Mentor: Nicole Achee

Location of Research: Notre Dame

Category of Research: Microbiology

Working Title: Adaptation of dengue virus vectors to environmental stressors

Topic/problem: ‍the problem is that people are getting infected with this virus that can be deathly in many cases, therefore in order to reduce mosquito population my research is simulating a wild environment that consist of different container types and temperatures that allows me to keep track of mortality and survival rates that may be affecting the larvae life cycle due to the environmental stressors being tested in the lab.

Research Plan:

Background. Dengue virus is an emerging disease of concern in the Americas, and other regions. Estimates of dengue causes are 2000 annually deaths worldwide with the disease representing a major health problem in 19 countries. There are four serotypes of dengue virus all of which cause dengue fever. The virus is a single stranded positive sense RNA virus of the family Flaviviridae, genus Flavivirus.

Dengue fever also known as “break-bone” fever causes symptoms which include fever, headache, muscle and joint pains, and a characteristic skin rash that is similar to measles. In a small proportion of cases the disease develops into the life-threatening dengue hemorrhagic fever (DHF), resulting in bleeding, low level blood platelets, and blood plasma leakage, or into dengue shock syndrome (DSS), where dangerously low blood pressure occurs

Dengue virus is transmitted to humans by the bites of mosquitoes within the genus Aedes, principally Aedes aegypti and Aedes albopictus. Container cleaning campaigns (the removal of breeding sites for Aedes mosquitoes) are routinely conducted for dengue fever prevention. This might cause behavioral changes in the vector as they must adapt to less optimal breeding sites.

Because dengue vectors typically lay their eggs in clean water, an important research question to ask is whether female mosquitoes could lay their eggs in polluted water, and if so, do larvae survive in polluted water? What about if the water is cold? Or if particular container types may effect developing larvae and therefore the fitness of adult female mosquitoes? Previous studies have begun to explore these topics for both Aedes aegyptiand Aedes albopictus. The current project will perform similar experiments under laboratory conditions.
The title of my project is: Adaptation of dengue virus vectors to environmental stressors”. The goal is to determine whether mosquito larvae of two important dengue vectors – Ae. aegypti and Ae. albopictus - can adapt to specific environmental conditions. Because dengue is a major health concern, and is getting worse in many countries, describing adaptations of dengue vector larvae can help guide the scientific community as to how to take action to prevent the growth of vector populations and therefore also reduce dengue fever virus exposure in humans. Objectives include exposing dengue vector larvae to different water conditions and observing their development, survival and mortality behaviors.

The project will occur in three Phases, each of which will be conducted to determine differences in time for larval development, survival and mortality. If time allows, fitness of adult females will also be measured.
  • Phase I will explore effect of water temperature. Usually the larvae of Aedes aegypti and Aedes albopictus live in tropical temperature areas, but global climate change is causing mosquitoes to expand to more temperate environments [ref]. This Phase of the project will evaluate both Aedes aegypti and Aedes albopictus larvae under a ‘warm’ and ‘cold’ water temperature condition.
  • Phase II will explore effect of water quality. Evidence exists that the expansion of brackish water bodies in coastal zones can lead to an increase in densities of salinity-tolerant mosquitoes of medical importance like Anopheles sundaicus (a malaria vector) and Culex sitiens (a vector of lymphatic filiariasis). Similar adaptations of fresh water mosquito vectors like Aedes aegypti and Aedes albopictus to salinity is also a possibility. This Phase of the project will expose the larvae of both species to different water qualities – a polluted and a ‘clean’ water condition.
  • Phase III will explore effect of container type. It is known that Aegypti aegypti can lay eggs in waste containers (i.e. soda cans) that might have been discarded outdoors but still contain water. In the laboratory, standard protocols have been developed to raise mosquito populations that use ‘rearing’ cups. The color of the cups is white and high survival occurs. This Phase of the project will attempt to rear larvae in containers with different color, sizes, and/or construction material (i.e., clay, plastic, metal).
Each Phase will have a unique hypothesis to test if water temperature, water quality, and container type will have an effect on Ae.aegypti and Ae. albopictus larval development, survival and mortality behavior.
  • Phase I: water temperature; my expectations for this phase is to observe the mortality of larvae in warm and cold temperature, I expect more larvae to die in cold water than warm.
  • Phase II: water quality; from this phase I expect more female larvae to die in polluted water, and more larvae to survive in clean water. I am also looking for a new adaptation in polluted water.
  • Phase III: container type; my expectations for this phase is to use different container material, I expect more larvae to die in metal material than clay (or other).

Similar hypothesis will be tested for both Ae. aegypti and Ae. albopictus. In addition comparison between species will also be conducted from experiments using both species at the same time.

Hatching eggs:

- Hatching the necessary number of eggs, by cutting a dried oviposition egg paper, label the hatching cup with the date harvested and hatch date.

- Fill the rearing cup containing eggs strips with 450 ml tap water.

- Make sure paper is fully submerged.

- Place under vacuum for 15-20 minutes.

- After eggs have vacuum – hatched feed larvae with crushed fish food pellets and a pipette.

- Place one scoop of ground fish food into rearing cup with pipette.

- After the first day post-hatch transfer eggs strips into a new hatch cup and larvae overnight for delayed hatching.

Larval Sorting:

- After hatching, larvae are sorted into individual rearing cup in groups of 50.

- Place the cup into labeled trays.

Monitoring of Larval Development and Survival:
- One scoop of finely crushed fish food pellets is added to each larval cup the first 2 days post-hatch.

- On day 3 and 6 post-hatch one whole pellet is placed into each cup.

- Drained water from larval cups until 1 inch of liquid remains.

- The refill with 450 ml tap water.
- Count the number of larvae and pupae each day.

- Remove pupae and place into individually label cups.
Identifying Adult Gender:

- Before sorting pupae, larvae are removed from rearing cups using a pipette.

- Once pupae has emerge as adult, adults are placed in the freezer until killed

- Identify and count male and female adults, using morphology
After finishing phase I of my project which regards to water quality I came with the conclusion that temperature does have a big effect on Aedes aegypti mosquito, by giving the larvae different environmental behaviors. I was able to test my hypothesis true, there certainly is more mortality rate in colder temperature, however, some larvae where able to survive in cold temperatures with less humidity, even though it took them longer periods of time to fully grow and develop to become a pupae. which lead me to the conclusion that the more time they take to develop and grow the bigger the pupae and the adult will become which gave me the idea that the bigger the mosquito is, the more blood they will need, which will also give them a longer life cycle, and it will cause more virus among people.
I am now in Phase II of my project testing mortality and survival rates of Aedes aegypti among different container types as said before.


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