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Gambusia holbrooki Blackspot Influence
on Red Blood Cell Circulation Rate
Dr. Alan Gillen, Lab Manual Reading 5
Ann Varghese, Nicole Buckley
Department of Biology and Chemistry,
Liberty University
Spring 2023
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ABSTRACT
Gambusia holbrooki, also known as Eastern Mosquitofish, were taken from the lake primarily
from the Kingfisher Pond, and additionally from Lake Hydaway in Rustburg, Virginia. The fish
that were gathered were observed in a tank maintained at an average of 22 degrees Celsius. To
carry out the experiment, ten fish, five males and five females, were removed from the tank and
put under the influence of McKesson Normal Saline. When the caudal tails of Gambusia were
studied under a microscope, the red blood cell circulation in regions with a high count of
blackspot was compared to the red blood cell circulation in regions with very little blackspot
over the course of one minute in increments of 15 seconds. According to the findings of our
study, the red blood cell circulation in the capillaries of Gambusia holbrooki was significantly
faster in areas where there was very little to no blackspot as compared to areas with a significant
amount of blackspot.
INTRODUCTION
Gambusia holbrooki have been collected primarily from the Kingfisher Pond, and
additionally from Lake Hydaway in Virginia. The fish were analyzed, and black spots were
noticed under the microscope leading us to conduct an experiment to see the red blood cell
circulation of this specific Gambusia that are affected by Black Spot Disease. Black Spot
Disease (BSD) is caused by the metacercariae of a trematode, Uvulifer sp., which induces the
production of black spots on the body surface of the fish (Bush et al. 2001). Specifically,
Uvulifer ambloplitis is common in freshwater fish. At the larval stage, this parasite burrows into
fish’s skin, inducing the formation of a black pigment (Kirse, 2010). While normal pigmentation
is also present in fish, the two can be distinguished under the microscope, as the BSD
pigmentation is more dense and darker. Furthermore, U. ambloplitis then dwells in the tissues of
the fish, which serves as the 2nd intermediate host in its life cycle (Kirse, 2009). While BSD
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does not pose any fatal threat to the fish, this study seeks to further understand the impact of
BSD infection on the circulatory system of the fish. We hypothesized that the fish with heavy
blackspot will have slower red blood cell circulation than the fish with little to no blackspot.
Some factors that may have impacted the outcome are the sex of the fish, temperature of the
water in the tank and container used during the experiment and lastly if sedation caused the
blood circulation to slow down. A total of 10 fish were attained: 5 males and 5 females, with the
temperature ranging from 18 to 24 degrees Celsius. Gambusia are known to survive in
temperatures ranging from 0.5 to 40 degrees Celsius (33-104 F), making them more resilient and
hardier than the common guppy which is more tropical. They can be somewhat aggressive and
hardier than both native fish and common aquarium tank fish. They do make excellent mosquito
biological control in local ponds and farm water tanks. Caution should be made when
introducing to non-native watershed systems because they can become invasive.
MATERIALS AND METHODS
Liberty faculty and students collected Gambusia holbrooki, commonly known as
mosquitofish, primarily from the Kingfisher Pond, and additionally from Lake Hydaway. The
fish were subsequently relocated to an indoor fish tank within the laboratory. A critical note was
made of their habitat temperature in the tank, at 23.3 degrees Celsius. A mason jar was utilized
to transfer water from the tank to an empty container, which served as a temporary habitat for
fish subjected to observation, and the fish were transferred into the jars using a fishnet. Across
two different observation days, a total of 10 fish were subjected to observation, to include 5
female Mosquitofish and 5 male Mosquitofish. Small fish nets were utilized to transfer fish from
the fish tank and into the container. Prior to observing each fish, the temperature of the water in
the temporary habitat was recorded as well as the size of each fish. As each fish was extracted,
they were sedated with McKesson Normal Saline (Isotonic Irrigation Solution, 0.9% sodium
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chloride) and measured with a ruler. The length of each fish was recorded in millimeters, and the
surface anatomy of each fish was observed to determine and record the sex of the fish. The fish
were then wrapped in damp gauze or cheesecloth and kept moist with water from their habitat by
carefully using a sterile dropper to minimize any potential discomfort to the fish. The fish were
positioned in the gauze so that their tail was left exposed for observation under the Leica ICC50
HD microscope. First being placed on a glass slide, a small coverslip was then placed over the
tail of the fish. To achieve proper focus, the microscope was adjusted under a magnification of
40X. Fish circulation was then observed under 100X magnification, and video was recorded
using an iPhone. After data collection, each respective fish was immediately returned to the fish
tank.
After data collection was obtained from all 10 fish, the video data was observed. Two regions of
each fish’s tail were marked for observation, one termed “Heavy Blackspot” to indicate a
portion of the tail infected by Blackspot Disease and one termed “Less Blackspot” which likely
includes the fish’s normal pigmentation with possibly less Blackspot, or none present. Manual
handheld counter clickers were then utilized as a tool to measure the rate of circulation over 15
second readings, with three readings for each of the two regions of the fish’s tail, with a total of
6 readings per fish, and 60 readings overall. The data was then subjected to a two-tailed t-test for
statistical analysis, to compare the rate of circulation between the regions with “Heavy
Blackspot” and “Less Blackspot.” The null hypothesis stated that there was no significant
difference between the rates of circulation in the two sample groups. The alternative hypothesis
stated that there was a significant difference between the rates of circulation in the two sample
groups.
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RESULTS
Fish Size (mm) Temp (C) Sex
1 30 20 Female
2 25 19.5 Male
3 30 19.5 Male
4 25 18 Male
5 25 24 Male
6 25 24 Male
7 30 24 Female
8 23 24 Female
9 28 22 Female
10 26 22 Female
Figure 1. All 10 fish with individual characteristics recorded such as sex, size and temperature of
the water when fish were observed during the experiment.
Trial Heavy Blackspot
(RBC in
capillary/minute)
Less Blackspot
(RBC in
capillary/minute)
1 41 67
2 53 66
3 52 57
4 43 60
5 40 66
6 41 69
7 32 49
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8 32 50
9 34 53
10 46 58
11 46 60
12 45 58
13 66 81
14 63 78
15 70 80
16 49 87
17 54 87
18 52 92
19 66 80
20 66 75
21 71 75
22 56 68
23 55 65
24 55 62
25 46 66
26 45 65
27 45 66
28 48 64
29 47 66
30 46 63
Figure 2. The rate of red blood cell circulation in the Gambusia capillaries with
Heavy Blackspot versus Less Blackspot.
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Figure 3. An observable comparison between a region with heavy blackspot, as seen on the left,
and less blackspot as seen on the right, and each group's respective means of circulation rates.
Heavy Blackspot Low Blackspot
Mean 49.83333333 67.76666667
Standard Deviation 10.67411737 10.87129512
Sample Size 30 30
Significance Level 0.05 0.05
Degrees of Freedom 58 58
t-Value ≈ -6.45 -6.45
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Normal Blood Circulation
Parasite in Blood Circulation
irc ulation
Metacercaria in Blood Circulation/Blackspot Formation
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DISCUSSION
Ten (10) Gambusia holbrooki were subjected to data collection and observation (Figure 1),
including their length (mm) and sex recorded. Although no observable trends were noted
regarding these characteristics, it ensured consistency in the experiment, in that five fish of each
sex were observed, with male fish averaging 26 mm, and female fish averaging 27.4 mm.
However, temperature may have had an impact on the rate of circulation. It was thought that fish
kept in warmer temperatures would have higher rates of circulation as compared to fish kept in
colder temperatures. We observed that Fish #4, with the lowest recorded temperature prior to
observation, yielded the slowest rates of circulation in both the Heavy Blackspot region and the
Less Blackspot region (Figure 2). While the present study did not study the statistical significance
behind this finding, this could be an area of future research.
While performing the observations under the microscope, we made note of critical
variables in experimental technique. In the earlier stage of the experiment, we found that at
times, it was difficult to observe the caudal tail circulation. With proper focus and magnification,
and a living fish, it was questioned why the circulation was difficult to be seen. However, we
later discovered that it is critical for observation to focus on a more proximal region of the tail.
Previously, the tip of the tail was being observed. We changed this for further observation and
were able to observe the circulation with much more ease and clarity. Additionally, prior to
experimentation, we wondered if sedating the fish would significantly impact their observable
circulation. We opted to first try to observe the fish without sedation. However, this proved to
be quite difficult, as the fish were put under stress that made it difficult for observation under
the
microscope. Therefore, we elected to sedate them, and this did not seem to have a significantly
negative impact on our data collection.
After data collection was performed, each respective fish was immediately returned to the
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fish tank. All fish but Fish #1 survived experimentation. However, we were able to derive
circulation data from Fish #1 before it died. Data collection entailed looking at the recorded
videos on the iPhone and taking three counts from each fish using the clicker, for a total of 60
counts overall. This was conducted to achieve a statistically significant dataset of 60 counts.
Following data collection, statistical analysis was performed. Upon establishing the null and
alternative hypotheses, an independent two tailed t-test was performed. As seen in Figure 3 and
the statistical calculations, for the “Heavy Blackspot” group, the sample size (n) was 30, the
mean was 49.83, and the standard deviation was found to be 10.67. For the “Less Blackspot”
group, the sample size (n) was 30, the mean was 67.77, and the standard deviation was found to
be 10.87. The level of significance used was 0.0001. After this data was established, the degrees
of freedom and t-value were calculated. The degrees of freedom were found to be 58, and t-
value, ≈ -6.45. Using a t-distribution table, the p-value was found to be <0.0001. Since the p-
value is much smaller than the level of significance of 0.05, the null hypothesis was rejected.
Therefore, there is a statistically significant difference between the means of the two sample
groups, indicating a difference in circulation rates between regions of Heavy Blackspot and Less
Blackspot.
In previous research (Tobler and Schlupp, 2008) of Gambusia Blackspot infections and other
related parasites, parasite infections with Blackspot was found to interfere with Shoaling Behavior.
This included Social/Schooling Parasites can fundamentally altering the cost–benefit ratio of living
in a group, e.g. if infected individuals increase the predation risk of shoal mates. In their research,
they found the effect of an infection with a trematode,LUvuliferLsp. (Diplostomatidae) on the
shoaling behavior of female western mosquitofish,LGambusia affinis. In our study of male and
female eastern mosquitofish, Gambusia holbrooki, informally, we observed similar behavior.
Tobler and Schlupp (2008) demonstrated the parasite causes a direct phenotypical change of the
host by forming black spots on its body surface. When given a choice between a stimulus shoal and
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no shoal, shoaling tendencies were significantly reduced in infected focal fish. In another
experiment, Tobler and Schlupp (2008) tested for association preferences relative to the infection
status of the stimulus fish. Given the choice between an infected and a healthy stimulus fish, both
infected and healthy focal fish preferred to associate with non-infected stimulus fish. Tobler and
Schlupp (2008) results suggested that (1) the cost–benefit ratio of shoaling might be different for
infected and non-infected individuals. Infected fish may be more affected by competition for food
within a shoal. (2) Associating with infected conspecifics appears to be costly for female
mosquitofish, maybe due to increased predation risk. Blackspot in Gambusia affects mate choice
(Females don’t want males).
We also believe that lesser RBC Circulation (fewer red blood cells through a capillary) over time
may lead in the caudal tail could leads to Fin Rot and Disease and more disease. In Fancy Guppie
with large tails: Fin rot can be the result of aLbacterial infection (ex. Pseudomonas fluorescens),
which causes a ragged rotting of the fin), or as a fungal infection. Poor circulation in the fish tails
may lead to a fin rot and a bacterial/fungal infection of the fins that virtually eats away at them. At first
glance, a fish's fins or mouth may look damaged. This could have been the first problem that led to
infection. Fins, mouths, and tails, on the other hand, degrade and rot away with time. If a fungal
infection isn't visible, the fish is most likely suffering from fin rot It is typically brought on by poor
water conditions that stress the fish and lower their immune system's ability to fight other diseases
beyond blackspot.
Conclusions
More study is needed to determine the effects of poor circulation, the presence of parasites on
survival, rapid adaptation to new environments in Gambusia and its relatives in the Poeciliidae
family.
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REFERENCES
Bush AO, Fernández JC, Esch GW, Seed JR (2001) Parasitism: the diversity and ecology of
animal parasites. Cambridge University Press, Cambridge.
Kirse, Sarah C., "Parasite Ecology of Fish with Black Spot Disease" (2010). Senior Honors
Theses. 184.https://digitalcommons.liberty.edu/honors/184
Shubel, S. 2006. Aquarium Care of Fancy guppies. Animal Planet. Tropical Fish Hobbyist TFH
Publications. Neptune City, NJ.
Tobler, M., Schlupp, I. Influence of black spot disease on shoaling behaviour in female western
mosquitofish,LGambusia affinisL(Poeciliidae, Teleostei).LEnviron Biol FishL81, 29–34 (2008).
https://doi.org/10.1007/s10641-006-9153-x
Figure 4. Calculated data contributory to statistical analysis. An independent two-tailed t-test
was performed to compare the two sample groups.
Appendix 1
STATISTICAL ANALYSIS CALCULATIONS
(1) Mean and Standard Deviation derived using Microsoft Excel
(2) Calculate the degrees of freedom (df):
df = (n1 - 1) + (n2 - 1) = (30 - 1) + (30 - 1) = 29 + 29 = 58
(3) Calculate t-value
t = (M1 - M2) / sqrt[(SD1^2 / n1) + (SD2^2 / n2)]
t = (49.83333333 - 67.76666667) / sqrt[(10.67411737^2 / 30) + (10.87129512^2 / 30)]
t = (-17.93333334) / sqrt[(113.738592 / 30) + (118.336627 / 30)] t =
(-17.93333334) / sqrt[3.7912864 + 3.94455423]
t = (-17.93333334) / sqrt[7.73584063] t =
(-17.93333334) / 2.78003367
t ≈ -6.45
(4) With the calculated t-value (-6.45) and degrees of freedom (58), a t-distribution table was used to
find the p-value.
P-value < 0.0001
(5) 0.0001 < 0.05
The null hypothesis (H0) is rejected.
