Blades Report

### Investigations of environmental cues and physiological processes that regulate larval diapause in Calanus finmarchicus.

    INVESTIGATORS:

    Pamela Blades-Eckelbarger
    University of Maine
    Darling Marine Center
    Walpole,  ME  04573
    Pamela@maine.maine.edu (Internet)

    Nancy H. Marcus
    Dept. Oceanography
    Florida State University
    Tallahassee,  FL  32306
    Marcus@Ocean.fsu.edu (Internet)

    GRANT PERIOD:  September 1, 1993 to September 1, 1997

### STATEMENT OF OBJECTIVES:

The major objective of this research is to develop a better understanding of the environmental cues and physiological processes that regulate larval diapause in Calanus finmarchicus. Our approach is to conduct experiments on individuals that have been cultured in the laboratory under controlled conditions simulating seasonal changes of temperature and photoperiod. The immediate objective for year 1 was to design, construct and test such a culture system. The goals for the following three years are to conduct experiments on lab-raised generations of C. finmarchicus designed to elucidate the factors (photoperiod, temperature, diet) responsible for the induction and termination of diapause; and to use light and electron microscopy to examine and compare the morphology of the reproductive systems of juveniles and adults from both lab-raised and field populations.

### SUMMARY OF KEY FACTORS:

Three generations of Calanus finmarchicus (G1 to G3) were raised between January and December of 1994 in the unique, flow-through culture system that was developed during the first year of the project. The system was designed to simulate ambient temperatures and provide natural-light photoperiods. The diets of the G1 (mid-January to mid-June) and G2 (mid-June to mid-August) populations were supplemented with three species of lab-cultured dinoflagellates, whereas the G3 population was reared on natural food items entering the culture tanks via the flowing sea water system. By early December 1994, most of the G3 population had matured to adults, but approximately 1/3 of the individuals remained in the CV stage. These did not molt into adults until early January 1995. Within the CV group, two distinct morphs were identified by their gross morphology. CVs with very large lipid sacs and small gonads were categorized as "fat" CVs, those with little or no lipid storage and medium to large gonads were called "thin" CVs. These two morphs have also been observed in field populations by us and other GLOBEC PIs conducting field collections.

Our evaluation of the culture system at this time is that it is successful to a limited degree. The animals grow very well from CI to adult (eggs to CI were raised in an incubator separate from the culture tanks), however, we experienced the classic problem of a skewed sex ratio with relatively few males emerging from the terminal molt. The sex ratios for the G1 and G2 generations were 1: 20 and 1: 16 respectively. There were no males in the third, and thus last, generation. Even though the sex ratio appeared too low, the males in the first two generations inseminated enough females to produce each subsequent generation. We concluded that if the sex ratio of the culture system is not representative of field populations, then one or more factors relevant to the normal physiological state of Calanus are altered or missing in the culture system. We were concerned that the generations reared in the tanks were not growing under truly "natural" conditions, and felt that this may bias the planned diapause experiments. Based on results of the culture work, we modified the research plan for year 2, and directed more of our efforts towards achieving a better understanding of the factors that influence sex determination in C. finmarchicus, and that ultimately control their basic life history.

In addition to continued laboratory work, field collections were made every 4 to 6 weeks at a 50 meter deep site in the Gulf of Maine, off the mouth of the Damariscotta River. Morphological observations were made on individual, live C. finmarchicus from both field-collected and laboratory-reared populations with respect to the relative size of the gonad in stages CIII through adult, as well as the presence of "fat" and "thin" CV morphs. Results of these analyses will reveal the extent of variability between individuals within the same population and life stage. Specific details of the projects undertaken up to the present time are described below.

#### Sex determination in Calanus finmarchicus

##### Fine-tuning the culture system.

During the first year, there was concern that stages younger than NVI could not swim against the outflow current in the culture tanks. To insure their survival, we reared Calanus from egg to NVI or CI in 4 liter containers held in incubators that were kept at ambient temperatures but did not provide the animals with a natural photoperiod. The copepods were released into the flow-through culture tanks upon attaining the NVI or CI stage. The fact that relatively few males molted in these populations suggested that factors involved with sex determination may act upon the NI to CI stages.

To test this assumption, another culture was started in February 1995 with eggs spawned from field females collected in late January 1995. This time however, the early stages were raised in the culture tanks while contained in 1.5 liter, clear plastic jars fitted with 100 micron mesh caps. As before, the cohorts were set free in the tanks when the NVI and CI stages were reached. Hence, all stages from egg to adult were raised in the same conditions of natural-light photoperiod, temperature and diet. Inventory of the tank was taken in early June and revealed mostly females, some males (a 1:13 ratio), CVs and fewer CI, II and CIII. The surprise was that the tank also contained several hundred NI through NV, mostly NIII, indicating that reproduction was occurring in the tank and that the younger stages were able to avoid the outflow current. All of these copepods were returned to the tank to allow this new generation to grow. We will follow its development throughout the summer. It is clear from this experiment, nevertheless, that raising all stages under the same conditions did not greatly increase the number of males, at least in this one instance.

##### Determination of developmental stage at which sex is fixed.

In order to better understand the process of sex determination in Calanus, we initiated experiments with field collected individuals that are designed to establish at what developmental stage the gonad is programmed as a testis or ovary and is no longer influenced by external factors. We collected CVs from the field on three occasions (September 1994, March and April 1995) and held them in 4 liter containers at ambient temperatures, with no food, until they molted to adulthood. The September collection was particularly interesting in that the majority of the CVs were "fat" with very large lipid stores and no visible gonad. They all had molted to adults three weeks later with a sex ratio of 1:3. Both "fat" and "thin" CV morphs were found in the March and April 1995 tows. Both morphs in each collection carried very small gonads, and all molted within a month to adulthood. The sex ratio for the March group was 1:1.3 and 1:2.5 for the April group. The results of all three molting experiments imply that sex is fixed during or before the CV stage in the field. We are presently repeating this experiment with younger stages and will continue these experiments in year 3. The younger stages will be held in the culture tanks under the same conditions as those for rearing animals from the egg stage.

##### In search of an androgenic gland.

Concurrent with the above experiments, field-collected and tank-reared copepodites and adults have been fixed and embedded for light and electron microscopy to evaluate ontogeny of the gonad and attempt to identify an androgenic gland. In higher crustaceans this gland, often a group of distinct cells associated with the gonad, secretes a hormone that signals the gonad to become a testis. The gonad becomes an ovary in the absence of this hormone. More individuals will be fixed, and sectioning will be completed in years 3 and 4 to locate this gland, if it exists, and to compare gonad morphology between tank-reared and field-collected Calanus.

#### Developmental rate vs. food quantity at ambient temperatures.

A field collection of Calanus on September 20, 1994 was composed of stages from CIII to adult males and females, with the CVs comprising the major component of the population. As noted above, most of the CVs were "fat" and it was assumed that these must be from a population preparing to enter dormancy. We calculated that eggs spawned from the females would still have time to reach the CIV or CV stage in the Gulf of Maine in time to overwinter. Approximately 100 females were sorted and held in egg separation chambers that were kept in the incubators at ambient temperatures. The females were fed a mixed diet of dinoflagellates for maximum egg production. The eggs were reared through NVI or CI under the same conditions as the females.

##### Experiment 1: Ambient temperature, low food quantity (not supplemented)

During the last week of October and first week of November, the first cohorts were transferred as NVI and CI to the flow-through culture system. No supplemental food was added to the culture tanks so that this group of Calanus were allowed to grow under conditions simulating late autumn to winter photoperiod, temperature, and food. The tanks were periodically sampled to follow the development of the copepods. On December 20, the tanks contained stages CI, CII, and CIII. On January 17 1995, they were mostly CIV and a few CV (both "fat" and "thin" morphs). A full inventory on March 8 revealed 265 CVs and 262 females (no males). The majority of CVs were "fat" carrying a very large lipid sac, small to medium gonad and their guts were full. The CVs were returned to the tank until March 27 when a count of 202 females, 1 male and 34 CVs was taken.

##### Experiment 2: Ambient temperature/ high food quantity (supplemented)

The very last cohorts from the females collected in September were spawned during the second week of November. These were reared through adulthood in the 4 liter containers held in the same incubators as the females. Conditions in the incubator simulated ambient temperature but not photoperiod. These cohorts were fed a super-saturated diet of mixed dinoflagellates. Approximately 4 weeks after they hatched, the cultures contained stages CIV, CV and adult females. By the first week of January 1995, all had molted to females, who were observed to be in stage 6 of oocyte maturation.

Summarizing these two experiments: It took approximately 25 to 27 weeks for cohorts spawned in mid- to late September and reared in the flow-through culture system to reach the adult stage. It took only 6 to 8 weeks for eggs from the same females, but raised in the incubators, to reach adulthood. Temperature conditions were the same between the two experiments, the differences being photoperiod and the quantity and composition of the food items. We do not consider the long period to adulthood in the culture tanks as representing a diapause state since the copepods were actively swimming and feeding, and there was little storage of lipids until "fat" CVs were found in March. We believe that the limited quantity and composition of food present in the incoming sea water at that time of year influenced the developmental rate. The animals appeared to have only enough food for maintenance and slow growth.

Our culturing data and field observations to date, suggest that, provided with abundant food, Calanus finmarchicus will continue to reproduce in the laboratory at any time of the year regardless of seasonal changes of photoperiod and temperature. In the remaining two years of this project we will focus on the effects of diet on the developmental rate, and the possible influence of diet on the induction of a dormant state in the late fall/early winter. We will continue with the new experiments initiated in year 2 that are designed to determine the critical stage for sex determination. Cohorts of specific stages collected from the field will be placed in the flow through system and reared to adulthood in a manner similar to the earlier (egg to adult) experiments, at which time the sex ratio will be determined. This approach should reveal the critical stage for sex determination in the culture system. Internal morphology of the reproductive systems will be compared between individuals reared in the laboratory and those collected from the field to assess the degree to which the laboratory conditions simulate the field. We will also obtain animals from Georges Bank populations, rear these in the tanks, and compare the results to our earlier work to determine if place of origin is an important factor to consider. As outlined in the proposal, laboratory experiments designed to assess the impact of temperature and photoperiod on diapause induction and termination will be conducted on selected developmental stages of Calanus reared from both Georges Bank and local populations.

#### Publications

The writing of two manuscripts is in progress by P. Blades-Eckelbarger. The first is paper is an ultrastructural study of the processes of oogenesis and vitellogenesis in Calanus finmarchicus. The second paper is a collaborative effort with Jeff Runge, in which we combine my observations of the vitellogenic stages with his index of reproductive maturity for assessing egg production in the field.