Miller Report

### U.S. GLOBEC: Copepod population biology on Georges Bank

    INVESTIGATOR:

    Charles B. Miller
    Oregon State University
    Corvallis, OR

    GRANT PERIOD: 1 September 1993 - 31 August 1996

#### STATEMENT OF OBJECTIVES

The original project summary reads as follows:

It is proposed to participate in the management and data analysis of the GLOBEC Georges Bank Broad Scale Survey. This will include (1) work on the formation and management of the Georges Bank Plankton Technical Group (GBPTG) at Woods Hole, and (2) studies of the population biology of Calanus finmarchicus, Pseudocalanus moultoni, and Pseudocalanus newmani using the very large sample set to be collected by the Broad Scale Survey. I will be a major author of the technical handbook for the GBPTG, and will participate in data development and analysis throughout the Georges Bank Survey. In specific studies of copepods I will use the broad scale survey preserved sample set, plus specially preserved samples and subsamples, to examine aspects of C. finmarchicus and Pseudocalanus spp. life history, growth, and production biology as functions of season, temperature and stratification, location over or off the bank, food availability, and hydrography. I will apply gonad analysis of late copepodites, vitellogenesis staging of females, trithek/quadrithek evaluation of females, stage- and size- frequency analyses, molt phasing of jaws, jaw development facies indicative of diapause and lipid (specifically wax ester/triglyceride) quantification. Goals will include comprehensive estimation of secondary production by C. finmarchicus and Pseudocalanus spp. to allow comparisons among seasons, years, and sites across Georges Bank.

#### STATEMENT OF WORK:

In Year 1 the PI and project technician (Ms Cheryl Morgan) participated in the May-June 1994 Process Cruise due to the delay of Broad Scale Survey sampling until 1995. Collections were primarily taken and used for a study of storage lipid accumulation in C. finmarchicus. A data report on our lipid results has been prepared and distributed to GLOBEC PI's. A reader of this summary can get a report by writing the PI. I believe it was also scanned onto the World Wide Web. In brief:

Lipids:

Statistical considerations forced us to move from analysis of C. finmarchicus older copepodites in groups of five to analysis of single individuals. Calibration curves for the Iatroscan flame ionization detector (FID) are non-linear when rod loading is light. We developed a system for using power functions as calibration curves (previously done but not specified by Ohman). Analysis of single individuals removed intrasample averaging, which allowed us to show an inverse relation between the fraction of storage lipids present as triglycerides (TAG) and total storage lipid weight, that is, TAG plus wax esters (WE). This suggested that the bulk of storage lipids in specimens with fully stocked oil sacs must be wax ester, and that all of the oil in the lipid sac probably is wax ester. This was also shown earlier by Benson (who published no supporting data, however). We demonstrated this partition of lipid types by drawing lipid from the oil sac with micropipettes before analysis. This greatly reduces up to ca. 95%, depending upon how cleanly the withdrawal is done) the amount of TAG and eliminates phospholipids relative to analysis of the whole body. Certainly, if the withdrawal could be done with no contamination from other body fluids, the oil sac would be found to contain virtually pure WE.

An additional finding from analysis of single individuals was a discrepancy between video estimates of lipid content (volume estimated from a measure of oil sac projected area, converted to weight by a normal lipid density) and Iatroscan estimates. Video estimates of WE in the oil sac were 1.5 to 2 times greater than Iatroscan estimates for the whole body. Video estimates were much closer, approximately correct, for lipids determined gravimetrically by extraction. This shed some doubt upon the standard Iatroscan method. Work is still in progress on this problem. We have shown that it is a difference in the response of the Iatroscan FID detector for different WE molecules. Iatroscan peak areas for WE have been calibrated by us and others against palmitic acid stearyl ester (16 carbon acid, 18 carbon alcohol, both saturated), which is cheap and readily available. Unfortunately, copepod waxes are mostly 20:5 acid and 22:1 alcohol, which are not commercially available. Longer chains give less FID area per unit weight. We are in the process of extracting and purifying the natural mixture of copepod waxes to use as an Iatroscan standard. This is a 'band-aid' measure to save our extensive data. All of the considerable literature data from this instrument calibrated with short, saturated waxes is almost certainly trash. In the end, studies of general lipid classes in copepods will have to be done with gas chromatography, relegating use of Iatroscan for this purpose to realm of good but flawed ideas.

In Year 2 the P.I. and Ms Morgan participated in seven cruises of the Georges Bank Broad Scale Survey. The first was a training cruise in November 1994. Others on the survey itself were one cruise in each month from February through July (July cruise departs Woods Hole on 10 July). The PI was chief scientist on the March cruise aboard R/V ENDEAVOR sailing from Rhode Island. In the laboratory ashore we have concentrated on (1) video and Iatroscan analysis of storage lipids in Calanus finmarchicus. Other work has been sample evaluation with emphasis on (2) the fraction of Calanus females with quadrithek antennae (see below), (3) jaw and gonad development staging of Calanus fifth copepodites (C5) as indicators of diapause and immediate maturation. None of the results is ready for distribution or entry in the Georges Bank data base. However, some preliminary results can be reported from each of these studies:

Quadritheks:

Samples from all seasons showed no difference in the fraction of females bearing quadrithek setation of the first antenna. Fleminger suggested that this character could be evidence of switching by genetic males to female maturation, since the quadrithek pattern is borne by all males. The quadrithek pattern has two aesthetasc setae on segments 2b, 3, 5, 7 and 9, while the trithek pattern has only one aesthetasc on these segments. Hypothetical selective benefits of switching to female function in the adult phase all suggest that the fraction switching should vary with generation and, thus, with season. So far, we have not found such variation, the fraction of quadrithek females running 8-14%, mean of 11%, in all months. We have not looked yet at samples from the Georges Bank process study cruise of January 1995, which contained the females maturing earliest from the 1994 diapausing stock. Under the principal hypothesis for advantage, these females should show the most change.

Diapause entry:

Both in June 1994 and in May 1995, when the second generation of those years were finishing development, we found C5 taking two developmental routes. Some were entering diapause, some maturing directly. There is evidence of this split both in formation of new jaws (an indication of maturation occurring in a sizeable fraction of the surface stock) and retention of the postmolt facies (an indicator of diapause). Deep living C5 had heavily loaded oil sacs and tiny gonad anlagen. Some shallow living ones approximated the appearance of the deep ones, others had substantially enlarged and sexually differentiated gonads.

Most of the work from the end of the July cruise until the 1996 Broad Scale Survey begins will be devoted to study of sexual switching and stock division into maturing and diapausing groups at the ends of generations. We will also carry through an ovarian activity analysis for our collection of sorted females which is intended to expand the utility of egg production rate estimates produced by Dr. Jeffery Runge on the Georges Bank Process Study cruises. Our intention to work extensively with Pseudocalanus, as well as Calanus, has run aground on our inability to distinguish the two species known to be living on and around Georges Bank: P. moultoni and P. newmani. We can't even distinguish the adults of either sex on a reliable basis. The small urosomal hairs whose length distinguishes these species are too frequently rubbed off for reliable ecological work based upon them. After substantial effort to acquire a "gestalt" by which the species might be distinguished, we have given up. We await a molecular probe, although that may not allow sufficient counting to be ecologically useful.

The PI and project technician (to be appointed; Ms Morgan intends to quit on 15 August) will participate in all 1996 Georges Bank Broad Scale Survey cruises. We strongly urge NSF and NOAA to sustain the intention to field a Broad Scale Survey in both 1996 and 1997. The continuity of our time series will be extremely valuable to the study of Calanus, as well as to the group effort on cod and haddock. In any case we will continue to work on aspects of later life history in Calanus finmarchicus. We will work on Pseudocalanus biology as opportunities present themselves.

Finally, a peripheral effort is intended to illuminate distributional data for C. finmarchicus adults when it becomes available. This is a study of how copulation is accomplished in Calanus spp. Apart from demonstrations that pheromones are important in copulation, the mechanics are unknown. Using a 1.3 m plankton "Kreisel", the PI and Dr. Atsushi Tsuda of the University of Tokyo have succeeded in observing the male search patterns and mode of approach to females.

Summary of Key Findings: The foregoing is brief enough.