finaldraftppt.pptx
Week 4 Discussion
April 15, 2022MovieannotationworksheetMod51.docx
April 15, 2022Priya Patel
Life cycle of Wolbachia
Introduction to Wolbachia
Wolbachia are primary reproductive parasites.
Their effects on hosts include inducing parthenogenesis, male killing and feminization2.
Effects of Wolbachia on insects species include:
Manipulating reproduction cycle.
Establishes stable association via vertical transmission.
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Impacts of Wolbachia on Human Health
Wolbachia impacts on insects directly affects human beings.
When they manifest in insects that live with humans in the same environment, chances of disease causes or transmission is high.
Lymphatic filariasis: Caused by nematodes which are roundworms under family Filariodidea.
human onchocerciasis: Also caused by nematodes.
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Rationale for Project Participation
GSU participates because in order to;
Expand their professional field and knowledge
Strengthen their careers
Enhance their academic performance and contribution.
As an individual the project will help improve my collaborative skills while mentoring me to joining the right field1.
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Experiment Flowchart
| Make observation |
| Construct Hypothesis |
| Report Results |
| Drawing Conclusion |
| Test Experiment |
| Order of activities |
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Date and Photograph
Insect I collected is ANT.
Date of collection: 24th January 2022, Monday
Time of collection: 12:00 am.
Location of collection: I found on the floor, may be about to get the food which somebody dropped it in the kitchen of my house.
Environment of collection: Ants may be found near their food sources, moisture and in hidden, protected places like wall voids, under appliances, behind window frames and beneath floors. Ants are commensal pests, meaning they like to live close to people and eat some of the same things we do – sweets, meats, starches and liquids.
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Orthoptera drawing and label
Two pairs of fully developed wings, which extend, thick and veiny
Back legs for jumping
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Orthoptera
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Orthopterans can be found in many types of terrestrial environments. Depending on the species, they are usually seen in conjunction with plants at all levels of the vegetation hierarchy, from the ground to the canopy, in burrows in the soil, or migrating through open ground. Among the types of shelter used by orthopterans are general hiding spots among living plant foliage and dead leaves on the ground, as well as unique buildings.
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Habitat
Low lying plants
Grass filed
Diet
Plants (Flowers, grasses, wheat, corn)
Unique trait
They can jump u to 20 times the length of their body
Wolbachia infection
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With the information collected from last year and my new knowledge contributed too by the journal articles here is my prediction. The presence of Wolbachia in the Orthoptera I collected at aromstrong campus will undergo a DNA testing after this process the Orthoptera will not contain Wolbachia.
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Prevalence 74.5% (Bugrov et al., 2016)
Habitat- tall grassy plains
Phenotype- male killing and feminization
Prevalence 62%
Habitat
Grassy areas
Phenotype
Pathogenesis of hosts
Male killing
cytoplasmic incompatibility
Prediction
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Method of testing
DNA
Prediction
The orthoptera will not have Wolbachia
This due to;
lower number of infected hosts
Complete population replacement
INSECTS DETAIL TABLE
| Sample ID | Insect specimen identification | Insect specimen order name | Extracted DNA concentration | A260 | A280 | Extracted DNA ratio | Quality hint |
| MAC02FSP22-12 | Grasshopper | Insecta orthoptera | 800bp | 3.4 | 1.97 | 1.72 | Clean sample |
| MAC02FSP22-12 | Aphid | Aphis fabae | 700bp | 2.5 | 1.77 | 1.41 | Clean sample |
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The sample is clean because the DNA results are large due to the presence of large genomes in grasshoppers with an average value of 9Gb due to huge value of DNA that tends to be repetitive.
Besides DNA, chromosome differentiation can be performed and also abdominal use such as removal.
Both insects tend to have large genosomes due to the large DNA.
Muscle tissue extraction from hind legs can also be applied.
Abudance in genomes especially the nuclear genome and chromosomes are also major requirements alongside the DNA.
The samples are clean because large DNA with a high A260/A280 ratio in the tissues that can also be noted f PCR is carried out.
Proteins should also be abundant in the sample since they tend to determine the A260/A280 ratio.
Nucleic acid and protein are very significant.
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Bacterial cell
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Animal cell
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DNA Extraction and Quantification of Sample
| Sample ID | Order Name | [DNA] (ng/uL) | A260/A280 | Good Quality?(Yes/No) |
| RICOO3Sp21-21 | Dermaptera | 170.05 | 2.27 | Yes |
| RICOO3Sp21-22 | Blattaria | 7.5 | 1.66 | Yes |
Sample 21 may have a high level of RNA in the sample due to ratio being greater than 2.0.
Sample 22 rounds to 1.7, so both samples are good.
Ratio maybe due to a higher level of protein contaminates in sample.
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DNA isolation can be extracted from different sources such as bacteria, plant, fungi and animal cells. Methods for the extraction of the DNA from the above-mentioned cells are different.
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PCR Design and Sample Preparation
| Tube # | Name of the sample/control | Template DNA(insect sample /type control/none) | Master mix? ( CO1 and 16S rRNA primers)(Yes/No) | Insect CO1 Gene | Wolbachia 16S rRNA Gene (uL) | Template DNA (insect sample/ type of control none) | PCR Grade water | Master Mix | Final Volume |
| 1 | RICOO3Sp21-21 | Negative control | Yes | + | – | 2uL | 0uL | 23uL | 25uL |
| 2 | RICOO3Sp21-22 | Negativecontrol | Yes | + | – | 2uL | 0uL | 23uL | 25uL |
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The first step to proceed with a standard PCR is the primer design. You will need to determine which fragment of the DNA template you are looking to amplify, which will require you to know the DNA sequence of the template.
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GEL Electrophoresis and Unknown Sample Result
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Gel electrophoresis is a technique by which DNA fragments get separated according to size (KB). The process of gel electrophoresis first requires a an agarose gel to be made. Generally 1% gel is made . The gel is made to separate fragments according to size which is then visualized by auto radiography.
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Reflection
Wolbachia has several useful effects; thereby making it an ideal candidate for dengue control.
The bacterium increases the vigour of the female Aedes mosquito thereby enabling the infection to spread to virtual fixation.
This is the reason why Wolbachia is often referred to as the selfish bacterium.
Besides, as Wolbachia infects a wide variety of hosts, one strain of the bacterium isolated from a particular species may be introduced into another species.
In addition, Wolbachia produces a range of effects that can be exploited for disease control.
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Wolbachia bacteria live inside the cells of many insects and some crustaceans and nematodes (roundworms), notably inside ovary and testis cells. Wolbachia survives by being passed down through insect generations within the egg cytoplasm. The result is that any Wolbachia that ends up in the body of a male insect will die when the insect host does.
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References
Ross, P. A., Turelli, M., & Hoffmann, A. A. (2019). Evolutionary ecology of Wolbachia releases for disease control. Annual review of genetics, 53, 93-116.
Tantowijoyo, W., Andari, B., Arguni, E., Budiwati, N., Nurhayati, I., Fitriana, I., … & O’Neill, S. L. (2020). Stable establishment of wMel Wolbachia in Aedes aegypti populations in Yogyakarta, Indonesia. PLoS neglected tropical diseases, 14(4), e0008157.
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References
Bugrov, A. G., Ilinsky, Y. Yu., Strunov, A., Zhukova, M., Kiseleva, E., Akimoto, S., & Tatsuta, H. (2016). First evidence ofWolbachiainfection in populations of grasshopperPodisma sapporensis(Orthoptera: Acrididae). Entomological Science, 19(3), 296–300. https://doi.org/10.1111/ens.12187
Ilinsky, Y., Demenkova, M., Bykov, R., & Bugrov, A. (2022). Narrow Genetic Diversity of Wolbachia Symbionts in Acrididae Grasshopper Hosts (Insecta, Orthoptera). International Journal of Molecular Sciences, 23(2), 853. https://doi.org/10.3390/ijms23020853
Jeong, G., Ahn, J., Jang, Y., Choe, J. C., & Choi, H. (2012). Wolbachia infection in the Loxoblemmus complex (Orthoptera: Gryllidae) in Korea. Journal of Asia-Pacific Entomology, 15(4), 563–566. https://doi.org/10.1016/j.aspen.2012.07.002
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Reference
McNulty, S. N. (2019). Exploring the role of Wolbachia Endobacteria in the biology of filarial nematode parasites.
Hoy, M. A. (2018). Insect molecular genetics: An introduction to principles and applications. Academic Press.
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