Allele shuffling of single gene traits in Drosophila
Species Management Plan for Rana pipiens and Rana catesbeiana on the Durango Nature Studies Nature Center Property
India Waller
Durango, CO
2012
Purpose:
The purpose of this lab is to investigate whether the shuffling of alleles is random using Drosophila.
Introduction:
When choosing the organism to be study, it is a good idea to get something small, easy to keep, has a short generation time, and produces a lot of offspring. The fruit fly Drosophila melanogaster is an organism suggested by scientist and fits all the acquirements above making it a great model organism for genetic research. Drosophila, through research, has helped establish many facts about genetics.
Null Hypothesis:
If the p-value is greater than 5% than allele shuffling can be said to be random.
Materials:
Sorting brushes, sorting cards, fly morgue, stereomicroscope, petri dish of wild-type flies, petri dish of mutant-type flies, petri dish of F1 flies, F1 cross vial, culture vial with medium, foam plug, Fly Nap Kit, Instant Drosophila Medium, yeast, alcohol.
Methods:
The safety concern for this lab is over inhalation of Fly Nap.
1. Parent Generation was anesthetized by Fly Nap.
2. Wild-type and mutant-type were observed using sorting brush, sorting card and stereomicroscope. Wild-type and mutant-types were compared. Eye color and wing shape were noted.
3. Table was filled in with mutant-type and wild-type phenotypes.
4. The practicing sexing of fruit flies occurred.
5. The flies were then disposed of by means of the morgue. The morgue is a small container with a snap top and is filled with alcohol.
6. F1 flies anesthetized by Fly Nap.
7. The phenotype and sex of each fly was established recorded.
8. The culture vial for the F1 cross was set up. The Instant Drosophila Medium was prepared by mixing instant mash potatoes and water, when it solidified it was put into vial and sprinkled with 4-7 grains of yeast.
9. Then six males and six females of the F1 generation were placed in the vial. The vial was labeled with the appropriate date, parental strained, name and name of person executing experiment.
10. Punnett squares were then made to predict the phenotypes of the offspring of the F1 generation.
11. The F1 parents were removed from vials and put into morgue seven days after the cross was set up.
12. The F2 generation in larvae form was observed.
13. When the F2 generation emerged, they were anesthetized. Then the F2 flies were sexed and the phenotype of each was established and recorded.
14. Then they were put into the morgue.
15. The data was recorded.
16. Chi-square value statistics were calculated based on class data.
Results and Analysis:
For cross 1, sepia-eyed and wild-eyed, the F2 generation consisted of 871 Drosophila for the phenotype wild-eye and 326 for the phenotype sepia-eye. The total F2 generation from this cross was 1197 Drosophila. The expected amounts according to our punnett square prediction was 897.75 wild-eyed and 299.25 sepia-eyed. Example calculation: 1197x0.75=897.75. 1197 being total and 0.75 being the percentage of wild-eye offspring predicted. The chi-square value is a statistical test used to determine how close the observed amount is to the expected amount. The chi-square value for the wild-eye was 0.7970620997, for the sepia-eye it was 2.391186299, the sum of the two was 3.188248399. The sum of all three had a p-Value of >5%.
Chi-Square Equation:
For cross 2, between the phenotypes vestigial-red, vestigial-sepia, wild-red, and wild-sepia, the F2 generation consisted of 197 vestigial-red, 231 vestigial-sepia, 708 wild-red, and 240 wild-sepia. The total F2 generation from this cross was 1376 Drosophila. The expected amounts according to our punnett square prediction were, 258 for vestigial-red, 86 vestigial-sepia, 774 wild-normal, 258 wild-sepia. The chi –square value for the vestigial-red phenotype was 14.42248062, vestigial-sepia 244.4767442, wild-red 5.627906977, and wild-sepia 1.255831395. The total was 265.7829632. The p-Value of vestigial-red, and vestigial-sepia was <5%. For wild-red, and wild-sepia the p-Value was >5%. The total for all four phenotype chi-square values had a p-Value of <5%.
For cross 3, between the sex trait phenotypes white-eye male, white-eye female, wild-eye male, and wild-eye female, the F2 generation consisted of 228 white-eye male, 184 white-eye female, 240 wild-eye male, and 214 wild-eye female. The total of Drosophila for this cross was 866. The expected amounts according to our punnett square predictions were that there would be equal probability of each trait being inherited, therefore each phenotype was expected to have 216.5 Drosophila. The chi-square value for each phenotype was 0.6108545035 for white-eye males, 4.90655583 for white-eye females, 2.527977839 for wild-eye males, and 0.031209603 for wild-eye females. The total of all four chi-square values was 8.076597776. All the phenotypes had a p-Value of >5%. The total had a p-Value of <5%.
Analysis:
The total chi-square value for cross 1 is >5% which means that these results are not significant so the hypothesis is accepted. The total chi-square value for cross 2 is <5% which means that these results are significant so the hypothesis is rejected. The total chi-square value for cross three is also >5% which means that these results are significant so the hypothesis is rejected. Because of the two significant results another experiment is suggested to compare to the results found during this experiment. There were many variables involves in this experiment that could explain these results. The temperature in the room could affect the breeding of the flies. For cross 2 where vestigial wing was a phenotype it is probable that the mangling of the flies by mash potato and rough handling could be interpreted as a vestigial wing. Human error is also a major factor that could be accountable for error in our results. The data suggest that alleles are not shuffled randomly.
| Cross 1: Sepia & Wild Eye | |||||
| Ss X Ss | |||||
| Phenotype | Observed | Expected | Chi-Square Value | p-Value | Significant (Y/N) |
| Red-Eyed | 871 | 897.75 | 0.7970621 | >5% | N |
| Sepia-Eyed | 326 | 299.25 | 2.391186299 | >5% | N |
| Total | 1197 | 1197 | 3.188248399 | >5% | N |
| Cross 2: Vestigial/Wild & Sepia/Wild | |||||
| Vv/Ss X Vv/Ss | |||||
| Phenotype | Observed | Expected | Chi-Square Value | p-Value | Significant (Y/N) |
| Vestigial Red | 197 | 258 | 14.42248062 | <5% | Y |
| Vestigial Sepia | 231 | 86 | 244.4767442 | <5% | Y |
| Wild Red | 708 | 774 | 5.627906977 | >5% | N |
| Wild Sepia | 240 | 258 | 1.255831395 | >5% | N |
| Total | 1376 | 1376 | 265.7829632 | <5% | Y |
| Cross 3: White-Eyed And Wild-Eye | |||||
| Ww X Ww | |||||
| Phenotype | Observed | Expected | Chi-Square Value | p-Value | Significant (Y/N) |
| White-Eye Male | 228 | 216.5 | 0.610854504 | >5% | N |
| White-Eye Female | 184 | 216.5 | 4.90655583 | >5% | N |
| Wild-Eye Male | 240 | 216.5 | 2.527977839 | >5% | N |
| Wild-Eye Female | 214 | 216.5 | 0.031209603 | >5% | N |
| Total | 866 | 866 | 8.076597776 | <5% | Y |
Species Management Plan for Rana pipiens and Rana catesbeiana on the Durango Nature Studies Nature Center Property
India Waller and Robin Austin
Durango, CO
2011
Field Condition Form: Robin Austin
Durango Nature Studies would like to manage its 140 acres at the Durango Nature Center to promote native ecology, specifically native northern leopard frog populations (Rana pipiens) through the elimination of invasive species, such as the bullfrog (Rana catesbeiana), which the center started seeing three years ago (Figure 1). Bullfrogs negatively impact diversity, specifically in amphibians, because they compete for the same food, have more of a defense against natural viruses, and may cause mortality in other tadpoles through skin excretions as well as changing the natural patterns of the larvae. Durango Nature Studies hosts many school and community programs that focus on natural systems, so would like to address the arrival of the invasive species R. catesbeiana in this management plan.
The tenth grade biology class at Animas High School conducted a visual encounter study of bullfrogs and northern leopard frogs on the Durango Nature Studies property (Figure 2). These surveys allowed for the estimation of where the populations stand now, so we can understand what management practices are needed. In addition to taking stock of the amphibian population, studies were also conducted on the water quality, land use, and animal signs.
Management practices can be done anywhere on the property except where it would interfere with and affect research. Durango Nature Studies is willing to spend $2,000 a year to manage this property.
Plan Development: Robin Austin
Northern leopard frogs have different habitat needs depending on their developmental stage and the time of year. During the early development of the leopard frog, the preference of mating leopard frogs is a pond 30-60m in diameter, about 2m deep, as well as ponds that do not support fish populations, and have an open canopy. The ponds must be at least semi-permanent, with a predilection for permanent ponds that dry every few years. After reproducing, the adults of the species move to wet meadows with grasses from several inches to a foot. In the winter, they migrate to sites where they can hibernate under the water. They prefer to be in a river or stream, or near an inlet where oxygen saturation is high because many winter deaths are caused by oxygen depletion. As Smith and Keinath said in their article, R. pipiens prefer meadows, wetlands, or riparian areas. They require multiple ponds with migration corridors open between them to ensure species diversity as well as backup habitat should something happen to the main habitat area. They have a home range of 68-503 m2. Tadpoles are mainly herbivores but they will eat any dead matter. Adult R. pipiens are mainly carnivores and will eat anything that is small enough to fit in their mouth, with their main prey being insects (Smith and Keinath, 2004).
The American bullfrog adapts well to many different climates. This causes it to be quite invasive, and dangerous to natural populations of amphibians. The bullfrog originated in eastern North America, and has since spread to much of the rest of the country (Figure 3), as well as being introduced in many other places in the world. The bullfrog prefers to live in large, deep bodies of water with dense vegetation. The tadpoles require more than one year for metamorphosis, and so the ponds must be permanent. The adults overwinter in the water. They do not seem to be negatively affected by alien fish in the habitat. Male R. catesbeiana are aggressive and one pond can only support between 2 and 4 of them. Cannibalism also contributes to low population densities. Young bullfrogs mainly eat invertebrates, but adults will eat anything that they can catch, from invertebrates to rodents and other frogs. They prefer a temperature range between 8⁰ and 37⁰ C. They are very competitive and can take over a trophic level of the ecosystem. Bullfrog larvae also have a skin excretion that causes deformities and even possibly death in other amphibian species. R. catesbeiana also is much more resistant to and serves as a vector for the most threatening disease that amphibians face, caused by the fungus Chytridiomycosis. This is the main reason that bullfrogs are listed on the list of ‘100 of the World’s Worst Invasive Alien Species’. R. catesbeiana has also been spread by humans as a food source, to many places around the world (Spitzen, 2004)
Durango Nature Studies is 140 acres of wild land under a conservation easement. The Florida River runs through the property. Upstream of the property there is Lemon reservoir and dam, as well as cattle waste runoff and fish stocking, which all can contribute to the downstream habitat. Many varied types of habitat can be found on the property, including riparian, meadows, oak woodlands, pinion juniper forests, and desert arroyos (Durango Nature Studies, 2010).
The tenth grade Biology class at Animas High School performed various tests to assess the quality of the water habitat on the Durango Nature Studies property. The results of these tests can be found in Figures 4 and 5. The tolerance indices of the macro invertebrates found in the pond and river were sufficiently high to indicate fairly healthy conditions. (Figure 6) However, stoneflies, which have a tolerance index of 1 on a scale of one to ten, were not found at all, which shows that there is some pollution affecting the ecosystem. The results of the Shannon-weiner index showed very low biodiversity for both the pond and the river, with the river being slightly higher at 1.02. This scale goes from 0-5 and anything below a one is unstable diversity. Obviously we have a problem in terms of the biodiversity of the water habitats at DNC. The levels of phosphorus were much higher than is to be expected, which is probably due to runoff from upstream. The pH of the pond and river were between 8 and 9 (slightly alkaline) which is the preference of amphibians. The nitrates were at 2ppm, a low value, which is good. Anything above 4 is considered not great, and above 40 is unsafe for drinking. The dissolved oxygen levels were at 8mg/L for the river, and 6.5mg/L for the pond. These are excellent levels for these habitats. Coliform was also found at both locations, but it is uncertain as to the levels. However, high levels of Coliform can cause deformities in the growth of frogs, so it should be monitored (Dunning, 2011). All of these results for the water quality of these areas paint a picture of a reasonably well off habitat, but possibly with higher levels of pollution, as well as the possibility of high Coliform. Other than that, the quality of water at the DNS property is excellent.
Plan Implementation: India Waller
The northern leopard frog is a native species of Colorado that is considered a species of special concern by the Colorado Division of Wildlife. To promote growth of the R. pipiens population on the Durango Nature Studies property their habitat should be improved. The best way to promote the native leopard frog population is to eliminate bullfrogs. The methods for this are outlined in a later section. Improving water quality, lowering phosphate levels in the pond and the river is another way to promote leopard frog populations. Too much phosphate has potential to cause an algal bloom, which would result in reduced dissolved oxygen levels (Smith & Keinath, 2004). Bluegills (Lepomis macrochirus) were introduced to the pond in 2001. L. macrochirus are predators of the northern leopard frog; therefore their population in the established ponds should be monitored and controlled. Another pond should be built on the property as a breeding pond that is void of predatory fish (Figure 7). This will give the R. pipiens another breeding ground where the bluegills will not be a threat (Smith & Keinath, 2004).
To lower phosphate levels an investigation must first be done to find the main sources of phosphate along the Florida River upstream of Durango Natures Studies. Once main sources are located, mediation with the owners of the property can be attempted to reduce phosphate use and/or reduce phosphate run-off into the Florida River. Initial research to locate main sources will be done in September, 2012 by two Animas High school students.
To build the breeding pond a contractor should be hired. The pond should be 0.032ha because it is thought to be an adequate size for the breeding of the northern leopard frog and fits into the budget allowed. The construction of the pond including manual labor will cost $360. For a rubber lining to prevent the water from permeating into the soil there is an additional $130.83 added (Figure 8). Water will have to be delivered and will cost around $180 (Burton, 2005). The combined cost of this project is $700.93, well in the $2,000 budget of the Durango Nature Studies. Since northern leopard frogs tend to return to the pond they were born in to breed, we should also transport larvae from the inhabited pond on the property to the new breeding pond. Animas High School students and/or volunteers will do the transportation of the larvae in the spring, 2013. The pond should be built in September, 2012 so an ecosystem can be started before the larvae are transported in the spring or summer. These seasons are when there would be an abundance of larvae. Half the larvae mass from the establish pond should be transported to the new pond; this way the R. pipiens population will be evenly distributed between the two. The bluegills will be managed and monitored by the fishing activities already established on the property.
In the interest of promoting R. pipiens population it is crucial to monitor and manage the invasive R. catesbeiana population. To do this the following is suggested: monitor bullfrog population, and remove the bullfrogs that are there now. Doing these tasks will control the invasive bullfrog population and therefore, since bullfrogs can be harmful to the native species of northern leopard frog it will promote R. pipiens population. September was chosen because this is when the Animas High School students are available.
The monitoring of the R. catesbeiana population at the Durango Nature Studies pond should be done by mark recapture study or by observation, by Animas High School biology students, each year in September. This study should be done on the pond and river on the Durango Nature Studies property. The removal of all the bullfrogs should be done using direct removal, by Animas High School, also in September. The direct removal will be done by means of pitfall trap arrays. For this five shovels are needed. This will cost $29.90, with a cost of $5.98 per shovel. Pitfall trap arrays will be placed around the existing pond in fall, 2012 prior to construction of the new pond. This will be done 0.5 to 3.0km around the pond and river on the Durango Nature Studies property (Smith and Keinath, 2004). Pitfall trap arrays will be used again in 2014 around the newly established pond in order to estimate the new leopard frog population and check for existing bullfrogs. All removed bullfrog will be given to Animas High School and/or to Mountain Middle School to be used as class pets or dissection.
Plan Evaluation: Robin Austin
Durango Nature Studies promotes community understanding of our native southwest environment. It has various programs to educate the community about their surroundings, including Children Discovering Nature, adult and family programs, research projects, and a range of other seasonal programs. The proposed plan will disturb these programs because of the unnatural upsetting of the habitat. However, because the most disruptive part of the plan (the construction of the pond) is being done in September, it will be less troublesome for the programs, as they run primarily in the winter, spring, and summer (Durango Nature Studies, 2010). The interference with the habitat is more severe, as the building of a pond will require bulldozers and other construction equipment. Due to the time it is being put into action, however, it is unlikely to be irreparably damaging to the habitat. The species of concern, Rana pipiens, is not likely to be disrupted because this activity will not greatly upset the water habitats of the area (Smith and Keinath, 2004).
In order to keep tabs on the populations of R. pipiens, the Animas High School Biology class will perform a visual encounter survey in September. They will keep a record of the numbers found, and make sure that the leopard frogs are steady or increasing in population. To monitor R. catesbeiana populations, Animas will capture any sighted and they will give them to schools for instructional purposes. If there are none who are interested, then we will dispose of them in a cooler full of CO2. This will give the nature center more information about the current situation of the amphibians of their property.
To ensure that the management plan is working as planned, Animas will continue to keep records of population sizes. If there has not been a bullfrog sighted or caught for 5 years, then we can consider the area free of bullfrogs, but Animas High School will continue to monitor the population sizes. If there are more sightings of R. catesbeiana, Animas High School will resume the removal of the frogs. This plan is intended to sustainably increase the numbers of R. pipiens. Animas High School will keep records of the results of the visual encounter survey, and make sure that numbers of R. pipiens increase in the projected way.
Figure 1
Figure 2
Figure 3
Pond Levels
|
Analysis
| |
pH
|
8 or 9
|
more alkaline
|
Nitrogen
|
1ppm
|
unpolluted
|
Dissolved Oxygen
|
6.5mg/L
|
high
|
Phosphorus
|
2ppm
|
high
|
Coliform
|
positive
|
existing
|
Figure 4
River Levels
|
Analysis
| |
pH
|
8 or 9
|
more alkaline
|
Nitrogen
|
2ppm
|
unpolluted
|
Dissolved Oxygen
|
8.5mg/L
|
high
|
Phosphorus
|
1ppm
|
high
|
Coliform
|
positive
|
existing
|
Figure 5
DNS Pond
|
Florida River
| |
Damselfly
|
53
|
0
|
Mayfly
|
41
|
125
|
Midge
|
5
|
15
|
Caddisfly
|
2
|
120
|
Dragonfly
|
1
|
0
|
Blackfly
|
0
|
5
|
Coleoptera
|
0
|
5
|
Stonefly
|
0
|
0
|
Total Number
|
102
|
270
|
Diversity Index
|
0.98
|
1.02
|
Figure 6
Figure 7
Vendor
|
Description
|
Cost per Unit
|
Quantity
|
Total Cost
|
Contractor
|
Construction of a Pond
|
2.50 per yd.3
|
0.032ha
|
$360
|
Water Delivery
|
Water
|
$90 per 0.016ha
|
0.032ha
|
$180
|
Contractor
|
Rubber liner
|
$0.50 per ft.2
|
261.67 ft2
|
$130.83
|
Home Depot
|
Shovel
|
$5.98
|
5
|
$30
|
Animas High
|
Net
|
0
|
3
|
$0
|
Total
|
$700.73
|
Figure 8
Reference List
Burton, T., 2005, How Much Does It Cost to Build a Proper Koi Pond, Mid-Atlantic Koi Club, http://www.makc.com/pondconst_tb.pdf (October 17, 2011)
Durango Nature Studies, 2010. Durango Nature Center. Durango Nature Studies. http://www.durangonaturestudies.org/center.htm
Smith, Brian E. and Keinath, Doug. 2004. Species Assessment for the Northern Leopard Frog (Rana pipiens) in Wyoming. United States Department of the Interior. Cheyenne, Wyoming.
Spitzen – van der Sluijs, A. M. & R. Zollinger, 2010. Literature review on the American bullfrog Rana catesbeiana (Shaw, 1802). Stichting RAVON, Nijmegen, the Netherlands
Project Reflection Management Plan
India Waller
For this project our task was to create a management plan to promote the native northern leopard frog population and to eliminate the invasive bullfrog on the Durango Nature Studies property. First we had to acquire a better understanding of these two species by means of readings and research. During this project we conducted field work on the DNS property to better understand the northern leopard frog and bull frog habitat that we were dealing with. We were going to do a mark recapture study on the bullfrogs but we couldn’t so instead we did a visual encounter survey. This basically means you go out on the property for a curtain amount of time and count how many bullfrogs you see and this is your estimated population. This was also done on the northern leopard frogs. We also did a wildlife inventory as well as water quality tests on the Florida River and pond and macro invertebrate sampling to assess pollution and biodiversity. We took the data that we gathered and analyzed it. With the macro invertebrate sampling we were able to assess pollution by looking at the pollution tolerances of the bugs found or not found in each body of water. Also with the macro invertebrate sampling we used the Shannon-Weiner index to find the biodiversity. We then created charts and graphs in Excel to communicate this data in a clearer more organized way. All this data and information gathered and read was then used to make a feasible management plan for these two species.
We read an article “Time to stop worrying about invasive species?” that talked about how it’s normal for species to move from theirs habitats. The main point of this article is to show you that just because a species wasn’t there a year ago doesn’t necessarily mean it doesn’t belong there. I feel that invasive species are still a global issue. Yes it is true that some invasive species increase biodiversity but there are also those that are detrimental to the native species of the land they are infiltrating therefore decreasing the biodiversity. I think this article is right is saying that invasive species should be assessed individually. It is natural for more adaptable species to spread, but when this spread is assisted by humans it becomes not natural and can be harmful especially when people are destroying habitat of the native less adaptable species. It becomes a competition for habitat. For these reasons I feel our management plan is still use full. Invasive species are a problem.
In this project we not only learned about the northern leopard frog and bull frog but we also learned how to write technically. This is really important because we are used to write very descriptively and very subjectively, but in a scientific paper you want objective straight forward writing. We also learned about ecology and how different habitats successes are so reliant on all the biotic and abiotic things involved. One of the main ideas that Colleen drilled into our head was that diversity equals sustainability. This project also gave us an opportunity to learn how to wade through a bunch of scientific mumbo jumbo and create something that makes sense to us mere mortals. We also got the chance to do some field work and then analyze this data we gathered. We learned how to use an identification key to identify a species. We also had to make a power point and present our management plan to biologist who knew a lot more about what we were talking about then we did. This improved our professional presentation skills. This helped us become more savvy with power point and also excel where we had to make graphs and data tables. Over this project covered a lot of basic skills that will benefit us as well as more specific knowledge.
