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SPECIFIC REQUIREMENTS


3. Laboratory cage trial using punctured fruit

The following basic components are required to conduct a laboratory cage trial:

3.1. Adult fruit flies

Adult fruit flies should be obtained from laboratory colonies. The laboratory colonies of multivoltine species used should be no more than one year old or, if older than one year, they should have been replenished with wild flies at least once every 12 months. Records of colony performance and replenishment will be required in addition to host status results.

3.2 Fecundity test

Prior to conducting host status trials, a fecundity test should be conducted on gravid females from the laboratory colonies. This allows the estimation of the potential oviposition load to which the replicates of the test fruit may be exposed.

At least five replicates, each of 10 gravid females per cage, should be used for the fecundity tests. Cages should have fine mesh of minimum dimensions of 300 mm x 300 mm x 300 mm. Measures should be taken to prevent access by ants and Drosophila spp. Each cage should contain a source of sugar and water.

Oviposition receptacles can be either a hollowed, punctured dome of a known host or an artificial egging device. If a dome is used, its edges should be sealed to prevent flies from getting under the dome. Oviposition receptacles should be exposed to gravid females for a period of 24 hours.

After 24 hours exposure, the eggs should be washed from the dome or the artificial egging device. Those embedded in the dome should be carefully eased out of the fruit tissue and washed from the dome. The eggs should then be placed on moist filter paper, counted and held for a sufficient period to determine egg hatch. This allows the calculation of the mean number of viable eggs per gravid female over a 24-hour period.

The number of gravid females to be used per replicate should be adequate to ensure that each replicate is exposed to a potential oviposition load of a minimum of 1 000 viable eggs.

3.3 Fruit flies used in the trials

Each fruit fly species for which host-status studies are required should be tested separately.

The determined number of gravid females should be caged with test fruit for 24 hours. The trial will consist of 5 replicates each with the same number of gravid females per cage.

Gravid females for the laboratory cage trials should be obtained from the same cage of flies used in the fecundity test. Flies should be at their peak fecundity.

3.4 Test fruit

The host status of each fruit variety should be tested separately. A variety may be described formally in an application for proprietary rights[2] or, where this is not the case, a variety should be described including distinctive commodity characteristics when present. Colour photographs of the trial commodity are required if a variety has not been formally described under proprietary rights.

Test fruit of the described variety should be grown under conditions that exclude the use of chemicals that may deleterious to fruit flies (e.g. insecticides, miticides).

Test fruit should be collected at the stage of maturity accepted for export harvest. The stage of maturity should be described by the grower/supplier.

The trials should be replicated three times with trial fruit sourced from different producers for each replicate. For each replicate five batches, each with a minimum of 500 g of fruit, should be used. Whole fruit should be used, irrespective of the weight of individual fruit. The weight and number of fruit used per replicate should be recorded just prior to exposure to the flies.

A control using a minimum of 500 g of a known primary/preferred host should be run concurrently with the 5 trial replicates. This provides evidence that the experimental procedures adopted do not prevent the successful emergence of fruit flies. The control replicate should be exposed to the same number of gravid females as determined in section 3.1.

Before exposure of a fruit to female flies, the skin of the trial fruit and control fruit should be punctured 50 times penetrating through and puncturing the pericarp of the fruit using entomological pins of size 3. The punctures should be distributed evenly across the surface of the fruit. When placed in the trial cage, fruit should be randomly orientated (e.g. stem end up, blossom end up) in a single layer. Fruit should remain in the cages for a period of 24 hours.

Cages should have minimum dimensions of 300 mm x 300 mm x 300 mm and be covered with a fine mesh. Measures should be taken to prevent access by ants and Drosophila spp. Each cage should contain a source of sugar and water.

Trials should conducted under optimum conditions for fruit fly activity. The minimum and maximum temperatures and relative humidity should be recorded during the period of caging.

At the end of the 24-hour period the number of dead flies per cage should be recorded. High adult mortality may indicate unfavourable conditions (e.g. excessive temperature) or contamination of trial fruit (e.g. insecticides).

3.5 Fruit holding

After exposure to gravid females for 24 hours, the fruit should be removed from the cage and held over a suitable pupation medium. Sawdust, sand or vermiculite may be used. The medium should be obtained from untreated sources and be sterilised (e.g. 120×C for a minimum of two hours).

Each replicate of fruit should be held separately so that the number of pupae and adults emerging can be recorded per weight of fruit for each replicate.

Fruit that breaks down rapidly (such as eggplant, bitter gourd, cucumber, tomato, banana and most citrus) should be held above the pupation medium on a container covered by fine mesh which allows the passage of juice into the container but prevents larvae entering the container.

Each replicate should be held in individual containers that allow adequate ventilation yet prevent the access of ants and Drosophila spp.

The minimum and maximum temperatures and relative humidity should be recorded each day during the period of fruit holding.

After an appropriate holding period (which may vary with temperature and host) the pupation medium should be sieved to extract pupae. Fruit should be dissected (but not discarded) to determine the presence of larvae. If larvae are present, the fruit should be held until all larvae have pupated.

The numbers of pupae should be recorded and pupae held in a moistened pupation medium until eclosion. All emerging adults should be counted and identified after morphological characteristics have developed (teneral adults should not be used for identification).

3.6 Assessment and interpretation

If no adults emerge from the control replicate, the laboratory cage trial should be repeated.

If adults emerge from the control replicate and no adults emerge from the replicates of trial fruit, then the trial commodity at the described stage of maturity is regarded as a non-host to the fruit fly species tested.

If one or more adults are reared from the trial replicates, then the commodity is considered to be a potential host. A laboratory cage trial using unpunctured fruit should be undertaken.

4. Laboratory cage trial using unpunctured fruit

A laboratory cage trial using unpunctured fruit should be conducted if flies have emerged from the punctured test fruit in the laboratory cage trial described in section 3. Trial methodology and procedures are identical to that described in section 3, except that fruits are not punctured.

Each fruit fly species for which host-status studies are required should be tested separately.

4.1 Assessment and interpretation

If no adults emerge from the control replicate, the laboratory cage trial using undamaged fruit should be repeated.

If adults emerge from the control replicate and no adults emerge from any of the replicates of trial fruit, then the trial commodity at the described stage of maturity can be regarded as a conditional non-host to the fruit fly species tested.

If adults of one or more of the fruit fly species to be tested emerge from trial replicates, then field trials should undertaken.

5. Field cage/glasshouse trials using unpunctured fruit

A field cage or glasshouse trial using unpunctured fruit should be conducted if flies have emerged from the undamaged test fruit in the laboratory trial described in section 4.

Trial methodology and procedures are basically similar to those described in section 3, except that fruits are not punctured and remain attached to the test host plant. The fruiting host plants may be exposed to the test fruit fly species either by caging fruit in the field or by using potted fruiting host plants in a glasshouse.

Each fruit fly species for which host-status studies are required should be tested separately.

5.1 Adult fruit flies

Adult fruit flies should be prepared as in 3.1.

5.2 Fecundity test

Prior to conducting host status trials a fecundity test should be conducted on gravid females from the laboratory colonies. Test should be made as per section 3.2 except that the exposure period is 48 hours.

The number of gravid females to be used per replicate should be adequate to ensure that replicates are exposed to a potential oviposition pressure of at least 1 500 viable eggs.

5.3 Field cage trial

The trials should be replicated three times. For each replicate five batches of approximately 500 g of undamaged fruit attached to the parent plant should be used. The plants should be grown under conditions that exclude the use of chemicals that may be deleterious to fruit flies.

A cage should be placed around the selected fruit be it a single fruit, group of fruits or a whole plant. A replicate of a minimum of 500 g of fruit may comprise more than one cage preferably on one plant but if not possible, on adjacent plants. Should the replicate be divided into multiple cages, the number of gravid females per cage should be evenly distributed between cages to maintain the potential oviposition pressure (1 500 viable eggs) as specified in 5.2.

A suitable cage shall consist of a supporting frame enclosed by a fine gauze cage with minimum dimensions of 300 mm x 300 mm x 300 mm. The mesh should be of a size to ensure containment of the flies and allow airflow.

Where the cage is in place on a tree/plant branch, the cage end(s) should be securely fastened around the branch or stem to prevent escape of the flies and the entry of ants and predators. A source of sugar and water should be provided in each cage for the gravid females.

The minimum and maximum temperatures and relative humidity should be recorded each day for the duration of the trial.

Gravid females for the trial should be obtained from the same cage of flies used for the fecundity test in section 5.2.

A control using approximately 500 g of a known host should be run concurrently with the 5 trial replicates and under exactly the same field conditions. Control fruit should be punctured as per section 3.4 whilst under the same experimental conditions and exposed to the same number of gravid females as the trial fruit as determined in section 5.2.

After exposure to gravid females for 48 hours, the fruit should be removed from the plant and each replicate weighed and the number of fruit recorded. The number of dead flies per cage should also be recorded.

5.4 Glasshouse trials

For glasshouse trials, test fruit should be grown in containers (e.g. pots) of a size that allows normal plant development, including fruit production. The plants should be grown under conditions that exclude the use of chemicals that may be deleterious to fruit flies.

Cages dimensions should be slightly larger than the height and width of the trial plants.

The frame of the cage should be covered by gauze fine enough to exclude Drosophila spp. and other fruit infesting insects. It should be constructed to ensure that flies introduced into the cage would not escape.

Plants in containers are placed in the cage immediately before the trial commences and should be protected from ants. Fruit should be at the described stage of export harvest maturity.

Five batches of approximately 500 g of whole fruit attached to parent plants should be used for each of three replicates. Each batch should be in separate cages. Whole fruit should be used, irrespective of the weight of individual fruit. The weight and number of fruit used per replicate should be recorded subsequent to exposure to gravid females and immediately after harvest.

Depending on the weight of fruit produced per plant, it may be necessary to use multiple plants/cages to achieve the minimum of 500 g of fruit per replicate. Regardless of the number of cages and plants used to house 500 g of fruit, the number of flies/replicate should be evenly distributed amongst the cages.

Gravid females for the trial should be obtained from the same cage of flies used for the fecundity test in section 5.2.

A control replicate using approximately 500 g of a known host should be run concurrently with the 5 trial batches and under exactly the same glasshouse conditions. Control fruit should be punctured as per section 3.4 whilst under the same experimental conditions and exposed to the same number of gravid females as the trial fruit as determined in section 5.2.

After exposure to gravid females for 48 hours, the fruit should be removed from the plant and each replicate weighed and the number of fruit recorded. The number of dead flies per cage should also be recorded.

5.5 Fruit holding

Fruit should be held as described in section 3.5.

5.6 Assessment and interpretation

If no adults emerge from the control fruit, the field or glasshouse trial using undamaged fruit should be repeated.

If adults emerge from the control fruit and no adults emerge from any of the replicates of trial fruit, then the trial fruit at the described stage of export harvest maturity is regarded as a conditional non-host to the fruit fly species tested.

If adults of the fruit fly species in the trial emerge from test fruit in any one replicate, then the fruit is considered a potential host for quarantine purposes.


[2] International Code of Nomenclature for Cultivated Plants 1980; International Union for the Protection of New Varieties of Plants 1991.

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