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.