Influence of body size on gametes
produced and released in the green
sea urchin, Stronglycentrotus
droebachiensis
Biology 4122- Advanced Topics in Marine Invertebrates
Memorial University of Newfoundland and Labrador
Submitted by: ????
DATE
Introduction:
Most benthic invertebrates move over vast distances in the search for food.
This enables animals to improve growth and reproduction, increasing their fitness
levels which are defined by the ability to produce viable offspring (Scheibling,
1981). The green sea urchin, Strongylocentrotus droebachiensis, usually occurs on
rocky substrates between the intertidal zone and 50 m in depth throughout its
circumpolar habitats (Scheibling and Hatcher, 2001) and can be found in various
sizes depending on a variety of factors including the stage of development,
environment, and available food resources.
Determining the relationship between size and reproduction potential can
provide insight into the lifestyle of a sea urchin. The sea urchin plays an important
role in many ecosystems, mainly feeding on seaweeds but will consume anything
from bivalves to small fish (Moore and Miller, 1983). The gonad size of a sea urchin
can be affected by the quantity or quality of food the urchin obtains (Lang & Mann,
1976), but also on the environment and age of the urchin. Experiments have shown
the relationship between growth of somatic and gamete tissues. The amount of
nutrients available for the urchins to use depends on rate of food consumption,
digestion, and absorption. The rate and efficiency with which animals acquire,
process, and allocate energy are important factors affecting fitness (Calow and
Townsend, 1981). A decreased intake of nutrients results in a lower number of
available resources for maintenance and production of body tissues, including the
gonads (Lawrence et al, 2003). In harsh conditions or starvation, gonad
development will not occur (Scheibling and Anthony, 2001).
Different sea urchins become reproductively active at different times of their
life cycle. Some species can reproduce at test diameters of 15-20mm while others
are delayed until they can reach a larger size (Kawamura & Taki 1965). Predation
and environmental cues can select for sexually mature smaller urchins (Estes et al,
1978), while increased resources can cause larger test sizes before becoming
sexually mature. Gonad size is a good indicator of the health of a sea urchin. Poor
nutritional states will result in poor to no development and extra food will increase
resources dedicated to reproduction (Pearse, 1980).
S. droebachiensis has a reproductive cycle with a major spawning event
occurring in early spring, around March/April (Himmelman, 1978, Keats et al.,
1984). The spawning events are coordinated by changing photoperiods in Autumn
which initiates gametogenesis through stored nutrients (Walker and Lesser, 1998).
Potassium chloride is a very effective non-toxic chemical to sea urchins, in low
dosages (<5%). Goto et al. (1990) used it to assist in detachment of urchins from
substrates in hatcheries but it can also induce spawning mechanisms when injected
into the coelom (Hinegardner &Tuzi, 1981). The subsequent contraction of smooth
muscle can expel immature and mature gametes from both male and female urchins
(Strathmann, 1987). This method was used for the part A of the experiment in order
to induce spawning to correctly measure the amount of gametes released and to be
able to compare this with the test diameter to see if there was a positive
relationship between the two.
In this experiment we aim to test the relationship between size and
reproductive potential. We aim to determine if size is an indicating factor for
gonadal development and efficency. In most animal species, the larger an organisms
becomes, the more resources it can focus into providing viable offspring. Food is
converted into somatic tissues, as seen by overall size of the urchin, or gonadal
tissue, demonstratedd by gonad size and weight. We want to determine if there is a
reproductive advantage to having a larger body size and therefore, if having a larger
body size means producing more gametes. Here, dissection can be used as an
important tool to separate the gonads from the test and weight them in order to
determine the relationship between the weight of the test and the weight of the
gonads (the gonadal index).
Methods:
Sixty green sea urchins were collected off the coast of the Avalon Peninsula
by the Ocean Science Center Dive Team in late January 2013 and kept in holding
tanks with a supply of kelp until February 6. At this time urchins were sorted into
three size classes based on their test diameter; Small (<4.5 cm), Medium (4.5 cm –
5.5 cm) and Large (>5.5 cm). Small and Medium groups were comprised of 20
individuals each while the Large group contained only 16 individuals due to a lack of
large urchins in the sample received. Each group was moved to a smaller tank and
supplied with food. On February 13th half of the individuals from each group were
subjected to spawning. Excess water was removed prior to weighing, and test
diameter was recorded. An intra-coelomic injection of 1mL of 0.5M KCl was used to
induce spawning. A 2010 study by Gago and Luis found that this method is
consistent in obtaining a large number of spawners and consistently large
spawnings. Urchins were then placed aboral side up on 50mL beakers and allowed 5
minutes to spawn. Sperm samples were rinsed into a graduated cylinder using a set
volume of 20mL of sea water which was later subtracted from the final volume to
give total sperm volume. Egg samples were rinsed into a graduated cylinder with
varying volumes of seawater and allowed time to settle before total volume of
settled eggs was recorded. Urchins that did not spawn were recorded as
unsuccessful and not included in the final results.
Dissections took place in two sessions over a course of two consecutive weeks. In
each session half of the remaining members of each group were dissected (5 small, 5
medium and 4 large) to eliminate error due to change in gonads between the two
sessions. Urchins were soaked in a 95% ethanol solution prior to dissection. In each
dissection a cut was made around the widest point of the test, near the border of the
aboral surface using dissecting scissors. Another circular cut was made around the
anus to allow easy separation of test pieces. Gonads were removed using forceps
and sex and wet weight were recorded. All living tissue was then removed and the
empty test weight was recorded. These measurements provided a gonad index
(ratio of gonad weight to test weight) for each urchin.
Individual test results within each size class were averaged to give a less
convoluted data set for comparison with individual urchin results.
Results:
Sea urchin diameter versus gamete volume produced
In comparing the diameter of the test and the volume of sperm released in
male sea urchins it can be concluded that the males in the “large” size category did
in fact produce a greater volume of sperm than those in the “small” category as seen
in table 1.1 and graphically in figure 1.1. Also, when comparing the diameter of the
test and the volume of eggs released, the volume of eggs released in those sea
urchins placed in the “large” size category was greater than the volume of eggs
released in sea urchins in the “small” size category as seen in table 1.2 and
graphically in figure 1.2. The comparison of average gamete volume in both of these
size classes for both sexes can be seen in figure 1.3. It may not be appropriate to
state that in both males and females the figures show a considerably linear
relationship in the positive direction due to the fact that the high number of urchins
that did not spawn created a bias in the results due to a lack of data points. These
results are based on a small number of individuals and therefore are not significant.
Sea urchin test weight versus gamete weight
In comparing the test weight with the removed and weighed gonads, it can
also be concluded that there is a slight correlation between the gamete weight and
the test weight for both sexes when comparing the three size categories (small,
medium and large). Generally, as the test weight increased, so did the weight of the
gametes as seen in table 1.3. Figure 1.4 and 1.5 show the values of gamete weight
versus the test weight for males and females respectively. For the male sea urchins,
the data only shows a slight positive linear relationship between increasing test
weight and gamete weight. When fitted with a trend line Figure 1.4 does show this
slightly linear relationship however the largest gamete weight did not belong to the
largest urchin nor did the smallest gamete weight to the smallest urchin. The
gamete weights showed much more variability with test weight in the males than in
the females. Figure 1.5 displays the gamete weight of the female gonads as a
function of the corresponding test weights. In the case of the females the graph
relates an exponential relationship unlike that seen in the males. This exponential
relationship is again seen in Figure 1.6, which shows the comparison of average test
weight and average gamete weight in three size classes of green sea urchin. This
figure also shows that in comparison with the female data the male data shows a
relatively small positive relationship. The results were however still expected in the
sense that size class “small” had the smallest amount of gametes produced followed
by the “medium” size class and finally, the “large” size class produced the highest
average amount of gametes (in both males and females).
Discussion:
Comparison of diameter with volume of gametes produced
The volume of released gametes in both male and female urchins showed an
increase with test diameter which would indicate that body size and reproductive
potential are indeed positively related. It is important to note however that
spawning was not successful in any of the medium size class and for 3 specimens in
the small class and 3 in the large class. The fact that some small urchins responded
to the KCl treatment shows that the lack of spawning – at least in the medium and
large size classes – is unlikely to be a result of immature gonads. The relatively small
proportion of successful spawners (less than half the sample) is made even smaller
when separated for sex and creates a bias in the data due to such a small data.
Therefore the results obtained from the induced spawning as well as the
conclusions inferred from them can be declared not significant. If the experiment
were conducted closer to the natural spawning event in the early spring the number
of successful spawners could have been increased. This could have reduced the
large proportion of urchins that did not respond to the KCl treatment since it is
likely that gametogenesis was not as far advanced in these individuals as in those
that responded to the treatment. Temperature, photo period, food availability and
water turbulence are just some factors which can have an effect on gametogenesis
so even if the experiment was conducted closer to the natural spawning date it is
likely that there would still be individuals that did not respond (Gago & Luis, 2010).
A study by Delgado et al in 2005 suggested that KCl injections should be altered
according to urchin size due to the fact the size of the urchin would effect the
internal concentration of KCl after injection.
Comparison of sea urchin test weight and removed gamete weight (gonadal index)
Test size and gonad production are two separate areas that various
resources can be attributed to. We determined the size of a sea urchin does give a
slight indication of reproductive ability. By comparing the wet gonad weight to the
corresponding test weight we found a slight correlation between size and gonad
weight meaning that usually urchins that are bigger are able to donate more energy
into developing their gametes. Somatic tissues and gonad tissue do not have to grow
at the same rate under varying conditions. McCarron et al (2009) determined that
small urchins focused more energy in growth of somatic tissues, where as large
urchins put more effort in gonad production. However, this is not always the case.
When resources are plentiful development of test size AND reproduction can occur.
This was demonstrated by Estes et al, (1918) who showed smaller urchins were
selected to mature at a younger age. The predation risk increased rapidly and thus it
was more beneficial to try and pass the genetic material at earlier times. Minor and
Scheibling, (1997) found decreasing food at the beginning of the annual
reproductive cycle had no effect on gamete production if well fed towards the end of
the cycle. They did not have much somatic growth throughout the year but given an
abundance of nutrients could develop normal gonads during the reproductive
season
There was a clear difference between gonad weights of females and males.
The females produced much more eggs at a greater test size than males. There was
an exponential relationship showing smaller females did not have the ability to
dedicate the same amount of resources to gonad development. The males’ results
were much more variable and showed fluctuations throughout all test sizes. They
also show little increase in the average gonad weight from small to large sizes
indicating size is not necessarily important for gonad development like it appears to
be in females. Overall size of a sea urchin should be an indication of age and how
well it is growing. We can normally expect larger sea urchins to produce more
gamete from larger gonads, but as demonstrated here, it is not an absolute
relationship. The largest male and female urchins did not produce the largest
gonads, implying there are many more factors at work. The male urchins small to
large were generally all over the place but females tended to follow a more expected
result. This could indicate it takes more energy to produce gametes in females than
males and a larger size is needed in order to obtain and store enough resources to
develop more gonads. The males may have an easier time producing sperm and thus
be able to produce them at younger ages and smaller sizes.
Implications and Improvements
If not constricted by time the experiment could have been improved by
performing a closer analysis of the gametes produced by the induced spawning. A
microscopic analysis could have given further information relating to the maturity
of the expelled gametes and individual gametes could have been counted to provide
a much more accurate measurement than volume alone. The amount of water in the
gonads during weighing may have skewed the results slightly as well but again the
time constraints prevented us from drying them out prior to weighing.
The results of this experiment have given us further understanding of
whether or not a larger body size is a reproductive advantage and thus selected for
over time by natural selection. If gonad size and output continue to increase with
body size we could assume that larger body sizes would be more beneficial. In the
case of the female urchins it is clear that a larger body size provides a reproductive
advantage and should therefore be selected for in the wild. The male urchin results
showed little to no reproductive advantage in having a larger body size however,
since there is no size difference between sexes in the wild, these results are not as
expected (if this was the case, then we would assume males would not be the same
size as females in the wild) and there are clearly other factors that should be taken
into consideration.
Conclusion:
Because the numbers in part A are based on only a few individuals (as a
result of low spawning rates), the results can not be viewed as significant in
indicating if diameter was positively correlated with the volume of gametes
produced upon spawning. In comparing the test weight with the gamete weight in
part B we can conclude that generally (on average), as the test weight increased so
did the weight of the gametes in both males and females. However, the results did
not display a clear and definitive answer as the smallest sea urchin did not produce
the smallest amount of gametes and the largest sea urchin did not produce the
largest amount of gametes for either sex.
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Appendix A
Tables and Figures:
Table 1.1: Comparison of diameter of test and volume of sperm produced after
spawning in two different size classes (small and large) green sea urchin,
Stronglycentrotus droebachiensis
Diameter of test (cm):
4.4
4.3
4.8
4.4
5.8
6.2
6.4
Volume of sperm (ml):
1.9
2.0
2.3
2.1
4.0
4.3
3.8
Size class designation:
Small
Small
Small
Small
Large
Large
Large
Table 1.2: Comparison of diameter of test and volume of eggs produced after
spawning in two different size classes (small and large) green sea urchin,
Stronglycentrotus droebachiensis
Diameter of test (cm):
4.7
4.9
4.7
6.3
7.1
Volume of eggs (ml):
3.6
4.1
4.2
5.5
7.9
Size class designation:
Small
Small
Small
Large
Large
No urchins of the medium size class underwent a successful spawning, nor did 3 of
the small size class and 3 of the large size class. Therefore these individuals could
not be sexed and therefore were not recorded.
Table 1.3: Comparison of size and gamete weight using gonadal index procedures in
the green sea urchin, Stronglycentrotus droebachiensis
Gamete
Weight (g):
0.7
9.8
3.3
2.7
1.9
0.5
2.4
6.1
2.6
3.5
3.5
4.0
1.4
5.0
2.3
3.6
6.7
6.3
1.8
10.3
18.9
11.6
9.8
5.5
7.5
5.1
5.7
5.5
Test Weight
(g):
14.9
28.0
25.6
16.7
18.9
15.3
26.1
22.9
20.4
17.3
26.4
36.8
24.9
27.4
31.5
25.2
55.0
39.6
25.6
24.8
47.3
48.1
35.9
38.5
24.0
32.6
40.3
52.8
Diameter
(cm):
4.6
5.2
5.3
4.6
4.5
4.3
5.5
5.0
4.6
4.5
5.6
5.9
5.5
5.5
6.2
5.5
6.5
6.4
6.1
5.9
6.7
7.7
6.6
6.5
6.5
7.5
6.6
7.2
Sex:
Female
Female
Female
Female
Female
Female
Female
Male
Male
Male
Female
Female
Female
Female
Female
Male
Male
Male
Male
Male
Female
Female
Female
Male
Male
Male
Male
Male
Size Class
Designation:
Small
Small
Small
Small
Small
Small
Small
Small
Small
Small
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Medium
Large
Large
Large
Large
Large
Large
Large
Large
5
4.5
Volume of Sperm (ml)
4
3.5
3
2.5
2
1.5
1
0.5
0
0
1
2
3
4
Test Diameter (cm)
5
6
7
Figure 1.1: Relationship between test diameter and total volume of gametes
produced in male sea urchin, Stronglycentrotus droebachiensis
9
Volume og Eggs (ml)
8
7
6
5
4
3
2
1
0
0
1
2
3
4
5
Test Diameter (cm)
6
7
Figure 1.2: Relationship between test diameter and total volume of gametes
produced in female sea urchin, Stronglycentrotus droebachiensis
8
8
7
Gamete Volume
6
5
4
Sperm
3
Eggs
2
1
0
Small
Large
Size Class
Figure 1.3: Comparison of gamete volume in two different size classes of both
female and male green sea urchins, Stronglycentrotus droebachiensis
8
Gamete Weight (sperm) (g)
7
6
5
4
3
2
1
0
0
10
20
30
Test Weight (g)
40
50
60
Figure 1.4: Correlation between gamete weight and test weight in male green sea
urchin, Stronglycentrotus droebachiensis
20
18
Gamete weight (eggs) (g)
16
14
12
10
8
6
4
2
0
0
10
20
30
Test Weight (g)
40
50
60
Figure 1.5: Correlation between gamete weight and test weight in female green sea
urchin, Stronglycentrotus droebachiensis
16
14
Gamete Weight (g)
12
10
8
Sperm
6
Eggs
4
2
0
Small
Medium
Size Class
Large
Figure 1.6: Comparison of average test weight and average gamete weight in three
size classes of green sea urchin, Stronglycentrotus droebachiensis