A bioassay is a chain of four decisions

A bioassay converts a biological question into a measurable response under defined conditions.

01

Define

State the contrast and one scoreable behaviour.

02

Match

Choose an arena that permits the required behaviour.

03

Protect

Identify the unit and control other variation.

04

Claim

Plan the analysis and limit the conclusion.

The question determines the arena. The arena determines the response. The response determines the claim.
Roberts et al., 2023, Entomologia Experimentalis et Applicata · Percie du Sert et al., 2020, PLOS Biology

Behaviour is an observable response

External stimulus

Odour · contact · light · humidity · host texture

Internal state

Age · hunger · experience · reproductive state · time

OBSERVABLE
RESPONSE
defined · detectable · repeatable
Operational definition

Define one response with explicit start, stop and classification rules.

Choice · latency · path · transition · duration · frequency

Variation in the stimulus or the insect's state can create bias or hide a real response.

The evidence ladder limits the claim

01Stimulus received

What reaches the insect?

→
02Detection

Can its sensory system register it?

→
03Behaviour

What does the insect do?

→
04Consequence

What changes afterwards?

ONE RESULT, ONE RUNG

A result supports the rung that was measured. Later rungs require separate evidence.

Roberts et al., 2023, Entomologia Experimentalis et Applicata

Validate the stimulus reaching the insect

Treatment labelApplied dose
≠
At the insectStimulus received
Sensory channelPreserve or validateResponse enabled
Airborne odourRelease rate, airflow, surface loss and source ageOrientation
Surface contactDose, texture, residues and access to plant tissueAcceptance
VisionSpectrum, intensity, contrast and geometryDirected movement
Touch and vibrationBench vibration and unintended air movementStartle or movement
Temperature and humidityThe gradient across the arena, not the room settingActivity
An uncontrolled sensory gradient becomes an alternative explanation.
Roberts et al., 2023, Entomologia Experimentalis et Applicata · Döring, 2014, Annals of Applied Biology · Powell et al., 2006, Annual Review of Entomology

Different paths can produce the same count

ORTHOKINESIS Speed changes

Move faster or slower as stimulus intensity changes.

KLINOKINESIS Turning changes

Turn more or less often, without steering towards the source.

TAXIS Direction changes

Steer relative to a directional cue or gradient.

ARRESTMENT Movement stops

Remain after encounter rather than actively orienting.

A terminal occupancy count cannot distinguish these processes. Record paths, speed, turning, transitions or latency.
Roberts et al., 2023, Entomologia Experimentalis et Applicata

State and history define the insect you test

CURRENT STATE
Development

Narrow age or moult-stage window

Form and reproduction

Sex, wing form and mating state

Feeding and activity

Deprivation, recovery and time of day

POPULATION + EXPERIENCE
Origin

Clone, population and collection history

Culture

Generation, density and rearing environment

Prior exposure

Host, cue and learning history

Keep these factors consistent or balance them among treatments. The tested population sets the boundary of the claim.
Ramírez & Niemeyer, 2000, Journal of Insect Behavior · McLean et al., 2009, Ecological Entomology

Handling can become the treatment

01 · TRANSFER

Use one defined method

Brush contact, aspiration and dislodgement can trigger immobility or escape-like walking.

02 · RECOVERY

Do not trust appearance alone

CO₂ or chilling can alter behaviour after movement appears to recover.

03 · ACCLIMATION

Pilot a fixed interval

Use blank trials to find when baseline behaviour becomes stable for this species and endpoint.

04 · RECORD

Make handling reproducible

Record tool, duration, time off host, recovery, temperature, light and operator.

There is no universal acclimation time. Pre-specify it and validate it for the assay.
Johnson, 1958, Animal Behaviour · Phelan et al., 1976, Annals of the Entomological Society of America · Powell & Bale, 2006, Physiological Entomology

Define one primary endpoint

INFORMAL OBSERVATION

“The aphid seemed to prefer the untreated leaf.”

↓
OPERATIONAL DEFINITION

Specify start and stop rules, observable criteria and treatment of ambiguous cases.

  • Observable: trained observers apply the same rule
  • Bounded: the window and zones are explicit
  • Aligned: the measure represents the tested process
Candidate endpointData structure
First option enteredcategory
Latency to contacttime until response
Residence timecontinuous
Response by the time limityes / no
Probe countcount
Turning ratecontinuous
Nominate the endpoint that answers the question. Treat the remainder as secondary or exploratory.
Percie du Sert et al., 2020, PLOS Biology

Choice and no-choice tests differ

Choice test

Relative selection

Which option is selected when alternatives are simultaneously available?

  • Tests discrimination among offered options
  • Responses within an arena are dependent
  • A dominant option can mask acceptance
No-choice test

Acceptance under constraint

Is an option accepted when no alternative is available?

  • Tests acceptance under confinement
  • Can reveal delayed acceptance
  • Confinement can exaggerate field use
Preference, acceptance and suitability are different claims.
Murray et al., 2010, Biological Control

Observation is part of the instrument

01

Record

Video or timestamped event log

02

Score

Explicit start, stop, zones and ambiguity rules

03

Validate

Blind rescoring and tracking checks

EVENT LOGPredefine what the observer can enter
00:00Releasedtrial starts
00:18First contactzone boundary crossed
01:42Settledcriterion held for 30 s
Reliability checkRescore a sample blind and report agreement
Automation increases throughput only when tracking and event classification are validated.
Noldus et al., 2001, Behavior Research Methods, Instruments, & Computers

Detection is not preference

01Stimulus received

Measure the cue

→
02Detection

Sensory evidence

→
03Behaviour

Preference evidence

→
04Consequence

Test separately

SENSORY TESTS

EAG · GC–EAD · single-sensillum recording

EAG sums electrical activity across the antenna. GC–EAD links separated chemicals to antennal responses. Single-sensillum recording tests individual receptor units.

BEHAVIOURAL TEST

Orientation, choice, settling or feeding

A sensory response does not establish attraction or avoidance. A negative whole-antenna recording does not prove that no receptor can detect the cue.

Revisit the same ladder: claim only the rung that the method measures.
Roberts et al., 2023, Entomologia Experimentalis et Applicata

The treatment assignment defines n

Illustrative simulation

Five dishes per treatment

Each dish receives one treatment and contains 20 aphids. The dish is the independently treated unit; aphids within it are subsamples.

162020187 versus 61571120

Numbers are responders out of 20. The simulated dish-to-dish variation is real variation in the design.

BEFORE THE REVEAL

What is n?

Choose the independent unit before looking at the p-value.

WRONG UNITn = 200 aphids

81% versus 59%

p < 0.001False precision from treating subsamples as independent
CORRECT UNITn = 10 dishes

Exact dish-level randomisation test

p = 0.26The evidence is weak once dish variation is respected
A more complicated model cannot create independent treatment assignments that the experiment never made.
Percie du Sert et al., 2020, PLOS Biology

Randomise, block and blind

Design actionWhat it protects againstBioassay example
RandomisePredictable treatment assignmentTreatment side and run order
BlockKnown temporal or apparatus variationDay, cohort, apparatus or source batch
BalanceChance imbalance in small experimentsEqual treatments within each block
BlindObserver and analysis expectationsCoded videos and treatment labels
Record the allocation and actual run order, including departures from the plan.
Percie du Sert et al., 2020, PLOS Biology

Non-response is part of the result

DEFINE BEFORE DATA COLLECTION

  • What counts as a response?
  • How will an insect that reaches the time limit be recorded?
  • Which equipment or handling failures justify exclusion?

REPORT IT, ALWAYS

  • Numbers released, responding and excluded
  • Reasons and rates for each treatment
  • Whether reasonable alternative rules change the result
Immobility, delayed response or dropping may be biology, not equipment failure.
Percie du Sert et al., 2020, PLOS Biology

Diagnose 60% non-response

ACTIVITY · 2 MINUTES

Choose the first two checks, state the evidence needed and define a stop/go rule.

01 · DELIVERYDid the cue reach the insect?

Check airflow, light, temperature, surface dose or source output.

02 · STATEWas the insect ready?

Check stage, feeding state, recovery, acclimation and activity window.

03 · ARENACould it perform the behaviour?

Check geometry, confinement, wall effects and unintended gradients.

04 · ENDPOINTDid the score miss the response?

Inspect paths, latency, dropping and the observation window.

05 · ASSAY CHECKCould the assay detect a response today?

Run identical blanks and a validated positive control where one exists.

A positive control distinguishes “no treatment effect” from “no functioning assay”. Increase replication only after diagnosis.
Roberts et al., 2023, Entomologia Experimentalis et Applicata

Preregister the plan and check permissions

TIME-STAMP THE DECISIONS

Preregister before collecting data

State the primary endpoint, experimental unit, sample-size rule, exclusions, allocation and analysis.

OSF Registrations · ARRIVE Study Plan

RESPONSIBLE PRACTICE

Legal scope is not the standard

UK law does not currently classify insects as protected animals under the Animals (Scientific Procedures) Act or within the Sentience Act's animal definition. Local ethics and professional responsibilities still apply.

PLANT HEALTH

Check status before obtaining insects

Ask the Animal and Plant Health Agency (APHA) before importing, moving or keeping regulated or unlisted non-native pests. Authorisation depends on pest status and proposed work.

Record deviations from the registered plan. Define containment, decontamination, disposal and incident procedures.
Nosek et al., 2018, PNAS · Percie du Sert et al., 2020, PLOS Biology · Home Office, 2024, GOV.UK · APHA & Defra, 2022, GOV.UK

Four questions before the first trial

1

What are you claiming?

Define the response, comparison, conditions and target population.

2

What is the experimental unit?

Follow treatment assignment and any grouping.

3

What else could explain it?

Control plausible alternatives through the design.

4

What would change your claim?

Decide the endpoint, exclusions and decision rule in advance.

The question determines the arena. The arena determines the response. The response determines the claim.

Design a Petri-dish escape bioassay

HYPOTHESIS

Predator-like brush contact increases the probability that a settled aphid leaves a field-bean leaf disc within 60 seconds, by dropping or walking off, compared with a no-contact brush approach.

SET-UP · 2 HOURS
  • One wingless adult in each 90-mm Petri dish
  • Raised leaf disc above damp filter paper so a drop is visible
  • One named clone per species; fixed settling rule
  • Record and constrain temperature and light
DESIGN + OUTCOMES
  • Pea aphid and black bean aphid × contact and no-contact control
  • Use each aphid, disc and dish once; randomise within blocks
  • Primary: left the disc, yes or no
  • Mode: dropped · walked off · remained
ANALYSIS + CLAIM
  • Binomial model: treatment, species and block
  • Report absolute difference with 95% confidence interval
  • Use a multinomial model only if category counts are adequate
  • Limit the claim to the tested clones and conditions
Deliverable: protocol · randomisation sheet · data table · planned analysis · claim boundary
Braendle & Weisser, 2001, Journal of Insect Behavior · Gish, 2021, Journal of Pest Science · Rasekh et al., 2010, Journal of Insect Science