The sera of lobsters are variably colored. Lobster sera are mostly colored light blue or light orange or are clear. The serum color is somewhat masked by translucence derived from blood cells which create points of light refraction. When freshly drawn lobster hemolymph is kept chilled and centrifuged quickly, the hemocytes are removed and a clear serum results which reveals its color, Fig 1. However some lobster serum is a darker blue, a medium orange to deep orange, or light to dark green. These colors are suspected to correspond to specific physiological states of the lobster. The dark blue would correspond to Hc charged with oxygen in a metabolically energized lobster; it has a dominant transmission at wavelength 490 nm, similar to the free cupric ion. Most often lobster serum is not blue (i.e. not oxygenated or lightly oxygenated) but the Hc can be turned to its maximal blueness by treating it with oxygenated buffer. This may allow us to develop a simple field absorbance assay for Hc. The deep orange serum is most likely associated with the transport of Vg through the serum en route to the ovary. Most Vgs carry carotenoids dissolved in their lipid moiety providing a yellow to orange color. In my experience from over 200 samples collected on two cruises aboard the Albatross IV in the Guff of Maine, a medium to deep orange color has been found most commonly in female lobsters which have well developed ovaries and is not found in females carrying eggs externally. Such orange sera have transmission spectra that have no maximum in the visible spectrum but exhibit a monotonicly increasing transmission from 400 nm up through 750 nm. On the other hand, it is suspected that green serum corresponds to a state of egg resorption since it has never been found by us in samples from lobsters freshly caught at sea, but has been observed in serum samples from a captive female, which did not eventually produce eggs. The green sera exhibit a transmission spectrum that has two peaks one in the green near 550 nm and one in the violet region near 400 nm. These striking color differences need to be related to the exact physiological and behavioral state that they reflect. It is possible that a simple color code could be developed to allow fishermen to avoid the critical berrying and molting phases based on serum color. Indeed the orange serum of a ripe lobster can, with a trained eye, be seen directly through the intersegmental membranes. Certainly the confusion between whether green or orange serum reflects a female close to berrying needs to be cleared up. More extensive sampling of females from trawls at different times of the year and from sequential sampling our PIT tagged lobsters, in close to normal growth conditions, should allow determination of whether green hemolymph is a normal phase of the lobster reproductive cycle.
 

Arthropod serum proteins tend to be large ranging from 400 KD to 1,000 KD. Separation of the proteins using gel permeation chromatography (Fig 2) is effective in demonstrating the major serum proteins and also in purifying  those proteins when used in combination with other techniques such as ion exchange chromatography (Kunkel and Pan 1976). Blue serum reveals primarily a large peak soon after the void volume (0.15 Kd in Fig 2) which engenders the blueness of the serum and is undoubtedly Hc, perhaps intermixed with the colorless (Cu free) PHc. Vitellogenic female, deep orange, serum exhibits a large peak which penetrates the gel to about 25% (0.25 Kd in Fig 2) of its volume. This 0.25 Kd protein is the correct size for lobster Vg. Green serum includes a peak which penetrates about 33% of the gel volume (0.33 Kd in Fig 2) and is likely the smaller sized Lv or a degradation product which has been released from, non-ovulated, involuting-eggs. The development of specific antibodies for these individual proteins will allow us to measure their relative and absolute titers in these complex mixtures. Vg and Lv are potentially made up of the same gene products perhaps modified by changes in peptide sizes due to modification by proteases, kinases, phosphatases and glycosidases. It must be established via standard biochemical and immunochemical methods whether the two known lobster Vgs can be distinguished from the serum form of Lvs that we propose are in green lobster hemolymph. Separation of the proteins on SDS-PAGE and probing with our antibodies via western blotting should establish the identities of the Vgs and Lvs.
 

The large proteins of arthropod serum are not well separated using polyacrylamide gel electrophoresis (PAGE) since their huge size does not allow adequate penetration even in relatively dilute acrylamide gels. Agarose horizontal or vertical slabs have been use effectively to separate the major serum proteins of insects (Duhamel and Kunkel, 1983). This methodology allows the identified native proteins to be further identified using antibodies raised against the purified forms of these proteins. A NOAA funded project is currently supporting the production of antisera against the major lobster serum proteins (Vg, Lp, Hc, PHc) to be used in this project for screening protein titers using quantitative immunoelectrophoresis (QIEP) (Kunkel, 1987) or ELISA depending on the quality of the developed antisera.
 

The rigid lobster exoskeleton grows in discrete stages interspersed by molting, but the growth of tissue is more continuous and depends upon the availability of food and nutritional reserves. Nonetheless a newly molted lobster increases the blood space volume in order to inflate the new cuticle to its new size and therefore the weight of the newly molted lobster weight reflects its new blood volume rather than any tissue growth. During the soft phases of the lobster molting cycle the lobster does not tend to feed and will be depending on stored nutrient. This is a time, if lobsters are similar to insects, in which the serum storage proteins decline as they are used to provide the raw material for growing tissues despite feeding being curtailed. The production and extrusion of eggs are female specific phenomena, which are overlain on the molting background. The eggs grow gradually competing with nutrition for somatic growth of the female and displacing some blood volume.

Water relations during the terminal maturation of eggs may produce a stage of anomalous sudden heaviness when eggs in the females take up water just prior to extrusion. The absolute timing of this process is not established; however, a number of potential instances of this phenomenon have been identified in our data from Albatross IV and captive lobsters. If confirmed, this property of females may be a valuable indicator of imminent egg extrusion when combined with other diagnostic factors. The carapace length and weight of lobsters will continue to be important data to factor into our understanding of the molting and reproductive cycles.
 

We need a convenient source of lobsters used to correlate serum protein titer and ovarian and molt physiology. The National Marine Fisheries Spring and Fall Bottom Survey cruises, Fig 3, offer an opportunity to study the  serum, ovaries and pleopod morphology of lobsters captured by otter trawl which will allow us to validate the ovarian and cuticular molting stage of animals for which we have single serum samples. The third two week leg usually covers George's Bank and the fourth leg usually covers the Gulf of Maine. The short 30 minute otter trawls predictably collect large numbers of live healthy hard-cuticle lobsters. One benefit of the Albatross IV collection trips is the 34 years of continuous records of random sampling for lobsters in the Gulf of Maine study area. Another benefit is the scrupulous detail of the records collected on the NOAA National Marine Fisheries Service (NMFS) sponsored trips. A new shipboard data collection and management system (FSCS) was introduced and beta-tested during a recent NMFS Fall Bottom Survey cruise (AL0006) on the Albatross IV in which one of us (Joe Kunkel) participated as a volunteer scientist. Substantial steps in the data and sample collection and storage have been automated in the FSCS protocols, which will go into general use in the Spring or Fall of 2001. Each bar-coded sample collected, e.g. Fig. 3, can be referenced to a particular collection station whose latitude and longitude and time of sampling are maters of NMFS database records. Previous protocols required a recorder to prepare labels in pencil or pen under less than ideal weather and writing conditions.

Unfortunately, lobsters which are close to molting in either direction, soft shelled lobsters, are often damaged by the trawl process and are not able to be sampled accurately for correlating serum, ovaries and cuticle state. The sera of soft-shelled stages will need to be studied with the cooperation of commercial lobster trappers or through operation of a lobster boat that belongs to TLC. On these non-NMFS collection trips we will be more totally responsible for recording the time, physical location and conditions at the sampling site.
 

We have initiated a research database (Fig 4) of lobster information collected in our cooperative project. The database currently includes:

1. Records of serum sample and standard morphometric measures on lobsters from 2 Albatross IV survey cruises.
2. Records of serum sample and morphometric measurements from several data collection visits to the TLC’s Lobster Life Study Center at Friendship Long Island (Maine) and the Massachusetts Lobster Hatchery at Martha’s Vinyard (Massachusetts).
3. Lobster catch data from several Albatross IV survey trips extracted from NMFS survey database.
4. Equipment, supplies and methodology descriptions.

Currently a visitor can search online (Fig 4) for the lobsters sampled during a particular cruise or year or of a particular size range, sex or stage of reproduction. A list of all the lobsters in those categories are listed and the locations that they were caught plotted on a map of the Gulf of Maine/ George's Bank. This will allow lobster fishermen, volunteers and interested individuals to access survey information about lobsters in general as well as follow some of the growth, biochemical and behavioral cycles of individual lobsters in our lobster PIT tag project. This publication of the raw data will provide interested visitors and volunteers the type of data we collect. Perhaps some particular data in the browsed table or figure will have been worked on by the browser as volunteer. This sharing the ownership of the project is an important aspect of getting students excited about doing research. It will be necessary to involve the fishermen and public more intimately in this project if our findings are to have a useful effect on the industry and attitudes toward protecting this species for the future.

More elaborate password protected queries and maintenance pathways to the database tables (Fig 5) are available to the principle participants in the current project. Administrators of the database have the online option to create new  database tables, records, fields and relations. Menu driven options allow the administrator to establish new database tables which might contain the behavioral observations that we are making on the PIT tagged lobsters which have been recently deployed in the Lobster Life Study Center lobster pound. The database is made a living document by providing a way for collaborators to post and share new data with each other as immediately as they desire. The public is allowed access to only that data which is targeted by the proscribed SQL queries. This developing database structure will be available for further elaboration by natural science and IT students and will be a method of introducing the needed technical expertise into the discipline through its continued maintenance at the University of Massachusetts which has an active IT research and education program. With several hundred lobster serum samples already taken during our preliminary phase we must prepare for the flood of samples we will be taking when our diagnostic antisera are available and our data collection phase starts in earnest.
 

Lobster size limit regulations include both a minimum (83 mm CL) and maximum (127 mm CL) size at which lobsters may be harvested. Growth parameters, including molt increment (stepwise increase in size at ecdysis) and molt interval (duration of time between ecdysis) are lacking in lobsters larger than the maximum legal size.

We have modified a procedure developed to internally tag salmonoid fishes for use in lobsters. Passive integrated transponder (PIT) tags are microchips with a 10 digit alphanumeric identification code. We are injecting PIT tags into the muscle tissue of the distal segment of the first right walking leg. The size of the lobster were are able to tag is therefore dependent on the diameter of periopods – and corresponds to lobsters of minimum legal size

In 1999 and 2000, we tested PIT tag retention and measured molt increment in 15 lobsters measuring between 83 and 145 mm CL and weighing from 478 – 3824 g. None of the 1999 lobsters molted. In 2000, five individuals molted and retained their PIT tags (Table 1). These animals were held in cages for close observation.
 

Table 1. Growth of PIT tagged lobsters retaining the PIT tags through a molting cycle.
 

PIT Tag ID	Sex	Date of Ecdysis	Premolt CL	Postmolt CL	Molt increment	% length increase
414808010F	male	Nov 8, 2000	132.65	144.2	11.55	8.7
41394E2457	male	Sep 26, 2000	85.2	90.1	4.9	5.8
413944791F	female	Oct 16, 2000	92.4	94.7	2.3	2.5
411F322343	female	Oct 8, 2000	83.3	89.5	6.2	7.4
4138734E0F	female	Oct 26, 2000	90.1	100	9.9	11