Joseph G. Kunkel^*, Josef Idoine^a,
Jeff Xu^* and Diane Cowan^b

^* University of Massachusetts Amherst
^a National Marine Fisheries, Woods Hole
^b Lobster Conservancy, Friendship ME

Lobster adult physiology is dominated by the alternation of molting and reproduction. The commercial harvesting of this valuable resource must navigate the imposed fishing regulations and market forces that change the value a lobster in hand. It is illegal to have a "egger" lobster in your possession in the USA or Canada. Lobsters carrying eggs are to be left at sea and if they extrude eggs while in your possession they must be released or the possessor faces a tax. A newly molted lobster is recognized by its thin and soft-shell. Such soft-shelled lobsters have less meat and are full of haemolymph; they are sold at a deep discount to the more mature lobster, which has filled in the blood space with lobster meat. For this reason the prediction of the lobster’s stage within the molting/reproductive cycle could be extremely useful to the commercial lobsterman. If the procedure for establishing the stage were simple and cheap enough it could help establish a more accurate grading system which might leave more lobsters at sea, helping to maintain the species’ population.

To develop this stage discrimination procedure we have combined the resources of the Lobster Conservancy Friendship Lobster Laboratory, the National Marine Fisheries Northeast Bottom Survey and a University of Massachusetts Amherst biochemical laboratory. Each component provides a critical resource to the project. It seems critical to establish the properties of molting and reproductive cycles of natural populations rather than the behavior of laboratory maintained animals. In natural populations it will be essential to be able to identify individual animals prior to and after molting. For that reason we are using PIT tagging to locate and identify animals in a lobster pound environment. Physical and biological information is being accumulated to accompany each identified animal.

The information to be used to stage a lobster will include the traditional measures including weight, length and cuticle color and texture. These methods have been used in the traditional grading system. We have added the use of haemolymph properties to the traditional list, including color spectrum and immunological assay of serum components. Only longitudinal studies will tell whether any of these properties are useful to predict the animal’s stage of reproduction or molting.

Initial results suggest that the color of the haemolymph will be able to aid in predicting the reproductive stage of females. Females developing eggs internally have been shown to have orange haemolymph, Fig 1, which most likely corresponds to accumulating vitellogenin (Vg) which is often a carotenoid containing lipo-protein. In addition different degrees of blueness can be perceived in lobster hemolymph which may correspond to the titer of the serum protein hemocyanin (HbCy), Fig. 1.

The significance of the differences in the serum spectra is that they provide the potential for predicting the stage of the lobster in any molting/reproductive cycle. The orange serum was obtained from a lobster whose ovaries were clearly well developed with approximately 1-mm oocytes clearly visible in the transected ovary. This spectrum has no visible peak in the blue region, which is marked by the cupric chloride spectrum. This dark orange serum’s spectrum does have an increasing ramp of transmission toward the red wavelengths, which is what results in its visible orange color. The dark blue serum has the most distinct blue transmission peak and the least amount of the ramp toward red. It is not clear if this dark blue serum spectrum is actually free of a Vg component or if the ratio of HbCy:Vg is high enough to mask any visual trace of orange to the eye. A lighter orange serum’s spectrum has a small transmission peak in the blue region indicating that the two colors can coexist in one serum's spectrum. It is to be determined if the spectrum can be decomposed by careful construction of color standards to which it can be compared by a trained eye. Prior to that eventuality the HbCy and Vg need to be purified and have their inate spectrums determined. In addition antisera to the principal serum proteins will be made so that their titer can be estimate immunologically and correlated with the color transmission properties.

Figure 1. Lobster serum transmission spectra. Female lobster serum was obtained from three lobsters, which represented points in a continuum of serum colors. One extreme serum was visibly dark orange. Another extreme had bright blue serum. An intermediate light orange serum clearly had some blue and orange combined. Spectra of a colorless bovine serum albumin solution (1 mg/ml) and a cupric chloride solution (0.1 F) were provided as controls.

Another consideration in the development of this analytical approach will be the ease of obtaining the sample. If the method is not simple and convenient, it will have trouble being introduced into the routine of processing large numbers of lobsters. Inexpensive 1 ml syringes are available for obtaining serum. The serum clots extremely rapidly at some stages of lobster development. The NOAA Ship Albatross IV does the Spring and Fall Bottom Surveys of the prime lobster territory of the Gulf of Maine and George’s Bank, Fig 2. On board there was convenient space and electricity to allow a portable microfuge to be used to quickly separate the serum from the blood cells, which are the source of the major clotting components. This was found to be a completely effective method of preparing clear serum to evaluate for its visible light transmission. However, requiring a centrifuge in order to prepare serum in the field was considered a complication and expense that might make the procedure impractical. As an alternative to centrifugation was to separate cells from serum by forcing the haemolymph, obtained as above via syringe, through a syringe filter soon after the haemolymph was drawn. This method was also found to be an effective method for producing a clear serum from which color transmission could be evaluated.

Samples of the collected hemolymph were run on a gel filtration to determine the sizes of the molecules involved at particular stages and serum colors (blue, green and orange), Fig. 3.  Further research is necessary to confirm the identity of the orange and green serum proteins which seem to be diagnostic of imminent berrying (deep orange) and involution of retained eggs (green).