To evaluate insect behavioral response (attraction/repellency) to odor sources using a Y-tube olfactometer under controlled airflow conditions.

• Y-tube olfactometer (glass/acrylic)
• एयर delivery system (pump + flow meters)
• Activated charcoal filters (for clean air)
• Humidifier / water wash bottles
• Odor source containers (glass chambers)
• Tubing (inert, e.g., PTFE/silicone)
• Insects (uniform age, sex if required)
• Stopwatch / timer
• Data recording sheet (your Excel sheet)

3.1 Cleaning

• Wash Y-tube with neutral detergent → distilled water → ethanol
• Air dry completely
• Avoid residual odor contamination

3.2 Airflow Setup

• Maintain equal airflow in both arms
  • Typical: 200–500 ml/min per arm
• Ensure:
  • Smooth laminar flow
  • No leakage

3.3 Odor Preparation

• Place:
  • Test odor in one arm
  • Control (clean air/solvent) in the other
• Randomize left/right placement

• Temperature: 25 ± 2°C
• Relative humidity: 60–80%
• Light: uniform, no directional bias
• Avoid external odors (perfume, chemicals)

• Use healthy, active insects
• Standardize:
  • Age
  • Feeding status (e.g., starved 4–24 hrs depending on species)
• Acclimatize insects to lab conditions before testing

Step 1: Start airflow

• Run clean air through system for 2–5 minutes before introducing insect

Step 2: Release insect

• Introduce a single insect at the base of the Y-tube

Step 3: Observation

• Allow insect to move freely
• Record behavior using defined criteria

✔️ Valid Choice

• Insect crosses decision line (≈ 1/3–1/2 arm length)
• Remains oriented in that arm

❌ Not a Choice

• Insect stays at junction
• Moves slightly into arm and returns without crossing decision line

✔️ After crossing decision line

• Choice is final, even if insect comes back

• Max observation time: 3–5 minutes per insect
• If no choice within time → record as:
  • “No Choice” / Non-responder

Optional:

• Record time to first choice (seconds)

• Minimum: 20–50 insects per treatment
• Perform:
  • Multiple replicates
  • On different days if possible

• Switch odor arms after every 5–10 insects
• Rotate Y-tube (if possible)
• Run blank test (clean air vs clean air)
  • Expect ~50:50 distribution

Record for each insect:

• Choice (Left / Right / No Choice)
• Time to choice (optional)
• Notes (hesitation, unusual movement)

Use your prepared Excel sheet for:

• % choice
• Chi-square test
• Statistical validation

• Exclude or report non-responders separately
• Use:
  • Chi-square test (Left vs Right)
• Significance:
  • p < 0.05 → meaningful preference

• After 5–10 insects:
  • Clean Y-tube OR
  • At least flush with clean air
• Between treatments:
  • Full cleaning required

• ✔️ Control test ≈ 50:50 response
• ✔️ Consistent trend across replicates
• ✔️ Low % of non-responders (<30% ideal)
• ✔️ Stable airflow throughout experiment

• Unequal airflow ❌
• Recording choice at junction ❌
• Not switching odor sides ❌
• Using stressed or inactive insects ❌
• Contaminated glassware ❌

• Decision = crossing marked line
• Time limit = 3–5 min
• Sample size = 20–50 insects
• Validate with control test + chi-square