Test method for determining resistance to insecticide in the rust red flour beetle, tribolium castaneum (HERBST)

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The test method has three phases.

  1. Establishment of base response data from known susceptible strains.
  2. Use of discriminating concentrations to screen samples for resistance.
  3. Complete definition of resistance by comparison of graded series of concentration mortality responses of susceptible and resistant strains.

Type of Test

The type of test used is continuous exposure of adults, without food, to filter paper impregnated with insecticide. Insecticides are dissolved in a 3:1:1 mixture (by volume) of petroleum other (60-80C b.p.), acetone and Risella 17 oil to which 2 parts of dibutyl phthalate is added in the case of carbaryl. Aliquots of 0.5 ml of the solutions are spread with a pipette on Whatman No. 1,7 cm filter papers. For convenience, depostits are referred to as concentrations of solution rather than as dose/unit area of filter paper. Impregnated papers are allowed to stand overnight on sheets of plate glass. Glass rings, 5 cm dia. and 2.5 cm high are used to confine test insects on the treated surface. The criterion of response used is knockdown, defined as inability of insects to stand and walk. A gentle push forward with forceps is usually sufficient to determine in which category an individual is to be classed.

Papers are labelled with pencil before impregnation. To prevent loss of the insecticide to the substrate during application, papers are supported on 3 fine points or on small glass rings. They are allowed to dry for approximately one minute and then transferred to the plate glass sheet.

The 0.5 ml aliquots of insecticide solution are spread on the filter paper circles with 1 ml pipettes using a progressively decreasing circular pattern to ensure even distribution. Care should be taken to select pipettes with fine points and suitably sized outlets.

To prevent any change in concentration from evaporation of the volatile solvents, never take aliquots of solution for application to papers from the test solution flasks if the solutions are to be retained for later use. Always transfer twice the required voluem to a separate narrow-neck flask and pipette from this, discarding the remainder. The outside of the pipette should be wiped with a tissue after each paper is prepared.

Fresh test papers should be made daily and unused papers discarded. The test solutions should be kept in a refrigerator and under such conditions can have a storage life of several years (this should be checked periodically be chemical analysis). solutions should be allowed to come to room temperature before using.

Collection of Samples

A sample of insects, preferably adults, should be collected from each different commodity or habitat where infestations are detected. Samples must be kept separate and labelled. In general, a sample of 100 insects will be adequate - otherwise as many as possible. Plastic vials, approximately 3 cm diameter and 8 cm high with a perforated lid are convenient containers.)

Insects are collected by:

  1. Sieving of the commodity.
  2. Brushing from floor, walls or surfaces of bags.
  3. Selectively taking small quantities of the commodity or residue in a spoon.
  4. Using an aspirator.

If the insects are substantially separated from the commodity a small quantity of flour should be added to the container. Samples should not be allowed to stand in the sun or be otherwise exposed to extreme conditions and should be despatched as soon as possible to the testing centre.

Receipt of Samples

On receipt in the laboratory, the insects should be separated from any commodity or medium and placed in breeding containers in a medium of flour + yeast. A minimum of 2 9 of medium should be allowed per insect. Samples are placed in an incubator at 30C and 70 percent relative classified "no resistance detectable" and the

Preliminary Discriminating Tests

A minimum of 80 insects in two batches of 40 are used per sample if sufficient material is available. Otherwise all available insects are used. The test insects are removed from the breeding media which is then replaced in the incubator. The insects are counted into vials and held for 1 hour without food at 25C and approximately 70 percent relative humidity. They are then transferred to the filter papers impregnated with insecticide at the discriminating concentration (Appendix 4) and the time of commencement of exposure recorded. After the exact exposure period, the response and total number number of insects used is recorded on the resistance testing data sheet (Appendix 5) and the collector advised.

If there are no survivors, the sample can be classified "no resistance detectable" and the medium in which the test insects were held placed in a hot air oven to destroy the culture. If survivors are recorded beyond the exoposure period, comparisons must be made of the responses to a graded of doses, of the suspect strain and a known susceptible strain using laboratory standardised material. This is done either at the laboratory carrying out preliminary tests or at a central laboratory. Additionally if a low proportion only of insects survive the discriminating dose, the insects not knocked down are removed immediately from the test filter papers and placed in media to breed. Their progeny are tested at the discriminating concentration. If the surviving insects represent the upper range of normal susceptibilitty, all progeny can be expected to respond at the discriminating dose. If, however, they represent true resistant phenotypes, a lower proportion of the progeny will be knocked down than responded in the original sample. This test provides valuable confirmation of resistance in doubtful cases.

Where tests are being made for malathion resistance, it is useful to determine whether the resistance detected is malathion-specific or of a more general type. Extra batches of insects are exposed to papers impregnated with the 1:20 malathion:synergist mixture at the discriminating concentrations of 0.5 percent malathion, 2 percent triphenyl phosphate. If limited material only is available (80 adults) one half of the test insects are exposed at the discriminating concentrations of malathion and triphenyl phosphate to determine whether the resistance is suppressible. Thus if malation resistance is present, an immediate statement can be made of the type of resistance probably present.

Breeding Laboratory Standardised Material

Sufficient insects must be produced to allow a graded concentration-response test to be conducted. Each test requires a minimum of 4 concentrations ( + control) and 3 replications with 40 insects per batch. If the original sample contained 100 insects then the holding period of 4 days will have allowed ample egg production but if the sample contained less than 40 insects the required 600 test insects will not be available and the stock must be increased for a further generation. In general, adult insects are allowed to oviposit for 1 week in media (maximum density 100 insects/200 9 medium) after which they are removed by sieving. The first progeny will appear after approximately 23 days with the peak emergence over the 26-30 day period. All progeny are removed from the culture after 40 days and held for a further 2 weeks in flour before use. The bulk of the progeny will then be 3-5 weeks old adults and for practical purposes individuals with all development times will be represented.

Graded-concentration Response Tests

Test insects are removed from the holding medium one hour before use and counted into 15 (treatment x replications) batches of 40 insects. The batches are assigned to concentrations and replications by overall randomisation. A parallel test with a known susceptible strain should also be included.

With resistant strains the range of higher concentrations must be extended to give a more complete concentration-response curve. The response at the discriminating concentration will assist in determining the range required - alternatively a preliminary assay may be carried out using one replication and full range of concentrations to determine the range required.

A provisional LC50 is assigned by plotting percentage responses, corrected by Abbott's formula, on log-probability paper and eye-fitting a concentrationresponse regression Iine. Results are expressed on a basis of percentage concentration of solution used to impregnate the filter paper circles.

The response data and particulars of the test should be recorded on the Resistance Testing Data Sheet (Appendix 6).

Information That Should be Recorded When Collecting Samples of Beetles for Resistance Tests

A. General

  1. sample number
  2. date
  3. collector's name

B. Location

  1. state (country)
  2. town (nearest post-office)
  3. latitude and longitude

C. Details of site

  1. structure of building (floor, walls, ceiling)
  2. other storage use
  3. insecticide treatment
  4. general infestation level

D. Commodity

  1. variety
  2. origin
  3. time of storage (weeks)
  4. aeration
  5. drying
  6. moisture content
  7. temperature
  8. contamination
  9. insecticide treatment

E. Infestation

For each pest present

  1. species
  2. degree of infestation

Appendix 1 Rearing Temperatures, Media and Approximate Times of Development for the Test Species Rearing times

Insect Rearing
Rearing medium

Rearing times

Sitophilus oryzae 25C Wheat 35 36-43 63
S. zeamais 25C Wheat 35 36-43 63
S. granarius 25C Wheat 34 36-43 63
Rhyzopertha 30C Wheat + broken wheat 35 36-43 63
dominica   (3 1)      
Tribolium 30C Whole wheat flour + yeast 23 26-30 42
castaneum   (12 1)      
T. confusum 30C Whole wheat flour + yeast 27 29-32 42
    (12: 1)      
Oryzaephilus 25C Broken wheat + rolled 26 28-32 42
surinamensis   oats + yeast (5: 5: 1)      
O. mercator 25C Broken wheat + rolled 26 28-32 42
    oats + yeast (5 : 5 : 1)      

Some Response Data From Susceptible Beetles and Suggested Discriminating Concentrations for Detecting Resistance


Effective concentrations

KD50 KD99 KD99.9
Sitophilus oryzae 6 0.89 1.38 1.60 1.5
S. zeamais 6 0.35 0.69 0.85 1.5
S. granarius 6 0.63 1.25 1.57 1.5
Rhyzopertha dominica 24 0.41 1.42 2.15 2.5
Tribolium castaneum 5 0.15 0.32 0.41 0.5
T. confusum 6 0.24 0.43 0.52 0.5
Oryzaephilus surinamensis 5 0.28 0.62 0.80 1.0
O. mercator 5 0.10 0.66 1.20 1.0


REPORT FORM FOR MULTIPLE-DOSE TESTS FOR RESISTANCE OF STORED PRODUCT PESTS TO PESTICIDES FORM 2 (i.e., for basic susceptibility data or for investigations of suspected resistance)

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