I have a some specific questions for a research project. I am a starting researcher with close to no experience other then what I learned at my university so it could be that what I am saying is completely unfeasible or unlogical, therefore I would like some advice on how to make it feasible.

Within the field of Entomology there is a growing interest for measuring seasonal environmental related changes within the insect microbiome. I am planning a gut microbiome time series study to investige the relationship of specific bacterial taxa in Lu. longipalpis and Ae. aegypti to the seasonally changing environmental conditions they are living in. To make a model for this I thought of following theoretical concepts and would like some help in whether or not I am considering enough/to much factors to make the project feasible and advice on how to make it better.

Research question : How does the gut microbiome of Lu. longipalpis and Ae. aegypti contribute to their ability to transmit leishmaniasis and dengue fever, and how do changes in the microbiome over time affect the risk of transmission of these diseases?

The study will be performed within a broader context of a One Health approach conducted in an entire city where spatial (precise insect collection sites and characteristics of location), environmental (temperature, humidity, rainfall, daylight hours) and epidemiological (VL cases in both humans and canines and dengue cases in humans) data are available as a part of the routine surveillance. Furthermore a lot of laboratory studies have already been conducted on the influence of the microbiome on insect immunity and there is also information available on microbiome interspecies competition and continuously more information pooring in as well.

Field conditions at moment of collection:

Blood meal: Yes/No

Precipitation: Yes/No at moment of collection

Humidity: % at moment of collection

Temperature at moment of collection

I want to make the following assumptions (2-3 days between collections)

Mean precipitation between two collection rounds

Mean temperature between two collection rounds

Host age: assuming generation times as acording to average temperature for each insect species.

Average daylight-hours change between 2 collections.

Insects will be collected within a niche within the city and both insect vectors have a quiet sedentary lifestyle (Aedes aegypti flies a max of 50m, 49% of Lutzomyia longipalpis do not fly further then 50m, 48% not further then 300m and 3% fly not further then 500m from their birthplace during their lifetime) so all collected specimens of the specific insect species will be considered to be affected similarly at the gut microbiota level within a generation time.

Sample size:

10 insects of Aedes aegypti and 10 insects of Lutzomyia longipalpis will be captured and identified (only females) at the same location.

For Aedes aegypti: 40-60 insect specimens per generation (2-3 weeks life cycle)x 2 collections per week

For Lutzomyia longipalpis: 80-120 specimens per generation (4-6 weeks life cycle)x 2 collections per week.

Duration: 2 to 3 years of study

Aedes aegypti: +- 45 generations

Lutzomyia longipalpis: +-25 generations

Sequencing method: 16S rRNA sequencing

Possible seen changes on the microbiome:

Growth (birth): specific taxa grow in abundance

Elimination (death): specific taxa reduce in abundance

Colonization (immigration): new taxa are introduced affecting composition

Dispersion (Emigration): specific taxa are lost, affecting composition