1. Habitat Selection by the Burrowing Brittlestar Ophiophragmus filograneus in
the Banana river Lagoon, Florida.
Indira Brown, Department of Biology and Marine Science, Jacksonville University.
Abstract
Brittlestars are motile Echinoderms inhabiting the oxidized subsurface layer of soft-bottom
substrates in every marine environment. Burrowing brittlestars typically have a patchy
distribution that may be influenced by sediment organic content or presence of sub-aquatic
vegetation. Few studies have been undertaken to determine specific substrate preferences.
Ophiuroids from the Banana River Lagoon near Merritt Island, FL were collected to determine
their substrate preferences based on four sediment grain sizes. In controlled laboratory tests of
grain size preference, brittlestars preferred to burrow in the very fine and fine grained
sediments (N=48). Subsequent experiments testing preference between these two grain sizes
revealed a preference for the very fine sediments. Further experiments of grain size with
vegetation and artificial vegetation were performed. Results indicated Ophiophragmus
filograneus’ patchy distribution in the natural environment may correlate with very fine
sediment, seagrass and structural distribution.
KEYWORDS: Ophiophragmus filograneus, brittlestars, sediment preference.
Introduction
Benthic invertebrates play a vital role in the physical, chemical and biological structure of their
sedimentary environment. They are known to recycle nutrients and detoxify pollutants through
burrowing, feeding, and respiring (Peterson and Quammen 1982; Fukuyama and Oliver 1985;
Kvitek et al. 1992; Micheli 1997). The mobility of sediment-associated cadmium (Cd), for

2. example, may be increased during resuspension of sediments by a shift from reducing to
oxidizing conditions and by altered pH (Khalid et al., 1981; Förstner, 1987; Peterson et al.,
1996). Ophiuroids are one of the numerous benthic invertebrates that contribute to the
oxidization state of the subsurface sediment layer in the marine environment. They are also an
important component of the marine trophic system in shallow waters (Turner and Meyer 1985).
Ophiophragmus filograneus belongs to the Family Amphiuridae, which is also known as
the burrowing brittlestars. O. filograneus burrow in the sediment 5-10 cm deep. They are one
of the smallest known brittlestars with disk diameters of approximately 1 cm, and arms up to
10 cm in length. O. filograneus are common in the marine littoral regions from the southern tip
of Florida to Pensacola Bay on the North West coast and to Cape Canaveral in the North East.
They have also been found in Cedar Key, Florida. O. filograneus are unique because they occur
in estuaries at reduced salinities and not in the open sea. In the past, distribution of
Ophiophragmus was assumed to be limited because it was known to adapt to environments
with reduced salinities. However, recent studies indicate O. filograneus functions better at
higher salinities and therefore its restriction to estuaries is probably due to other factors such
as food resources, sediment type, competition, or predation or some other factor (Talbot and
Lawrence 2006).
Within estuaries Ophiophragmus filograneus still shows patchy distribution. The factors
that define their irregular distribution are unclear. Some possible factors influencing their
distribution are sediment size and the presence of sea-grass, or other submerged aquatic
vegetation. The burrowing brittlestar Micropholis gracillima preferred to burrow in fine grained
sediment with some organic content (Zimmerman 1987). The goal of this study is to determine

3. the substrate preference of Ophiophragmus filograneus with regards to sediment grain size,
seagrass and artificial seagrass substrate. The proposed hypothesis is that Ophiophragmus
filograneus would prefer fine and very fine.
Methodology:
The collection site for this experiment was in the Banana River, near Melbourne, FL
(28o12’ N, 80o37’ W) (Figure 1). The salinity of the water was recorded at 24 ppt. Sediment was
collected by the shovelful in shallow water (no more than 3 feet in depth) and then sieved
through 0.15 cm sieve in order to obtained the specimens burrowed within the sediment. The
specimens were placed in a bucket with portable air pumps filled with natural seawater from
the site. The specimens were transported back to Jacksonville University’s wet lab. The
specimens were placed in two ten-gallon aquaria with a 2 inch deep layer of natural sediment
from the collection site and natural seawater at 24 ppt. The brittlestars remained in the holding
tanks for 24 hours to acclimate to their new environment before testing began. The salinity was
adjusted with distilled water as necessary to maintain a constant 24 ppt. Specimens were fed
every other day with finely powered fish food flakes.
The first experiment was designed to determine brittlestar preference when presented
with four sediments. The sediment sizes were very fine (50/140), fine (45/60), medium (30/65),
and coarse (20/30). Sediment was purchased locally from (Standard Sand & Company Services,
Jacksonville, FL) and was clean of debris and organic material. Finger bowls (8 inches diameter)
were used as individual testing arenas. Natural sea water was placed into each of the bowls to a
depth of 2 inches. Each bowl was divided into four pie-shaped sections with plastic dividers to
prevent mixing of the different sand sizes. Sand was placed into each section so that all four

4. sizes of sand were represented in each bowl. The size of sand in each section was indicated
along the outer perimeter of the finger bowls (Figure 2). For example, the coarse treatment was
placed between the very fine and fine at the southern perimeter of the bowl, while the medium
treatment was placed between the very fine and fine at the northern perimeter of the bowl.
The sediments were allowed to settle for 24 hours before placing brittlestars in each bowl.
An individual O.filograneus was placed at the center of its assigned bowl at 3:00p.m.
After placing the brittlestars in each of the 12 bowls, the blows were covered with a box to
prevent light penetration that might influence the specimens’ substrate preference. After 24
hours the boxes were removed and the location of the burrowed brittlestar was recorded as its
sediment preference (Figure 3). The specimens were then removed and placed into a separate
holding tank from the brittlestars that had not yet been tested. The 12 finger bowls were
cleaned in order to remove all scent of previous specimens tested. The finger bowls were then
reset with new sediments and allowed to settle for 24 hours before testing resumed. Each set
of 12 finger bowls tested is referred to as a block. The experiment was repeated 4 times for a
total N of 48. The data were analyzed with a Freidman’s Rank Sum test (Table 1).
A second set of experiments was done to determine the preference of brittlestars
between the two sediment sizes most often chosen in the first experiment, which were the
very fine and the fine (Figure. 4). The same exact method from the previous study was used,
except in this case the arenas were divided into 2 sections instead of 4. The data was also
analyzed with a Freidman’s Rank Sum test (Table 2).
The third set of experiments was performed to determine brittlestars preference
between the very fine sediment and seagrass (Holodule wrightii). The same exact method from

5. the second set of experiment was used, except the brittlestars remained in the holding tanks
for 48 hours to acclimate to their new environment before testing (Figure 4). In addition, the
seagrass rhizomes were submerged within the very fine sediment as its’ blades were above the
sediment submerge in water. Due to the irregularity of placing the sediment at the edge of the
arenas, the experiment was performed again. During the second set of testing, the seagrass
were placed in the center on its side of the arenas. The data were analyzed with a Freidman’s
Rank Sum test (Table 3).
The fourth and final set of experiments was set up exactly as the pervious experiment in
order to determine the preference of brittlestars between the very fine sediment sizes and
artificial seagrass (Figure 5). The artificial seagrass was constructed using Diamond coffee
stirrers and Berwick curling ribbons. The ribbon, which was seven inches in length, was folded
in half then tied around a coffee stirrer in an overhand bend knot. The same exact method from
the previous study was used. The data was also analyzed with a Freidman’s Rank Sum test
(Table 4).
Results
In the first set of experiments, the brittlestars within each block appeared to have
selected the very fine and fine sediments most often, with very few brittlestars selecting the
medium or coarse sediment. Block 1 is significantly different from blocks 2, 3, and 4, (Figure 6).
However, despite this anomaly, the overall trend was that the majority of animals preferred the
very fine and fine sediment size, and rarely preferred the medium and coarse sediment size
(Figure 7). In this case, 44% of the brittlestars preferred the very fine sediment, 40% preferred

6. the fine sediment, 6% preferred the medium sediment and 10% preferred the coarse sediment
(Figure 8).
In the second set of experiments, the brittlestars’ preference between the fine and very
fine sediments was tested. Once again block 1 showed a different trend from blocks 2, 3 and 4.
For example, in block 1, seven brittlestars preferred the fine sediment and five brittlestars
preferred the very fine sediment. However, in all of the other blocks the very fine sediment was
most often selected (Figures 9 & 10). In total, 69% of the brittlestars preferred the very fine
sediment and 31% preferred the fine sediment (Figure 11).
In the third set of experiments, the brittlestars’ preference between the seagrass and
the very fine substrate was tested. The brittlestars within each block appeared to have
preferred the seagrass in the very fine sediments most often. Very few brittlestars selected the
boarder or the region between the seagrass and very fine sediment. For example in block 1, six
brittlestars preferred the seagrass bed, four preferred the very fine sediment and 2 preferred
the border. Block 3 was slightly different since no brittlestars preferred the boarder (Figures 12
& 13). In total, in all the blocks the seagrass substrate was often selected. Therefore, 60.42% of
the brittlestars preferred the seagrass substrate, 29.17% preferred the very fine sediment and
10.42% preferred the border (Figure 14).
In the final experiment, brittlestars’ preference between the artificial seagrass and very
fine sediments was tested. Once again the brittlestars within each block appeared to have
selected the artificial seagrass and very fine sediments most often, with very few brittlestars
selecting the border or the region between the seagrass and very fine sediment. There was no
difference between the trends in each block. For example in block 1, seven brittlestars

7. preferred the fine sediment, four brittlestars preferred the very fine sediment and one
brittlestar preferred the border. In all of the blocks the artificial seagrass substrate was most
often selected (Figure 15 & 16). In total, 60.42% of the brittlestars preferred the artificial
seagrass substrate, 31.25% preferred the very fine sediment and 8.33% preferred the border
(Figure 17).
Figure 1. The collection site of O.filograneus in the Banana River is indicated by the red
Oval.

8. Figure 2. Photograph of experimental set Figure 3. Brittlestar burrowed in one bowl
Of testing arenas from the first set of experiments. Location
of the brittlestar is very obvious.
Figure 4. Photograph of experiments 2 through 4 testing arenas

9. Figure 5. Photograph of testing arina with artificial seagrass.
Table1. Statistical analysis of data from the first set of experiments. The numbers in the table
reflect the rank of choice within each block. A choice of very fine sediment was assigned the
value of 1, fine sediment was assigned the value of 2, medium sediment the value of 3, and
coarse sediment the value of 4.
Bowl Number Block 1 Block 2 Block 3 Block 4
1 3.5 3.5 8.5 3.5
2 8.5 8.5 3.5 11.5
3 3.5 8.5 8.5 8.5
4 9 3.5 3.5 3.5
5 11.5 9 8.5 3.5
6 3.5 8.5 8.5 8.5
7 3.5 8.5 3.5 8.5
8 11.5 8.5 9 8.5
9 3.5 8.5 8.5 3.5
10 11.5 3.5 3.5 3.5
11 3.5 3.5 3.5 8.5
12 8.5 3.5 11.5 8.5
Rank 81.5 77.5 80.5 80
Rank2 6642.25 6006.25 6480.25 6400
∑ Rank2 553.5208
∑ Ranks2/12 46.12674
H -0.51466
t 11 11 10 11
T 1320 1320 990 1320
sum of T 4950
adjusted H -3.50669

10. Table 2. Statistical analysis of data from the second set of experiments. The numbers in the
table reflect the rank of choice across the entire experiment. A choice of very fine sediment
was assigned the value of 1, and the fine sediment was assigned the value of.
Bowl Number Rank 1 Rank 2 Rank 3 Rank 4
1 41 17 41 17
2 41 17 17 17
3 17 17 17 17
4 41 17 41 17
5 17 17 17 41
6 41 17 17 17
7 17 17 17 41
8 41 17 17 41
9 17 17 17 41
10 41 17 17 17
11 17 17 41 17
12 41 41 17 17
∑ Ranks 372 228 276 300
∑Ranks^2 138384 51984 76176 90000
∑ Ranks^2/12 11532 4332 6348 7500
H 150.8418
t 33 15
T 35904 3360

11. sum of T 39264
adjusted H 233.9318
Table 3. Statistical analysis of data from the third set of experiments. The numbers in the table
reflect the rank of choice across the entire experiment. A choice of very fine sediment was
assigned the value of 1, seagrass was assigned the value of 2, and the broader between very
fine and seagrass was assigned the value 3.
Bowl Number Rank 1 Rank 2 Rank 3 Rank 4
1 7.5 29 29 7.5
2 7.5 29 7.5 7.5
3 29 7.5 29 7.5
4 29 29 7.5 46
5 46 46 7.5 29
6 29 29 29 29
7 7.5 29 29 7.5
8 29 7.5 29 29
9 46 29 29 46
10 29 29 7.5 29
11 29 29 29 29
12 7.5 29 29 29
∑ Ranks 296 322 262 296
∑Ranks^2 87616 103684 68644 87616
∑ Ranks^2/12 7301.333 8640.333 5720.333 7301.333
H 147.0221
t 14 29 5
T 2730 24360 120

12. sum of T 27210
adjusted H 195.0274
Table4 . Statistical analysis of data from the third set of experiments. The numbers in the table
reflect the rank of choice across the entire experiment. A choice of very fine sediment was
assigned the value of 1, artificial seagrass was assigned the value of 2, and the broader between
very fine and artificial seagrass was assigned the value 3.
Bowl Number Rank 1 Rank 2 Rank 3 Rank 4
1 46.5 7 7 7
2 7 30 30 30
3 7 30 30 30
4 30 30 30 30
5 30 7 30 7
6 7 30 46.5 30
7 30 7 7 46.5
8 30 46.5 30 30
9 30 7 30 30
10 7 7 30 7
11 30 30 7 30
12 30 30 30 30
∑ Ranks 284.5 261.5 307.5 307.5
∑Ranks^2 80940.25 68382.25 94556.25 94556.25
∑ Ranks^2/12 6745.021 5698.521 7879.688 7879.688
H 143.1424
t 15 29 4
T 3360 24360 60
Sum of T 27780
adjusted H 191.1886

13. 7
6
Number of Brittlestars
5
4 VF
F
3
M
C
2
1
0
Block1 Block2 Block3 Block 4
Figure 6. Results of sediment preference among four sediment grain sizes with each block
shown separately. VF = very fine, F=fine, M= medium, C= coarse.

14. 7
6
Number of Brittlestars
5
Block1
4
Block2
3 Block3
Block 4
2
1
0
VF F M C
Figure 7. Results of sediment preference among four sediment grain sizes grouped by grain
sizes. VF = very fine, F=fine, M= medium, C= coarse.
50.00%
45.00%
40.00%
35.00%
Percentage
30.00%
25.00%
20.00%
15.00%
10.00%
5.00%
0.00%
VF V M C
Figure 8. Results of sediment preference among four sediment grain sizes with
all blocks combined. VF= very fine, F= fine, M= medium, C= coarse.

15. 12
Number of Brittlestars
10
8
VF
6
F
4
2
0
Block1 Block2 Block3 Block 4
Figure 9. Brittlestar sediment preference between very fine (VF) and fine (F) sediment size
12
10
8
Block 1
Number of Briittlesars
Block 2
6
Block 3
Block 4
4
2
0
VF F
Figure 10. Brittlestar sediment preference by sediment grain size—second set of experiments.
Blocks are indicated by the different colors.

16. 80.00%
70.00%
60.00%
Percentage
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
VF V
Figure 11. Brittlestar sediment preference by sediment grain size with blocks combined. VF=
very fine, F= fine.
12
10
Number of Brittlestars
8
VF
6
SG
B
4
2
0
Block 1 Block 2 Block 3 Block 4
Figure 12. Brittlestar preference between seagrass (SG) and very fine (VF) sediment size.

17. 12
10
Number of Brittlestars
8
Block 1
6 Block 2
Block 3
4 Block 4
2
0
VF SG B
Figure 13. Brittlestar preference between seagrass (SG) and very fine (VF)-third set of
experiments. Blocks are indicated by the different colors.
70.00%
60.00%
50.00%
Percentage
40.00%
30.00%
20.00%
10.00%
0.00%
SG VF B
Figure 14. Brittlestar substrate preference between seagrass and very fine grain size with blocks
combined. VF= very fine, F= fine.

18. 12
10
Number of Brittlestar
8
VF
6
A
B
4
2
0
Block 1 Block 2 Block 3 Block 4
Figure 15. Brittlestar preference between artificial seagrass (AG) and very fine (VF) sediment
size.
12
10
Number of Brittlestars
8
Block 1
6 Block 2
Block 3
4 Block 4
2
0
VF A B
Figure16. Brittlestar preference between artificial seagrass (SG) and very fine (VF)-third set of
experiments. Blocks are indicated by the different colors.

19. 70.00%
60.00%
50.00%
Percentage
40.00%
30.00%
20.00%
10.00%
0.00%
A VF B
Figure 17. Brittlestar substrate preference between artificial seagrass and very fine grain size
with blocks combined. AF= very fine, VF= very fine.
Discussion
Although block 1 was statistically different from the other blocks in both experiments, the
results from the experiment indicated that Ophiophragmus filograneus preferred smaller grain
sizes, and specifically the very fine grain size in the absence of aquatic vegetation. The reason
for the difference in brittlestar behavior between block 1 and the subsequent blocks may have
been a result of handling time during experimental set-up. The process of transferring the
brittlestars from the holding tank to the bowls took longer for block 1 than for any of the
subsequent blocks. This trend was also true for the second set of experiments.
In addition, it was taken under consideration that brittlestars tend to be associated with
seagrass, (Halodule wrightii). This raises the questions “Do they prefer soft sediment or
seagrass substrate? Or do seagrass prefer the soft sediment? Due to the above mention quires,

20. further research in the laboratory and in the field was undertaken. The results from
experiments three and four indicated that Ophiophragmus filograneus preferred structure
substrate, especially in the presence of aquatic vegetation. The reason the third experiment
was performed a second time was due to the position of the seagrass within the bowl and the
difference in brittlestar behavior in block 1. In addition, the variation in block 1 may have been
a result of not having sufficient acclimation time. The process of transferring the brittlestars
from the holding tank to the bowls took longer for block 1 than the other blocks during
experimental set-up. The brittlestars were given an additional 24 hours to acclimate in
subsequent experiments. As a result there were no statistical differences between the blocks in
the third and fourth experiment.
Despite the anomaly of block 1, the overall trend was for brittlestars to prefer the
smaller grain sizes. In addition, the overall trend suggests that brittlestars prefer seagrass
substrate in the presence of aquatic vegetation. Just as important, brittlestars prefer structural
substrate in the absence of aquatic vegetation. This suggests that the hypothesis about
brittlestars sediment preference, seagrass and structural substrate were correct.
Ophiophragmus filograneus’ patchy distribution in the natural environment may correlate with
sediment distribution, seagrass distribution and structural distribution. This remains to be
verified in the field. However, It must be taken into consideration that the different sediments
were clean and major organic compounds were absent. Zimmerman et al 1987 showed that
organic content of the sediment is an important factor for other burrowing brittlestars.

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