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Keywords:

  • Ceratitis capitata ;
  • area-wide pest management;
  • field cages;
  • GRO exposure;
  • irradiation;
  • Sterile Insect Technique

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The exposure of sterile male Mediterranean fruit fly, Ceratitis capitata (Wiedemann) to the aroma of essential oil derived from ginger root Zingiber officinale Roscoe (GRO) has been shown in field cage trials to increase their mating success. This field cage study compared the mating performance of mass reared sterile Mediterranean fruit fly males prepared for ground release programmes under four different post-irradiation systems, two of which involved exposure of the flies to GRO aroma. In the first system, irradiated pupae were placed into non-vented 5-L paper tubs, individually aromatized by 20 μl of GRO (4 ml/m3) for 96–120 h until the adults were 2–3 days old. In the second system, irradiated pupae were placed in vented 5-L paper tubs, exposed for 24 h in a GRO aromatized room (0.5 ml/m3) when the adults were 3 days old and contained until required for mating tests at 5 days old. The third and fourth systems were the same as the first two systems respectively, except that the flies were not exposed to GRO aroma. Significant differences in mating success were found among treatments. The exposure of 2 to 3-day old flies to GRO aroma improved mating performance slightly compared with equivalent non-exposed flies, but it was still below the minimum value accepted under international standards, while 27% of unexposed 5-day-old sterile males mated and had competitiveness (Fried value 0.38) slightly above this minimum level. GRO exposed 5-day-old flies had the highest mating percentage (67%) and a level of competitiveness (Fried value 1.81) based on induced sterility above that of wild flies (Fried value 1). The results indicate that the effectiveness of sterile male ground release programmes can be increased significantly by releasing 5-day-old sterile males that have been exposed to GRO in an entirely aromatized adult fly holding room.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In Western Australia in 1978, the sterile insect technique (SIT) using flies of both sexes was used to eradicate an isolated population of Mediterranean fruit fly, Ceratitis capitata (Wiedemann) at Carnarvon (Fisher et al. 1985). The SIT was not used again until the genetic sexing strain (GSS) Vienna 7 ‘Mix’ 99 (Robinson et al. 1999) was imported in 1999 and used in a trial programme at Broome (Woods 2001). With this strain, exposure of eggs to high temperature kills all female zygotes allowing male only production (Franz et al. 1996). Since then, this strain has been continuously mass reared in Perth, Western Australia. Four Ceratitis capitata outbreaks in South Australia have been eradicated using the Vienna 7 ‘Mix’ 99 strain and it was also used in the area-wide control programme at Katanning, Western Australia (Jessup et al. 2007).

Production in the mass rearing facility at South Perth is maintained at two million sterile males per week, increasing to 6 million when sterile flies are required to eradicate outbreaks in South Australia or for use at Katanning. Quality standards for pupal weight, emergence and flight ability have been consistently maintained at well above recommended international standards (FAO/IAEA/USDA 2003). The flight ability and longevity of flies released in field programmes, both in South and Western Australia, was monitored by recapturing released sterile males using male lure traps.

The competitiveness of sterile insects is critical to their effectiveness in reducing populations of the target pest. Because of genetic drift and inadvertent selection, long periods of mass rearing can diminish competitiveness (Rössler 1975; Leppla and Ozaki 1991; Miyatake and Yamagishi 1993; Shelly et al. 1994; McInnis et al. 1996; Lance et al. 2000). The loss of competitiveness has been demonstrated in mass reared fruit flies and remains a constant challenge for programmes using the SIT. The recent tendency to rear ‘male only’ strains highlights this issue. These strains are difficult to genetically reinvigorate from wild stock, so facilities tend to rear the strains for as long as possible. A filter rearing system (FRS) is used in mass rearing facilities to maintain the integrity of the GSS in operational programmes (Cáceres et al. 2004). The FRS relies on the careful maintenance of a pure-breeding mother colony from which eggs are harvested. Following 3–4 generations of mass rearing, the resulting males are sterilized and released in the field. Insects that have been through mass rearing are never returned to the mother colony and therefore there is no accumulation of highly selected genotypes in the colony. Replacing a long-established strain or improving the environmental conditions under which a strain is maintained can improve mating competitiveness. The replacement of old strains is a reliable method to improve the quality of mass reared flies, although a loss of production capacity, at least initially, can be expected (McInnis et al. 2002). Another consideration is that quarantine issues may delay the import of new ‘improved’ strains even if this is the preferred option.

Genetic sexing strain Vienna 7/‘mix’ 99 Mediterranean fruit flies have been mass reared in Western Australia since 1999. Customarily, sterile males of this strain have been released into the field in Australia via a mobile ground release system 2–3 days after peak emergence when the majority are still sexually immature (Economopoulos et al. 1988). Concerns that the competitiveness of the strain was diminishing led to pursuing measures that would increase or maintain mating performance until a new strain could be imported.

The exposure of sterile male Mediterranean fruit flies to the aroma of ginger root essential oil containing the known attractant α-copaene (Flath et al., 1994a,b; Nishida et al., 2000) has been shown to significantly increase their mating success. Ginger root oil (GRO) offers the potential to reduce sterile fly release rates by improving mating competitiveness (Shelly et al. 2004a), as has the release of older more competitive sterile males (Paranhos et al. 2010). It was hypothesized as part of this study that combining exposure to GRO with the release of older, sexually mature flies may further increase mating success. In this study, sterile male Mediterranean fruit fly either exposed or not exposed to GRO aroma at 5 or 2–3 days old were used in outdoor field cage tests to compare their mating performance. The two age groups of flies used in this study simulated the established practice of using flies 2–3 days after peak emergence for mobile ground release systems in Australia and added a novel 5-day-old group for evaluation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Four post-irradiation systems, each with a different protocol (table 1), used in existing ground release programmes for sterile medfly were tested. In the first system, irradiated pupae were placed into non-vented paper tubs individually aromatized by GRO until the adults were 2–3 days old. In the second system, irradiated pupae were placed in vented paper tubs, exposed for 24 h in a GRO aromatized room when the adults were 3 days old and contained until required for mating tests at 5 days old. The third and fourth systems were identical to systems one and two respectively except that the flies were not exposed to GRO aroma.

Table 1. Details regarding age and ginger root oil exposure (GRO) or non-exposure (NON-GRO) of the sterile males Ceratitis capitata as well as the number of replicates for each of the four treatments where sterile males were competing in field cage tests with equal number of sexually mature wild males (11–15 days) for wild females (11–17 days)1
Age (days old)Mode of GRO exposureDose (ml/m3)GRO Exposure time (h)Replicates (field cage tests)
  1. 1For each replicate, a simultaneous control field cage was set up containing only 50 fertile wild males and 50 fertile wild females. Also for each replicate, a laboratory cage with irradiated mass reared males and unirradiated mass reared females was established to measure sterile male sterility.

2–3Oil-laden paper strip inside, non-vented tubs4.096–1208
2–3Non-GRO008
5Whole room aromatized, vented tubs0.5246
5Non–GRO006

Wild flies

Parental stocks were collected from infested peaches, plums and pears in metropolitan Perth, Western Australia. The infested fruit was set up in trays over a larger tray of vermiculite. Fully developed larvae popped into the vermiculite and completed their pupation. Pupae were sieved from the vermiculite and placed into a screened holding cage. Emerging adult flies mated and females oviposited into pesticide-free peaches. These were held over coarse white sand, where fully developed larvae pupated. Pupae were sifted daily from the sand and placed into laboratory cages. Within 2 days of emergence, the flies were immobilized and sexed in a refrigerated sea container operating at 5°C. After collection, males and females were kept in separate rooms at 24°C and 40–50% RH in ‘bulk’ cages. Dried figs, 1 : 3 yeast hydrolysate-sugar mix and water were provided ad libitum. Wild flies were collected from the sex-sorted bulk cages at <2 days old and transferred into smaller 7-L transparent acrylic cages in groups of 50. First-generation wild flies were used in this study. The flies were maintained on the same diet and under the same environmental conditions as for the bulk cages. All wild male (11–15 days) and female (11–17 days) flies were sexually mature at the time of their release into the field cages (Liedo et al. 2002).

Colony flies

Mass-reared males for the field cage tests were from a temperature sensitive lethal (tsl) GSS of Mediterranean fruit fly (Vienna 7 ‘Mix 99’). Female zygotes of the tsl strain are killed in a high-temperature (34°C) water bath leaving only a surviving male line of production for field use (Franz et al.1996). This strain has been continuously produced by the Department of Agriculture and Food, Western Australia, since October 1999. Approximately 150 generations had been reared at the time of this study. Adult flies were maintained in the mass rearing facility on a diet of yeast hydrolysate-sugar mix (1 : 3) and water at 21°C and 50% RH. The FRS (Fisher and Cáceres 2000) was used to maintain the genetic integrity and quality of the strain during mass production. Larval diet used for production was a mixture of wheat bran, cane sugar, torula yeast, hydrochloric acid, sodium benzoate and water. Larvae were collected daily in water and pupated in vermiculite. Pupae were kept in the mass rearing facility at 21°C, 80% RH until irradiation. In addition, some fertile mass-reared female flies were required for one component of the Fried test (FAO/IAEA/USDA 2003).

Irradiation

Male pupae from the GSS were collected for irradiation 2 days before emergence. Immediately before irradiation, 60 ml of pupae were placed into a small plastic cup and set up, by means of a jig, in the centre position of an acrylic canister. The canisters are normally used for nitrogen gassing and irradiating up to 1.2 L of pupae. The pupae were flushed in the same canister for 10 min with high-purity nitrogen gas at 5 l/min. Immediately afterwards the canister was placed into the sample chamber of a Gammacell 220 irradiator so that the pupae were located at the centre of the isodose distribution pattern, i.e. the locations within the irradiation chamber that receives 100% of the target dose. The pupae were given a 145 Gy dose of gamma irradiation from the 60Co source and a nitrogen atmosphere was maintained by delivering gas at 5 l/min to the sample chamber for the total irradiation time (18.7 min). Mass reared flies were irradiated on different days according to a schedule, so their availability, at the required ages of 2–3, and 5 days old, was synchronized with the field cage tests. For each field cage test (replicate) of each of the four treatments (table 1), two 60 ml lots were individually irradiated. The sterility of irradiated males was assessed in laboratory cages using a standard method described in the FAO/IAEA/USDA (2003) manual for quality control.

Irradiated males

Two variations of the same 5-L paper tubs were used as bulk holding cages to incubate and accommodate the sterile male flies. The tubs were either ‘non-vented’ or ‘vented’. Both types of tub had the same solid, push-on paper lid with a small tag stapled to the underside which protruded to the outside of the tub to facilitate opening. Non-vented tubs were the type commonly used in sterile Mediterranean fruit fly release programmes in Australia and were unaltered. ‘Vented’ tubs had been modified by punching out two, 10-cm circumference holes on opposite sides of the tub. The holes were then screened with 1 mm aperture, ant-proof mesh, glued to the paper tubs with white silicone gel. The inside of each tub was set up according to the method used in South and Western Australian sterile male release programmes. A lightweight brown paper bag measuring 115 × 125 × 75 mm containing a crumpled piece of paper was placed into each non-vented and vented tub. The crumpled paper was formed from a sleeve measuring 120 × 125 × 75 mm cut from the top of the originally taller bag. This increased the roosting area for the flies after emergence. A 100-ml container of scarified agar-sugar jelly (84.5% water, 15% sugar) was also placed into each tub. Each 60 ml batch of irradiated male pupae was placed into a paper bag, and the lid was pressed into place to close the tub. Sixty ml per 5 L tub (approximately 3600 pupae) is the volume used in sterile Mediterranean fruit fly release programmes in Western Australia.

The irradiated flies were incubated and held as adults in a constant temperature room at 25°C, 40–60% RH and a 12 : 12 (L:D) photoperiod of natural and artificial light. Depending on the treatment, sterile male flies were held as adults for either 2–3 or 5 days after peak emergence before being used in the field cages (table 1). Approximately 20 h before testing, the sterile flies were placed in a chiller at 5°C and ‘knocked down’ (immobilised) for sorting and marking. Fifty flies for each of the four field cages were removed from the holding tubs and set up in a new 5-L paper tub of the same type with agar-sugar jelly. At this time, some flies were also marked to designate their treatment with a dot of non-toxic, water-based, acrylic paint (Chromacryl Students’ Acrylics; Chroma Australia Pty Ltd, NSW, Australia) on the thorax prior to release in the field cages the following morning. Marking was alternated between irradiated and wild males for each replicate.

GRO exposure for 2–3 days release

Immediately after irradiation, pupae of the flies destined for release into the field cages at 2–3 days old were placed in a 5-L non-vented holding tub with a strip of blotter paper (80 × 30 mm) laden with of 20 μl GRO. Thus, the exposure period for pupae and adult flies to GRO aroma in the tubs (4 ml/m3 air) was 96–120 h. Each replicate was allocated two tubs containing approximately 3000 flying males each (60 ml pupae and 85% flight ability). One of these holding tubs was a spare. Approximately 20 h before release into the field cages, a sub-sample numbering 50 adult flies was taken from a holding tub and transferred to a new non-vented tub containing agar-sugar jelly. The oil-laden GRO strip from the original holding tub was also transferred. This occurred in the chiller when sorting, and when necessary, marking the adult male flies. The flies were marked with a dot of non-toxic, water-based, acrylic paint on the thorax again one full day prior to release in the field cages. The oil-laden GRO paper strip remained with the approximate 3000 flying adults until they were released into the field cages in groups of 50 sterile males. Thus, adult flies were exposed to GRO aroma from the time of emergence until they were used in the field cages, a maximum of 72 h. Corresponding sub-samples of irradiated non-GRO-exposed flies were removed from their holding cage, set up, and marked if necessary in the chiller prior to the GRO-exposed group. GRO-exposed and non-GRO-exposed flies were kept in separate rooms prior to release.

GRO exposure for 5 days release

Irradiated flies destined for release into the field cages when 5 days old were held in vented 5-L tubs. Two holding tubs, each containing approximately 3000 flying males were allocated respectively to each treatment (GRO and non-GRO). One of the tubs served as a spare. When the flies were 3 days old, both vented tubs were transferred at 8 am from a laboratory not affected by GRO to an aromatized, 28 m3 constant temperature room at 25°C, 40–60% RH and 12 : 12 (L : D) photoperiod. One ml of GRO was applied to each of 14 cotton wicks in small plastic cages hanging from the ceiling (0.5 ml GRO/m3). The tubs were exposed for 24 h with an oscillating fan used to circulate the air in the room and through the vented tubs. At 8 am the following morning, the tubs were removed from the GRO aroma and taken to the chiller for knockdown, where 50 flies for each replicate were removed from the holding tub. The 50 flies were marked, if due, and set up in a new vented tub with agar-sugar jelly and returned to the laboratory in preparation for their release in the field cage the following morning.

Field cages

Four cylindrical, custom built, field cages were used in each mating competitiveness replicate comparing irradiated and wild male performance. Each cage was constructed of white, 1 mm aperture nylon mesh and had a zippered entrance. The cages were 3 m in diameter, 2 m high and supported by a welded, external tubular steel (25 mm diameter) frame. Plastic ‘cable ties’ were used to secure the cage to the frame. There was a 1.8-m tall, artificial tree (resembling Ficus benjamina L.) in the middle of each cage. Artificial trees were used because they provided a chemically neutral substrate not containing compounds such as α-copaene that affects the sexual behaviour of male Mediterranean fruit fly while the tree structure facilitates the flies in carrying out their other natural activities (Shelly and Villalobos 2004; Shelly et al. 2004b).

Laboratory cages

Four cubical, transparent acrylic cages with sides measuring 20 × 20 × 20 cm, were set up as sterility tests for each replicate with irradiated sterile male flies and fertile colony females. The irradiated males were from the same batch of flies irradiated on that day for the field cage tests, but they were allowed to mature sexually before use. The eggs derived from the matings in these cages were required to provide sterility data for the Fried Test formula for competitiveness (C-value) (FAO/IAEA/USDA 2003). Each cage contained a yeast hydrolysate: cane sugar mixture (1 : 3) and water ad libitum from a 100-ml plastic tub reservoir with a flat sponge wick. One vertical side of the cage was modified by cutting a hole over which fine stainless steel mesh was secured to provide an oviposition site. The cages were slightly raised off the bench and a petri dish of water was placed under the mesh to collect the eggs.

Sterility tests

One laboratory sterility test was run for each replicate with 25 irradiated male and 25 unirradiated female colony flies placed in each of the four laboratory cages previously described. Eggs recovered from the matings in these cages were used to supply sterility data for the corresponding treatment under test. Eggs were recovered daily from water and set up on black filter paper in the same way as described for the eggs derived from the field cage matings. Egg hatch was expressed as the percentage of eggs hatched from the total number of eggs, normal in appearance and collected from a cage.

Experimental plan

All four treatments, consisting of combinations of sterile male age and GRO exposure and non-exposure, are shown in table 1. In general, eight and six replicates (field cage tests) were carried out for the treatments using 2 to 3-day- and 5-day-old sterile males respectively. Four field cages were simultaneously used to test sterile male Mediterranean fruit fly mating competitiveness. The treatments were rotated consecutively into another cage for the next replicate. Each cage contained sterile males and an equal number of fertile wild males and fertile wild females. Thus, each cage contained one irradiated male, one fertile wild male and one fertile female group (n = 50/group). At the same time, another field cage containing 50 fertile wild males and 50 fertile wild females was set up as a control for each replicate.

Field cage tests

At 10 am on the morning of each test day, 50 irradiated treated males and 50 fertile wild males were released into each cage. This was followed 15 min later by the release of 50 fertile wild females. During the field cage tests, all mating pairs were captured in vented and numbered 40 ml clear plastic vials and the participating males were identified as either irradiated or wild by the presence or absence of a paint dot on their thorax. The commencement and duration of mating was recorded along with ambient temperature, relative humidity and light intensity. The relative sterility index (RSI) was recorded as an indicator of sterile male performance (McInnis et al. 1996). The RSI equals the number of sterile matings divided by the number of all matings and is defined by the formula:

  • image

where SW and WW are the number of sterile male and wild male copulations with fertile wild females, respectively.

Egg collection

In the afternoon, after the mating pairs had separated in the plastic capture vials, the mated females were aggregated by treatment into small cages (BugDorm® 60 × 60 × 60 cm) for oviposition into pierced ‘Golden Delicious’ variety (Malus domestica L.) apples. Eggs were then dissected from the apples and collected in a small beaker of water. A fine glass pipette was used to place the eggs in lines onto black filter paper kept moist by a thin, wet sponge in a Petri dish. Eggs that were sound in appearance were kept for eclosion in a controlled temperature room for at least 96 h after which they were scored under a stereo microscope for the number of hatched and unhatched eggs. The small cages used for oviposition and the eggs in Petri dishes were kept in a laboratory at 25° and 60% RH. Eggs were dissected from the pierced apples in the small laboratory cages, and placed on filter paper kept moist by a thin sponge in a petri dish. The number of eggs hatched or unhatched (whichever was the lower) was recorded to provide a percentage egg sterility result. Male mating competitiveness based on egg hatch, C (Fried), was calculated using the formula:

  • image

where W is the number of wild males in the competitiveness field cage, S is the number of sterile males in the competitiveness field cage, Hw is the egg hatch percentage from wild females in the control field cage, Hc is the egg hatch percentage from wild females in the competitiveness field cage and Hs is the egg hatch percentage from GSS females in the laboratory control sterility cage (FAO/IAEA/USDA 2003).

Statistical analyses

Two-way anova was used to identify significant differences between treatments followed by Tukey’s honestly significant difference (HSD) test at P = 0.05 to identify significant differences in pair-wise comparisons between the different groups. Data from replicates with ≤10% of the possible matings were excluded from the analyses. Although this is less than the 20% recommended in the Product Quality Control Manual and Shipping Procedures for Sterile Mass Reared Tephritid Fruit Flies (FAO/IAEA/USDA 2003), it meant that much useful data in an experiment with a restricted, small number of replicates was retained.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Weather conditions

The ambient temperature range inside the field cages during the test replicates ranged between 14.7 and 35.6°C. Relative humidity ranged from 8% to 84% and light intensity from 400 to >5000 light intensity (lux).

Percentage of matings and RSI

Results regarding RSI and the number and average matings in each treatment are given in table 2. A significant difference in RSI was found among the four treatments (F3, 22 = 51.2, MSE = 0.017, P = 0.05, anova). Paired comparison showed no significant difference in RSI between 2 and 3-day-old non-GRO-exposed and both 2 to 3-day-old GRO-exposed and 5-day-old non-GRO-exposed sterile flies. However, 5-day-old GRO-exposed flies achieved a significantly higher RSI than all other treatments (P < 0.05). Of the 2 to 3-day-old sterile flies that were not exposed to GRO, 6 ± 5%, (n = 85) of the total matings were by 2 to 3-day-old irradiated males. GRO exposure increased the mating performance of 2 to 3-day-old flies to 12 ± 7%, (n = 103) of total matings, but this was still below the minimum acceptable RSI level of 0.2 (20% of total matings) for sterile male C. capitata in a cage with a 1 : 1 ratio of sterile : wild males (FAO/IAEA/USDA 2003). Five-day-old unexposed flies had 27 ± 12%, (n = 69) of total matings, which was slightly above the acceptable level. Only 5-day-old GRO aromatized flies demonstrated exceptional mating performance with 67 ± 3%, (n = 102) of total matings indicating an improved mating performance to that of wild flies and GRO exposed flies 2–3 days old.

Table 2. Relative sterility index (RSI) for sterile males of different ages that were either exposed or non-exposed to GRO. Six to eight replicates were run for each of the four treatments involving mating competitiveness of the sterile males competing against wild males for wild females
Age of sterile males (days)Mean RSI ± SE
Non-GRO exposedGRO exposed
  1. Different lower case letters to the right of the RSI values signify treatments that are significantly different (Tukey’s HSD test P = 0.05).

  2. Total number of matings is given in parenthesis. GRO, Ginger root oil; HSD, honestly significant difference.

2–30.06 ± 0.05 (85)a0.12 ± 0.07 (103)a
50.27 ± 0.12 (69)a0.67 ± 0.03 (102)b

Egg sterility

The percentage sterility of the various treatments can be seen in fig. 1. The mean egg sterility in the control field cages with fertile wild males and females was 29.3 ± 1.89%. Post hoc tests revealed that 5-day-old GRO-exposed sterile male flies induced significantly higher sterility (78.68%, n = 5) compared with all other male categories (F4, 28 = 7.45, P < 0.05). The mean sterility from fertile colony females and irradiated sterile males in the laboratory cages, used to provide data for the Fried formula as applied to the field cages, was 99.68 ± 0.05%.

image

Figure 1.  Mean percentage of sterile eggs (±SE) from matings with fertile wild female flies by sterile males of 2–3 or 5 days of age and exposure or non-exposure to ginger root oil and fertile wild male (Control) flies. A lower case letter above a bar signifies a treatment that is significantly different to all other treatments (Tukey’s honestly significant difference test P = 0.05).

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Mating competitiveness based on egg hatch

A Fried C value of 1 indicates equal competitiveness between sterile and wild males. Fried C values between 0.2 and 0.4 are considered normal for sterile GSS male Mediterranean fruit fly. Under normal circumstances, it is considered appropriate to discard negative values and values above 1.1, which would indicate greater competitiveness of sterile males than their fertile wild counterparts (FAO/IAEA/USDA 2003). However in this instance, where GRO is known to sometimes make treated sterile males more competitive than untreated wild males, negative C values and C values >3 were discarded for these analyses. This left C values from four replicates for 2 to 3-day-old GRO-treated flies, five replicates for 2 to 3-day-old untreated (non-GRO) flies, four replicates for 5-day-old GRO-treated flies and five replicates for 5-day-old untreated (non-GRO) flies. Mean C values obtained can be seen in fig. 2. The competitiveness as determined by Fried’s test was significantly different [F3,14 = 22.6, P = 0.05] among treatments. Again the male mating competitiveness based on egg hatch of 5-day-old GRO-exposed males was significantly higher than that of the other three categories of sterile males (P < 0.05), and also higher than that of wild males.

image

Figure 2.  Male mating competitiveness based on egg hatch (±SE) of sterile 2 to 3-day-old and 5-day-old sterile males either exposed or non-exposed to ginger root oil aroma against fertile wild male flies. A competitiveness value of 1 represents equal competitiveness with a wild male fly in mating successfully with a fertile wild female. A lower case letter above a bar signifies a treatment that is significantly different to all other treatments (Tukey’s honestly significant difference test P = 0.05).

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Mating duration

In this study, male fly age, GRO exposure, or radiation induced sterility in laboratory reared flies had no significant effect on mating duration when compared with fertile wild male and female matings (Tukey’s HSD test P = 0.05). The minimum mean mating duration for any treatment was 66 min and the maximum, 259 min.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Egg hatch measurements in outdoor field cage studies go further in assessing sterile male performance than RSI values as they include actual sperm transfer and insemination rates in wild females. Fried’s index uses egg hatch measurements to calculate the relative competitiveness of the sterile males in relation to wild males. A higher competitiveness (Fried’s C value) in sterile males means that suppression or eradication in the field will be achieved quicker and at lower cost (Robinson and Hendrichs 2005).

An RSI or Fried C value below 0.2 is considered reason for concern in male only GSS (FAO/IAEA/USDA 2003). As Shelly et al. (2006a) commented, ‘success of the SIT depends, to a large degree, on the ability of released, sterile males to attract and obtain matings with wild females. This factor is especially important for species, such as C. capitata, characterized by ‘complex’ mating behaviour (Lance and McInnis 2005), in which males produce multiple sexual signals using various modalities (visual, acoustic and olfactory), and females display a high degree of mate selection based apparently on male courtship performance (Whittier et al. 1992, 1994)’.

Taylor et al. (2001) showed that sterile male Mediterranean fruit fly typically become sexually active as early as 48 h after emergence. The longer a colony has been mass reared the greater the selection pressure for earlier mating (Miyatake and Yamagishi 1993). However, with a lek system of mating and female choice, sexual activity alone may not ensure successful mating. Liedo et al. (2002) demonstrated that wild females would not mate with 3-day-old laboratory males of a bisexual strain in field cages, and Economopoulos et al. (1988) showed that only 4–6 days after peak emergence sterile males do participate successfully in leks and achieve matings. Exposing young flies to GRO enables them to attract mates, whereas if untreated, this would not occur.

In our trials with GSS, 2 to 3-day-old males, either treated or not treated with GRO, did achieve some matings with wild females, but their mating performance and competitiveness was at or below acceptable levels. Although GRO exposure of 2 to 3-day-old males caused a slight increase in mating when compared with unexposed males of an equivalent age, their level of mating would not be acceptable for SIT field programmes.

Liedo et al. (2002) found that in field cages, 3-day-old laboratory males would mate with laboratory females, but wild females would not accept laboratory males as mates until they were 4 days old. Using irradiated GSS (Vienna 4/Tol-94) males 3–11 days old in field cages with wild females and without wild males, Taylor et al. (2001) showed that the probability of sperm storage decreased with male age. It appears that in a competitive situation, females prefer older wild males but if no wild males are available they will mate with mature, younger colony males.

Non-GRO-exposed 5-day-old flies still had an acceptable RSI (0.27) and a good C value (0.38), and as the GSS assessed still had very low genetic recombination and met or exceeded all the other key quality parameters, collectively these results indicate a lack of competiveness of young flies rather than any loss of quality induced by long-term mass rearing. GRO-exposed 5-day-old flies performed extremely well on both indices (RSI = 0.67 and C = 1.81), having more matings with wild females than wild males. The significant difference in egg sterility from matings by 5-day-old GRO compared with 5-day-old non-GRO-treated sterile male flies is indicative of the advantage to be gained through the exposure of sterile male flies to GRO aroma. In Western Australia, use of GRO and release of older sterile males has enabled continued effective use of a GSS which has been in mass production for 10 years.

At least in field cages, there is a significant advantage in releasing older GRO-exposed sterile males. It is difficult to prove whether this advantage is carried over to the field (Shelly et al. 2007). If it is, then it should be possible to reduce release rates and the overflooding ratio of sterile to wild field males from current levels and maintain the same degree of control.

Paranhos et al. (2010) showed that GRO can be used to treat sterile male Mediterranean fruit flies without interfering with their dispersal or survival in the field. Their study demonstrated that sterile male Mediterranean fruit fly, either exposed or not to GRO, exhibit similar dispersal behaviour and post-release survival. As the GRO exposure technique is improved and mating competiveness is increased by using older GRO-treated flies, it should be possible to reduce the fly release rate without reducing the effectiveness (Shelly et al. 2006b). Alternatively, one could maintain the same fly release rate and expect to reduce the wild population faster because of increased field effectiveness of the sterile males. For SIT against Mediterranean fruit fly to survive in Western Australia in a ‘user pays’ environment, it needs to be shared by a larger number of users to reduce production costs. The key to successful uptake of GRO is to aromatize entire adult fly holding rooms at a central facility. Large-scale aromatherapy followed by the shipment of chilled adult flies for release in the field would meet these requirements.

These experiments show that a GRO exposure regime and release at the age of 5 days produces sterile males that are, at least in field cages, more competitive than their wild counterparts. Using a bisexual strain, sterile males cannot be held for more than 2–3 days because matings are initiated between sterile males and sterile females in the holding cages before field release. However, in view of the absence of sterile females, using a GSS allows sterile males to be held longer and released at an age of increased sexual maturity. The challenge remains to develop a system for shipment and release of chilled GRO-exposed flies for release in rural areas. Aerial release is not a cost-effective option under Western Australian conditions and the development of effective chilled release machines for small-scale ground operation (e.g. 1 million flies per release) remains a high priority and one which should be addressed by agencies with expertise and resources in this area such as USDA/APHIS/Methods Development and the FAO/IAEA.

Additional prospects to further increase sterile male performance are to reduce the unnecessarily high irradiation dose (145 Gy), particularly for a GSS strain that carries male-linked translocations which already provide some degree of sterility (Steffens 1982) and also to include protein in the sterile male diet, which has been shown to significantly improve sterile male performance (Gavriel et al. 2009).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Department of Agriculture and Food staff for assisting with these experiments, Ian Lacey for helping in the field cage experiments and for collecting the wild colony, Phil Lawrence and Steve Gibellini for the production of quality sterile flies, Wayne Morris for providing innovative ideas and for modifying the field cages to make them easier to use, Terry Black for very ably organizing material requirements for the experiments and Rajendra Soopaya for his expertise with analysis of the data. We are also very grateful to Nikos Papadopoulos, Rui Pareira and Jorge Hendrichs for their many helpful comments on the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Cáceres C, Cayol JP, Enkerlin W, Franz G, Hendrichs J, Robinson AS, 2004. Comparison of Mediterranean fruit fly (Ceratitis capitata) (Tephritidae) bisexual and genetic sexing strains: development, evaluation and economics. In: Proceedings, symposium: 6th International symposium on fruit flies of economic importance, 6–10 May 2002, Stellenbosch, South Africa. Ed. by Barnes BN, Isteg Scientific Publications, Irene, South Africa, 367381.
  • Economopoulos AP, Hendrichs J, Wornoayporn V, 1988. Sexual activity of males from a mass-reared white-female-pupa genetic sexing strain when mixed with wild males and females in a caged-tree environment. Report 2, 1988. Activities of the Entomology Unit, FAO/IAEA Agriculture Laboratory, 4750.
  • FAO/IAEA/USDA, 2003. Product quality control and shipping procedures for sterile mass reared Tephritid fruit flies. Manual, Version 5. IAEA, Vienna, Austria. http://www-naweb.iaea.org/nafa/ipc/public/ipc-mass-reared-tephritid.html
  • Fisher K, Cáceres C, 2000. A filter rearing system for mass reared genetic sexing strains of Mediterranean fruit fly (Diptera: Tephritidae). In: Proceedings: area wide control of fruit flies and other insect pests, and the 5 International symposium on fruit flies of economic importance. Ed. by Tan KH, Penerbi Univesiti Sains Malaysia, Pulau Pinang, Malaysia, 543550.
  • Fisher HT, Hill AR, Sproul AN, 1985. Eradication of Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in Carnarvon, Western Australia. Aust. J. Entomol. Aust. Entomol. Soc.24, 207208.
  • Flath RA, Cunningham RT, Mon TR, John JO, 1994a. Additional male Mediterranean fruit fly (Ceratitis capitata Wied.) attractants from angelica seed oil (Angelica archangelica L.). J. Chem. Ecol.20, 19691984.
  • Flath RA, Cunningham RT, Mon TR, John JO, 1994b. Additional male Mediterranean fruit fly (Ceratitis capitata Wied.): structural analogues of α -copaene. J. Chem. Ecol.20, 25952609.
  • Franz G, Kerremans P, Rendón P, Hendrichs J, 1996. Development and application of genetic sexing systems for the Mediterranean fruit fly based on a temperature sensitive lethal. In: Fruit fly pests: a world assessment of their biology and management. Ed. by McPheron BA, Steck GJ, St Lucie Press, Delray Beach, FL, USA, 185191.
  • Gavriel S, Gazit Y, Yuval B, 2009. Remating by female Mediterranean fruit flies (Ceratitis capitata, Diptera: Tephritidae): temporal patterns and modulation by male condition. J. Insect Physiol.55, 637642.
  • Jessup AJ, Dominiak B, Woods B, DeLima CPF, Tomkins A, Smallridge CJ, 2007. Area-wide management of fruit flies in Australia. In: Area-wide control of insect pests from research to field implementation. Ed. by Vreysen MJB, Robinson AS, Hendrichs J, Springer, Netherlands, 685697.
  • Lance DR, McInnis DO, 2005. Biological basis of the sterile insect technique. In: Sterile insect technique: principles and practice in area-wide integrated pest management. Ed. by Dyck VA, Hendrichs J, Robinson AS, Springer, Dordrecht, The Netherlands, 6994.
  • Lance DR, McInnis DO, Rendón P, Jackson CG, 2000. Courtship among sterile and wild Ceratitis capitata (Diptera: Tephritidae) in field cages in Hawaii and Guatemala. Ann. Entomol. Soc. Am.93, 11791185.
  • Leppla NC, Ozaki E, 1991. Introduction of a wild strain and mass rearing of medfly. In: Proceedings, the international symposium on the biology and management of fruit flies. Ed. by Kawasaki K, Iwahashi O, Kaneshiro KY, University of Ryukyus, Okinawa, Japan, 148154.
  • Liedo P, DeLeon E, Barrios MI, Valle-Mora JF, Ibarra G, 2002. Effect of age on the mating propensity of the Mediterranean fruit fly (Diptera: Tephritidae). Fla. Entomol.85, 94101.
  • McInnis DO, Lance DR, Jackson CG, 1996. Behavioral resistance to the sterile insect technique by the Mediterranean fruit fly (Diptera: Tephritidae) in Hawaii. Ann. Entomol. Soc. Am.89, 739744.
  • McInnis DO, Shelly T, Komatsu J, 2002. Improving male mating competitiveness and survival in the field for medfly, Ceratitis capitata (Diptera: Tephritidae) SIT Programs. Genetica116, 117124.
  • Miyatake T, Yamagishi M1993. Active quality control in mass reared melon flies: quantitative genetic aspects. International Symposium on Management of Insect Pests: Nuclear and Related Molecular and Genetic Techniques, Vienna (Austria), 19–23 October 1992. Proceedings Series IAEA – SM-327/22, 201211.
  • Nishida R, Shelly TE, Whittier TS, Kaneshiro KY, 2000. A potential rendezvous cue for the Mediterranean fruit fly, Ceratitis capitata?J. Chem. Ecol.26, 87100.
  • Paranhos BJ, Papadopoulos NT, McInnis DO, Gava C, Lopes FSC, Morelli R, Malavasi A, 2010. Field dispersal and survival of sterile Medfly males aromatically treated with ginger root oil. Environ. Entomol.39, 570575.
  • Robinson AS, Hendrichs J, 2005. Sterile insect technique. In: Principles and practice in area-wide integrated pest management. Future development and application. Ed. by Dyck VA, Hendrichs J, Robinson AS, Springer, Netherlands, 742.
  • Robinson AS, Franz G, Fisher K, 1999. Genetic sexing strains in the medfly: development, mass rearing and field application. Trends in Entomol2, 81104.
  • Rössler Y, 1975. The ability to inseminate: a comparison between laboratory-reared and field populations of the Mediterranean fruit fly (Ceratitis capitata). Entomol. Exp. Appl.18, 255260.
  • Shelly TE, Villalobos EM, 2004. Host plant influence on the mating success of male Mediterranean fruit flies: variable effects within and between individual plants. Anim. Behav.68, 417426.
  • Shelly TE, Whittier TS, Kaneshiro KY, 1994. Sterile insect release and the natural mating system of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae). Ann. Entomol. Soc. Am.87, 470481.
  • Shelly TE, McInnis DO, Pahio E, Edu J, 2004a. Aromatherapy in the Mediterranean fruit fly (Diptera: Tephritidae): sterile males exposed to ginger root oil in prerelease storage boxes display increased mating competitiveness in field -cage trials. J. Econ. Entomol.97, 846853.
  • Shelly TE, Dang C, Kennelly S, 2004b. Exposure to orange (Citrus sinensis L.) trees, fruits and oil enhances mating success of male Mediterranean fruit flies (Ceratitis capitata [Wiedemann]). J. Insect Behav.17, 303315.
  • Shelly TE, Edu J, Pahio E, 2006a. Application of orange oil to pre-release holding boxes increases the mating success of sterile males of the Mediterranean fruit fly in field cage trials (Diptera: Tephritidae). Proc. Hawaiian Entomol. Soc.38, 7379.
  • Shelly TE, Holler TC, Stewart JL, 2006b. Mating competitiveness of mass-reared males of the Mediterranean fruit fly (Diptera: Tephritidae) from eclosion towers. Fla. Entomol.89, 380387.
  • Shelly TE, McInnis DO, Rodd C, Edu J, Pahio E, 2007. The Sterile insect technique and the Mediterranean fruit fly (Diptera: Tephritidae): assessing the utility of aromatherapy in a Hawaiian coffee field. J. Econ. Entomol.100, 273282.
  • Steffens RJ, 1982. The Combi-Fly, a new concept for genetic control of fruit flies. Naturwissenschaften69, 600601.
  • Taylor PW, Kaspi R, Mossinson S, Yuval B, 2001. Age-dependent insemination success of sterile Mediterranean fruit flies. Entomol. Exp. Appl98, 2733.
  • Whittier TS, Kaneshiro KY, Prescott LD, 1992. Mating behaviour of Mediterranean fruit flies (Diptera: Tephritidae) in a natural environment. Ann. Entomol. Soc. Am.85, 214218.
  • Whittier TS, Nam FY, Shelly TE, Kaneshiro KY, 1994. Male courtship success and female discrimination in the Mediterranean fruit fly (Diptera: Tephritidae). J Insect. Behav.7, 159170.
  • Woods W, 2001. Bugs by the million for medfly eradication. J. Agric. Western Australia42, 1923.