Improving Sterile Male Performance in Fruit Fly SIT programmes

Objective:

Reduce the cost and increase the effectiveness of SIT programmes by improving the performance of sterile males through manipulations implemented at the emergence and release facilities. Thus, the focus of the proposed CRP is the developmental period from the late pupal stage of mass-produced fruit flies through to field release. The proposed CRP focus on the ten tephritid fruit fly species currently the target of operational SIT programmes.

Activities:

a) The Abiotic Environment
From the time that the sterile fly pupae reach the distribution centres, they are subject to the abiotic environment into which they are placed. This environment includes the type and size of the fly holding container, and the specific ambient conditions of the holding room. The type of holding container will depend on the kind of fly release that will follow-either from the ground, or by air. Containers for ground release are typically small, either bags or boxes, and hold up to several thousand individuals. Fly density and the amount of surface area available for flies to rest on are important factors to consider in order to avoid overcrowding and stressing the flies. An advantage of ground-released flies is that once the fly pupae are set up they require no further handling, except perhaps to add additional food or water. Aerial release of flies is usually more complicated, as the flies must be transferred from initial holding containers to aerial drop boxes by temporarily anaesthetizing the flies, usually with cold. An exception to this procedure is the use of small bags or boxes, which can be loaded directly into plane and torn open at the time of release. The latter may be needed, or alternative methods of anaesthetizing the flies, in cases where a cold fly knockdown procedure is a problem, as recently described for 2 species of Anastrepha (ludens and suspensa) (P. Teal, unpub. data), and for Bactrocera papayae (Tan & Nishida, 1998) and B. carambolae (Wee & Tan, 2000). In these cases, male pheromone quantity or quality is affected by even brief cold treatments to young adults.

The ambient environmental conditions in the fly holding room are also potentially critical. Such parameters as light intensity, the light:dark cycle (if any), temperature, humidity, and barometric pressure may all play important roles in conditioning the flies for their vital role in the field: surviving and attracting, courting, and copulating with wild females. Much remains unknown about the effects of manipulating these environmental parameters, either separately or in combination, on subsequent male quality in the field.
b) The Biotic Environment
b.1. Hormones
Age is a significant factor affecting sexual signalling and reproduction in numerous tephritid species. For example, members of the Anastrepha genus typically require between two and three weeks to become sexually mature. Although mass rearing results in selection of strains which become sexually mature much earlier than wild flies, the most rapidly developing strains of A. suspensa and A. ludens still require more than 7 days to become sexually mature. This delay between adult emergence and sexual maturity poses a significant problem for SIT programmes because males must be held for a long period of time prior to release, or have to be released before becoming sexually mature, resulting in fewer surviving to maturity and copulation.

Clearly, development of cost effective methods to accelerate sexual maturity in released flies would have a significant positive impact on the efficacy of the SIT. Recent research on a number of tropical Anastrepha species (A. ludens, A. suspensa and A. obliqua) has shown that juvenile hormone is a critical hormone regulating sexual maturity and sexual signalling in these species (e.g. Teal et al., 2000). Topical application of juvenile hormone or the mimics methoprene or fenoxycarb, accelerates reproductive development and sexual signalling in sterile males by at least 5 days. Incorporation of methoprene into adult diets results in similar acceleration of development. Furthermore, sterile males fed methoprene release significantly more pheromone and are considerably more attractive to fertile females between the ages of 3-11 days than are sterile males denied access to hormone in the diet. Additionally, significantly more wild females are attracted to sterile treated males (6-10 days old) fed hormone than to wild males (16-20 days old) fed the same diet, except lacking hormone on each day of a four-day test. Effects of methoprene are optimal when as little as 0.05% (AI) is incorporated in the adult diet. This, coupled with the relatively low cost associated with the purchase of methoprene in a water-soluble formulation, indicates that incorporation of hormone supplements into adult emergence protocols may be a cost effective way to improve the efficacy of the SIT.
b.2. Nutrients
Both male and female tephritids are anautogenous, emerge as adults with undeveloped gonads, and relying on foraging during adult life to provide the protein needed for gonadal and accessory gland development (Yuval & Drew, 2000). In addition to protein, carbohydrates must be frequently ingested to fuel metabolic activities. Recent studies on species from several tephritid genera (Anastrepha, Bactrocera, Rhagoletis and Ceratitis) indicate that providing protein nutrition to males in the days following eclosion can enhance male reproductive success. These studies have been extended to sterile male Mediterranean fruit flies, Ceratitis capitata, establishing the potential for including protein in the diet offered sterile males in the release facility (Kaspi & Yuval, 2000), although the optimal dosage and form of presentation still needs to be established (Papadopoulos et al., 1998; Shelly & Kennely, 2002). Furthermore, recent studies indicate that several species of bacteria are common residents in the tephritid gut, and may make a significant contribution to fly fitness (Drew & Yuval, 2000; Lauzon et al., 2000). Currently, sterile males of most species are usually offered a pre-release diet of highly concentrated sucrose, presented in an agar block. The formulation and testing of optimal pre-release diets, containing sugar, protein and bacteria (and possibly other ingredients) in proportions that will result in enhanced sterile male performance in the field, will be developed in the proposed CRP.
b.3. Semiochemicals
Most pest species of Bactrocera are attracted to two major natural attractants, raspberry ketone (RK) and methyl eugenol (ME) (Tan, 2000 a & b; Tan et al., 2002). Species attracted to RK, such as B. cucurbitae and B. tryoni, sequester the chemical into the pheromonal system. However, the process of sequestration of RK has, thus far, not been observed to contribute significantly to the improvement of male mating or competitiveness in courting females.

Ingestion of ME by Bactrocera dorsalis has been studied intensively. ME is converted to two major booster sex pheromonal components - trans coniferyl alcohol and 2-allyl-4,5-dimethoxphenol in the crop (Tan, 2000). The metabolites are stored in the rectal gland and subsequently released during fanning performed during courtship. These components improved the mating competitiveness of males by at least three fold when compared with ME-deprived males. It is envisaged that providing sterile males with a source of ME to feed on before release will place them on at least an even playing field against wild males, thereby potentially reducing the number or frequency of sterile males released. To date, the role of ME as a precursor to boost sex pheromone in B. correcta and B. philippinensis is not fully understood nor whether improving male production mating competitiveness can be realized. Other advantages of ME feeding are related to survival. One of the components, 2-allyl-4,5-dimethoxyphe-nol, acts as a very potent allomone to deter vertebrate predators (Tan & Nishida, 1996; Wee & Tan, 2001). Furthermore, feeding on ME significantly reduces male response to ME in male annihilation traps, thus potentially allowing simultaneous application of the SIT and male annihilation methods.

Recent findings have shown that ginger root oil and the oil contained in the flavedo region (peel) of citrus fruit enhance considerably the mating competitiveness of wild or mass-reared C. capitata males (Katsoyannos et al., 1997; Papadopoulos et al., 2001; Shelly, 2002). Once exposed (by contact or vapour), males acquire a strong mating advantage over unexposed males and retain it for a number of days. Interestingly, commercial essential oils from citrus have a similar effect on males.

These oils are especially and at the same time very effective, so it would be worthwhile trying their potential in ongoing SIT programmes. If sterile males become more competitive following exposure to the oils a smaller number of them would be required to be released. One active ingredient in ginger root oil has been identified as ?-copaene, a potent male attractant. However, these oils contain a number of other volatile substances that may have similar enhancing effects on males. The composition of citrus peel oil and the effects of the various components must be examined. There are no known effective mating enhancing semiochemicals for Anastrepha. However, the dramatic mating enhancing effects seen with Bactrocera species and Ceratitis suggest that similar compounds may exist in the Anastrepha ecological sphere and await discovery.
c. Release Methods
Tephritid fruit flies can be distributed in the field by either aerial or ground release methods. Ground release methods are simpler but much less effective when large or topographically difficult areas need to be covered with sterile flies. Ground distribution can be accomplished by either point or roving releases of flies from containers designed to be handled easily by personnel. In the case of ground releases, flies are released from the same containers into which the pupae are packaged when they are received at the distribution facilities. Commonly, this means the use of buckets each containing several hundred up to a thousand individuals held in environmentally controlled rooms. Larger containers (e.g. PARC boxes) containing several tens of thousands of flies may also be used to make larger point releases in fly 'hot-spot' areas.

Aerial fly releases are necessarily used in large scale SIT programmes around the world. This method invariably means that the pupae arriving at distribution facilities will be held in temporary containers, such as PARC boxes or the more recently developed emergence towers, until just prior to aerial release. In the case of aerial releases, the flies must be extracted from the temporary containers through the use of cold fly knockdown methods, and then transferred to aerial drop boxes, which are transported to waiting aircraft. These methods, while operationally convenient, may not be always optimal in terms of the success of the SIT. For example, as noted above, in some species, (notably Anastrepha ludens, A. suspensa and several Bactrocera species), cold knockdown treatment adversely affects sexual competitiveness. Therefore, one of the goals of the proposed CRP is to integrate the release methods with the other manipulations discussed above, tailoring the release method according to the biology of each fly species.
Specific R&D Activities
R&D activities were focused on the ten tephritid species currently targeted by active SIT programmes in the different regions of the world. Consequently, basic research is conducted on those species for which the results of such pre-release manipulations are unknown or limited. Concurrently, for those species where promising applications have surfaced, these were applied, in pilot tests and eventually incorporated in standard procedures in ongoing SIT operations. In all these activities, the standard used was the international manual on fruit fly quality control (FAO/IAEA/USDA 2003) regarding male performance for the following traits: Survival, Dispersal and Copulation/Insemination.
1.a Fly holding conditions:
Several ambient environmental para-meters, including temperature, relative humidity, barometric pressure and illumination are varied in controlled experiments and the final field quality of the resulting sterile males assessed. The size (volume) of the containers and resulting fly density should be varied and tested as well. Experiments to pre-adapt late-stage pupae or adults to specific field conditions, e.g. high or low temperature, light intensity, barometric pressure, were carried out and compared to results from insects held under standard conditions. Of course, the addition of a supplemental treatment such as a hormone or semiochemical (see below) may affect the choice of the most efficient method by interacting with the fly holding conditions (e.g. the size of the container or the manner in which the supplement is applied to the flies).
1.b Fly release conditions:
If flies need to be transferred from holding containers to aerial drop boxes, standard and new ways to anaesthetize the flies are investigated (e.g. use of carbon dioxide or nitrogen gas instead of cold knockdown) for species known to be sensitive to cold treatments. For these species, alternatives to the use of anesthesia in the first place is investigated, i.e. transferring the flies directly to awaiting release aircraft, as is the case for ground releases. Since aerial releases are the principal means of sterile fly release in all large-scale action programmes, efforts are directed to find efficient means of aerially dropping flies for ongoing programmes in which only ground releases are currently operational.

After consuming methyl eugenol, Bactrocera fruit flies require 2-6 hours to convert methyl eugenol to its metabolite(s) and to transport them via the haemolymph to the rectal gland for storage and subsequent release during courtship. During this period, shifting the flies and transporting them for aerial release may affect the eventual sequestration of the metabolite - thus negating the mating competitive advantage of exposed released flies. As such, research should be conducted to: i) determine whether the process of booster sex pheromonal components reaching the rectal gland can be affected by chilling; and ii) determine the ideal length of time after post-treatment to chill (if any) and then release sterile flies (this may reduce unnecessary fly retention time in a mass rearing facility).
2. Hormonal R&D
Experiments on Anastrepha suspensa and A. ludens have shown the utility of incorporation of methoprene into the adult diet. Sterile males, between 3-11 days old, fed a methoprene containing diet, released significantly more pheromone and were significantly more attractive to fertile females than were sterile males denied hormone in the diet. The juvenile hormone and its mimics have some beneficial effects, but they have been known to induce morphological intermediates-larval-pupal and pupal-adult forms, when larvae and pupae are exposed at critical periods (just before and after pupation). As such, incorporating juvenile hormone or its analogue into larval diet is not feasible. To overcome this problem, one or two day old pupae can be treated with different residual doses of a juvenile hormone or its analogue. If the cost of juvenile hormone is high, the efficacy of some of its commercial analogues should first be analysed. Besides this, the cost effectiveness of residual treatment with the leaves of Cyperus spp.-weeds which contain substantial amounts of juvenile hormone III (Toong et al., 1988), an ideal substitute for juvenile hormone, was investigated. Should residual treatment of pupae with juvenile hormone appear not to be a viable option, incorporating juvenile hormone or its analogue into adult diet may be a viable option to induce early maturity in male flies.

This, coupled with the relative low cost associated with the purchase of methoprene, in a water soluble formulation, indicate that incorporation of hormone supplements into adult holding protocols may be a cost effective way to improve efficacy of the SIT. At this juncture, information available for A. suspensa and A. ludens is sufficient to allow for the :

  • Development of efficient large-scale methods for incorporation of methoprene into adult diets.
  • Conducting of pilot scale tests that allow assessment of efficacy of the methods.
  • Conducting of cost benefit analyses to determine feasibility of the method.

For other target species (A. obliqua, A. fraterculus, C. capitata and Bactrocera spp.) a significant amount of basic research was conducted prior to the implementation of hormone supplements into SIT release programmes. These studies include:

  • Behavioural and chemical assessments of the effects of methoprene to determine the age at which males become sexually mature and if the reproductive parameters are sufficiently improved to make the sterile males more competitive with wild flies.
  • Determine the minimum dose and length of time required to optimise reproductive performance and minimize time for maturation.
  • Assess the efficacy of the technology by comparing mating success of sterile males with wild females with the former either provided with hormone supplement or not.
  • Determine if hormone supplements should be incorporated into adult diets or provided in other ways (spray applications to pupae, provided in water provided to adults, etc.).
  • Develop efficient large-scale methods for incorporation of methoprene into adult diets.
  • Conduct pilot scale tests that allow assessment of the method.
  • Conduct cost benefit analyses to determine feasibility of the method.

3. Nutritional R&D
To date several experiments and observations have pointed to the beneficial effect of providing sterile males with protein before their release. The objective of the research proposed for this CRP is to establish the best source of this protein nutrition and the form in which it is presented to the sterile males. In light of the complex interactions between diet, sexual performance and survival, these studies strive to find the diet that provides the best cost effective balance between these three factors. In addition, the possibility of using inexpensive, locally available sources of nutrients (such as leftovers from fruit processing industries), to replace expensive yeast hydrolysates will be examined. As work with the medfly progresses towards implementation in field programmes, basic research on post-teneral diets and sexual performance of males in other species will be conducted. Tephritid fruit flies maintain intimate associations with a complex microflora resident in their digestive system. While this interaction is largely unknown, it has become evident that several dominant species (such as Klebsiella oxytoca and Enterobacter agglomerans) in this array of microorganisms may contribute significantly to fly health and reproductive performance. Due to the conditions in mass rearing facilities, these important bacteria are not present (at least not in large quantities) in the gut of sterile males at the time of release. One research component of this CRP investigated the utility of inoculating sterile males with bacteria, by quantifying the manner in which such inoculation affects subsequent survival and copulatory success.
4. Semiochemical R&D
4.a Volatile oils
Orange peel oil and ginger root oil exposure to males was found to enhance considerably C. capitata male mating ability and competitiveness compared to unexposed males. These findings may have an important impact on the success of medfly SIT programmes and merits further research. Future research should address:

  • The behavioural, physiological and ecological basis of the observed phenomenon.
  • The active compounds involved.
  • The parameters involved when applying such substances.
  • Ways of incorporating them into the pre-release treatments in SIT programmes.
  • Possible other effects on fly behaviour including longevity, dispersal and general performance.
  • The cost-benefit of incorporating these supplements into SIT operations.
  • Other plant derived chemicals with similar effects.

4.b Methyl eugenol
Methyl eugenol (ME) is not soluble in water. Since methyl eugenol is beneficial by increasing male sexual competitiveness and at the same time toxic at high concentrations, it is important to find an effective dose of methyl eugenol that simultaneously does not kill male flies and yet is sufficiently high to significantly reduce the tendency of flies to respond to ME sources (e.g. ME monitoring traps). To simulate a plant source of methyl eugenol, a suitable emulsifier (which does not induce mortality) needs to be identified. Emulsions of methyl eugenol of various concentrations can then be tested on male flies to determine the optimum concentration of methyl eugenol to achieve the desired mating competitiveness against wild males.

At sexual maturity, male flies are strongly attracted to and compulsively feed on methyl eugenol. The peak period of attraction of wild flies to methyl eugenol traps is between 0800 and 1100 hour for B. papayae (=B. dorsalis) and the feeding period lasts for 45 seconds to 7 minutes. These optimal ranges for feeding periods need to be determined for each target species that may differ from B. papayae. A suitable and cost effective absorbent for dispensing methyl eugenol emulsion needs to be identified through research. With the right absorbent, a proper method of dispensing the methyl eugenol into fly cages was developed in order to avoid contaminating male fly bodies (which may lead to death). The dispensing technology employed was for a short period of less than an hour.

Participants:

Fifteen Contract Holders from Argentina (2), Brazil, China, Croatia, Greece, Israel, Mauritius, Mexico (2), Philippines, Portugal, South Africa, Thailand, United Kingdom and six Agreement Holders from Australia (2), France, Spain, USA (2).

Reports: