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Priya 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.

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.

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.

Experiment Flowchart

Make observation

Construct Hypothesis

Report Results

Drawing Conclusion

Test Experiment

Order of activities

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.

Orthoptera drawing and label

Two pairs of fully developed wings, which extend, thick and veiny

Back legs for jumping

Orthoptera

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.

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

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.

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

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

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.

Bacterial cell

Animal cell

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.

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.

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

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.

GEL Electrophoresis and Unknown Sample Result

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.

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.

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.

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.

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

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.