmacology. Chapter 4, Vol.I, eds. GA Kerkut and LI Gilbert, Pergamon Press, pp 83-111.

Kunkel JG. 1986. Dorsoventral currents are associated with vitellogenesis in cockroach ovarioles. in Ionic Currents In Development ed. R Nuccitelli, Alan R Liss Publ., pp 165-172.

Kunkel JG, R Koenig, H Kindle and B Lanzrein. 1986. The role of ions in vitellogenesis and patterning in insect oocytes. Advances in Invertebrate Reproduction 4: 101-108.

Kunkel JG (1988) Analytical Immunological Techniques. Chapter I in Immunological Techniques: Arthropods. Edited by LI Gilbert. Springer Verlag, pp 1-41.

Kunkel JG. 1991. Models of pattern formation in insect oocytes. In Vivo 5: 443-456.

Telfer WH, and JG Kunkel. 1991. The function and evolution of insect storage hexamers. Annu Rev Entomol 36, 205-28.

Duhamel RC and JG Kunkel. 1983. Cockroach larval-specific protein (LSP), a tyrosine-rich serum protein. J. Biol.Chem. 258: 14461-14465.

Wojchowski DM, PA Parsons, JH Nordin and JG Kunkel. 1986. Processing of Provitellogenin in Insect Fat Body: a role for High-Mannose Oligosaccharide. Dev. Biol. 116: 422-430.

Koenig R, JH Nordin, CH Gochoco and JG Kunkel. 1988 Studies on ligand recognition by vitellogenin receptors in follicle membrane preparations of the German cockroach Blattella germanica. Insect Biochem. 18, 395-404.

Kunkel JG and E Bowdan. 1989. Modeling currents about vitellogenic oocytes of the cockroach, Blattella germanica. Biol. Bull.176(S): 96-102.

Anderson M & JG Kunkel. 1990. Cleaning insect oocytes by dissection and enzyme treatment. Tissue & Cell 22: 349-358.

Bowdan E & JG Kunkel. 1990. Patterns of ionic currents around the developing oocyte of the German cockroach, Blattella germanica. Developmental Biology 137: 266-275.

Kindle H, B Lanzrein and JG Kunkel. 1990. The effect of ions, ion channel blockers, and ionophores on uptake of vitellogenin into cockroach follicles. Developmental Biology 142: 386-391.

Siegel E, R Baur, JG Kunkel, H Kindle, and B Lanzrein. 1990. Demonstration of a voltage dependent calcium current in follicles of the cockroach, Nauphoeta cinerea. Invert. Reprod. Devel. 18: 159-164.

Zhang Y and JG Kunkel. 1992. High abundance calmodulin from Blattella germanica eggs binds to vitellin subunits but disappears during vitellin utilization. Insect Biochem. Molec. Biol. 22: 293-304.

Zhang Y and JG Kunkel. 1992. Program of F-actin in the follicular epithelium during oogenesis of the German cockroach, Blattella germanica. Tissue & Cell 24: 905-917.

Zhang Y and JG Kunkel. 1994. Most egg calmodulin is a follicle cell contribution to the cytoplasm of the Blattella germanica oocyte. Developmental Biology 161:513-521.

Anderson M, E Bowdan and JG Kunkel. 1994. Comparison of defolliculated oocytes and intact follicles of the cockroach using the vibrating probe to record steady currents. Developmental Biology 162:111-122.

Bowdan E and JG Kunkel. 1994. Ionic components of dorsal and ventral currents in vitellogenic follicles of the cockroach, Blattella germanica. J. Insect Physiol. 40:323-331.

Kunkel JG and PJS Smith. 1994. Three-dimensional calibration of the non-invasive ion probe (NVPi) of steady ionic currents. Biol. Bull.

Kunkel JG and E Faszewski. 1995. Pattern of potassium ion and proton currents in the ovariole of the cockroach, Periplaneta americana, indicates future embryonic polarity. Biol. Bull. 189:197-198.

Iyengar AR, and JG Kunkel. 1995. Follicle cell calmodulin in Blattella germanica: Transcript accumulation during vitellogenesis is regulated by juvenile hormone. Developmental Biology 170: 314-320.

Kunkel JG and EE Faszewski. 1995. Pattern of potassium ion and proton currents in the ovariole of the cockroach, Periplaneta americana, indicates future embryonic polarity. Biol. Bull. 189:197-198.

Hartling, RC, JJ Pereira, and JG Kunkel. 1997. Characterization of a heat-stable fraction of lipovitellin and development of an immunoassay for vitellogenin and yolk protein in winter flounder (Pleuronectes americanus). J. Exp. Zool. 278: 156-166.

Feijó JA, J Saínhas, GR Hackett, JG Kunkel and PK Hepler. 1999. Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip. J. Cell Biol. 144(3):483-496.

Cardenas L, Feijó JA, Kunkel JG, Sanchez F, Holdaway-Clarke T, Hepler PK, Quinto C. 1999. Rhizobium nod factors induce increases in intracellular free calcium and extracellular calcium influxes in bean root hairs. Plant J 19:347-52.

Roy SJ, Holdaway-Clarke TL, Hackett GR, Kunkel JG, Lord EM, Hepler PK. 1999. Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis. Plant J 19:379-386.

Hartling RC and JG Kunkel. 1999. Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus. J Exp Zool. 284:686-95.