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The Sterile Insect Technique (SIT) is widely used to suppress or eradicate infestations of the Mediterranean fruit fly, Ceratitis capitata (Wied.). In large-scale programs, sterile males are chilled (4°C) to allow their transfer and storage in the aircraft used for the releases. Sterile males may remain chilled for as long as 3 h prior to release. Here, we describe the results of experiments that assessed the effect of chilling on flight ability and mating competitiveness of sterile male Mediterranean fruit flies held under conditions of low (plastic buckets) or high (emergence tower trays) density. Males from both densities were subject to 0 (no chill), 1, or 3 h of chilling at 3 days of age. Chill treatment had no effect on flight ability for males held at low density. However, for males held at high density, chilling for 1 or 3 h significantly reduced flight ability below that observed for the no chill treatment. Consistent with the flight data, chill treatment had no effect on the mating success of males held at low density. However, among males held at high density, 3 h of chilling significantly reduced mating success below levels observed for unchilled males or males chilled for 1 h only in trials conducted 1 day after the cold treatment. An auxiliary experiment revealed that this reduction in mating performance was temporary: in mating trials conducted 3 days after 3 h of chilling, sterile males derived from tower trays had similar mating success as unchilled males. Implications of these findings for Mediterranean fruit fly SIT are discussed.
The Sterile Insect Technique (SIT) is widely used to suppress or eradicate infestations of the Mediterranean fruit fly, Ceratitis capitata (Wied.), a pest that attacks many commercially important fruits and vegetables worldwide (Enkerlin 2005). The SIT involves the production, sterilization, and release of large numbers of male Mediterranean fruit flies into the environment. Matings between sterile males and wild females result in the oviposition of infertile eggs, causing a decline of the wild population. Given this mode of population suppression, the success of the SIT depends to a large degree on the ability of sterile males to compete successfully against wild males in obtaining matings with wild females (Calkins 1984; Calkins and Ashley 1989).
Unfortunately, there is ample evidence showing that, with respect to procuring copulations, sterile males are competitively inferior to wild males (e.g., Rössler 1975; Lance et al. 2000). Reasons for this trend are not known with certainty, but several studies (Liimatainen et al. 1997; Briceño and Eberhard 1998) identified altered courtship behavior, presumably arising through artificial selection under crowded mass-rearing conditions, as a key factor. Other features of the production and release process may also constrain the performance of sterile male Mediterranean fruit flies. For example, the composition of the adult diet, specifically the presence or absence of protein, may influence the mating success of sterile C. capitata males (Yuval et al. 2007). Other studies have examined the potential influence of irradiation dose (Shelly et al. 2005) and atmosphere (Hooper 1971), dietary microbes (Ben-Yosef et al. 2008), and age at release (Liedo et al. 2002), on the sexual competitiveness of sterile C. capitata males.
In SIT programs against the Mediterranean fruit fly, sterile males are typically chilled to allow their handling and transfer into the aerial release machine and their storage within the aircraft used for the releases. The following protocol, which describes operations at the David R. Rumsey Fruit Fly Emergence and Release Facility, Los Alamitos, California, is typical of this procedure (USDA-APHIS 2009). Emergence towers (each containing ≈1.25 million sterile males) are wheeled into a refrigerated room (4°C), and after approximately 1 h the males are transferred to a metal release (or fly) box. This box is then transported to and placed in a small plane waiting in an adjacent airfield, and the craft departs immediately thereafter. The fly box is maintained at ≈4°C during flight. On an average, flies are released approximately 2 h after takeoff (E. Baltazar, personal communication), consequently the sterile males are chilled for a total of approximately 3 h before release.
A recent report (FAO/IAEA 2007) cited the need for additional studies on the potential effects of pre-release chilling on the performance of sterile C. capitata males. Here, we describe the results of experiments that assessed the effect of chilling on the mating competitiveness and flight ability of sterile male Mediterranean fruit flies. In addition, the possible effects of chilling were investigated for sterile males held at different densities. The implications of our findings for Mediterranean fruit fly SIT programs are discussed.
Materials and Methods
Mass-reared, sterile C. capitata males were obtained from the California Department of Food and Agriculture (CDFA) Hawaii Fruit Fly Rearing Facility, Waimanalo, Hawaii, which produces a temperature sensitive lethal (tsl) genetic sexing strain (VIENNA-7/Tol-99). This strain allows for male-only releases, as heating the eggs results in the selective death of female embryos (Franz et al. 1996). Flies were reared on standard larval diet (Tanaka et al. 1969), and pupae were then dyed (fluorescent pink), placed in a clear plastic bag (≈200 000 pupae), and irradiated 48 h before emergence (in hypoxia at 150 Gy of gamma irradiation from a 137Cs source). The pupae were removed from the plastic bag within 2–4 h of sterilization and placed either in screen-covered, plastic buckets or trays from emergence towers (see below). The subsequently emerged adults were held at 25–27°C, 50–90% relative humidity (RH), and a 12 : 12 h (L : D) photoperiod under artificial lighting. On any given day, we received only one bag of pupae for testing, and each bag was considered a distinct ‘batch’ of flies.
Owing to the low availability of wild flies, we used ‘wild-like’ flies from a laboratory colony in the mating trials. This colony was derived from 200 to 300 adults reared from coffee berries (Coffea arabica L.) collected near Haleiwa, Oahu. Adults were held in screen cages and provided with a sugar–protein (yeast hydrolysate) mixture (3 : 1 by weight), water, and an oviposition substrate (perforated plastic vials containing small sponges soaked in lemon juice). Eggs were placed on the larval diet (Tanaka et al. 1969) in plastic containers over vermiculite for pupation. Adults used in the mating trials were separated by sex within 24 h of emergence, well before reaching sexual maturity at 5–7 days of age (T.E.S., unpublished data) and kept in screen-covered, plastic buckets (5-l volume, 150–200 flies per bucket) with ample food (sugar–protein mixture) and water. Wild-like flies were maintained under the same conditions as the sterile males (described above). When used in this study, the wild-like colony was nine generations removed from the wild.
Sterile males were held under two different conditions with respect to crowding and diet. Under low-density conditions, 150 sterile pupae were placed in the plastic buckets (5-l volume) and provided a small block (5 × 2.5 × 1 cm, l : w : h) of sugar–agar gel (placed directly on the screen covering the bucket) and water. In the plastic buckets, male density relative to resting area (surface area of the bucket interior) was approximately 9 males per 100 cm2, while male density relative to food surface area was approximately 12 males per 1 cm2.
In the high-density environment, sterile males were held in trays from an emergence tower. These towers consist of 60 interlocking, screen-paneled aluminum frames or trays (76 × 76 × 2.5 cm, l : w : h) stacked on a wheeled base. In this study, we did not use entire towers but used only a single tray to hold pupae. Sterile pupae were placed in a trough around the edge of the tray (350 ml per tray, where 1 ml ≈ 60 pupae), and a block of sugar–gar gel (15 × 9 × 3 cm, l : w : h) was placed on a second tray positioned above the pupal-holding tray (i.e., emerging flies accessed the food from below). The amounts of pupae and food used per tray in our study were similar to those used in ongoing Mediterranean fruit fly SIT programs in California and Florida (USDA-APHIS 2009). The food was placed on the top screen at the time of pupal placement and was overlain with aluminum foil to reduce desiccation. In the tower trays, male density relative to resting area (surface area of upper and lower trays) was estimated at 182 males per 100 cm2, while male density relative to food surface area was approximately 155 males per 1 cm2.
For both low and high densities, sterile males were chilled at 3 days of age for either 0 (unchilled), 1, or 3 h. Chilling occurred in a walk-in, refrigerated room (4°C). For the low-density treatment, buckets (except the no chill buckets) were simply introduced to and removed from the cold room at the appropriate time. For the high-density treatment, we set up two pupal-holding trays per batch, one for the no chill treatment and one for the 1 and 3 h chill treatments, respectively. For the no chill treatment, we collected test flies by quickly lifting the upper tray and gently scooping flies into plastic buckets. For the chilled flies, we lifted the upper tray and used an index card to lift flies from the lower tray. After collecting flies at 1 h of chilling, we replaced the top screen and returned to collect flies from the same screen for the 3 h chill treatment.
Flight ability tests were conducted at the USDA-APHIS Hawaii Fruit Fly Production Facility, Waimanalo, Hawaii, during March–May, 2009. Tests of flight ability were conducted immediately after chilling and closely followed the standard procedure for measuring flight ability in sterile, mass-reared tephritid fruit flies (FAO/IAEA/USDA 2003). Procedures were identical for the unchilled sterile males, except that the males were placed in the flight tubes by gently aspirating them through a small hole drilled 1 cm above the base of the tube (the hole was blocked following introduction of the flies).
For a given batch of flies, flight ability was measured on the same day for sterile males held in plastic buckets or tower trays. For each batch, five replicates were conducted per chill treatment per holding condition (density), i.e., 30 flight tubes (5 × 3 × 2) were established per test day. Thus, for each batch, we established 15 buckets (5 for each of the chill treatments) corresponding to 15 flight tubes, and 2 tower trays, 1 of which supplied unchilled males for 5 flight tubes and 1 of which supplied chilled males for 10 flight tubes (5 tubes each for 1 and 3 h chilled flies, respectively). Flight ability was measured for seven different daily batches of flies.
Mating trials were conducted 1 day after chilling. Sterile males held at low density were simply kept in their original container after chilling, and those held at high density were transferred to plastic buckets (≈100 flies per bucket). Fresh sugar–agar gel was placed on all buckets. Sterile males initially held under high density were thus held under low density for 24 h immediately preceding the mating trials. While this protocol may have masked effects of the original high-density environment, we considered it unlikely that released males would engage in sexual competition immediately but would require hours or days to locate mating aggregations (or leks, Prokopy and Hendrichs 1979). In addition, by holding sterile males 1-day post-chilling, we provided an additional day for sexual maturation and thereby reduced age-dependent effects that may have contributed to lowered mating performance of the sterile males independent of any chilling effect (Shelly et al. 2007).
Mating trials were conducted in nylon-screen field cages (3.0 m diameter, 2.5 m high) located at the USDA-ARS Laboratory, Honolulu, Hawaii, during March–May, 2009. On each test day, trials were performed in three adjacent field cages, one cage for each chill treatment (i.e., 0, 1, or 3 h). The cages contained two artificial trees (each 2 m tall with ≈700 leaves resembling those of Ficus benjamina L.). In each cage, we released 50 sterile males, 50 wild-like males (7–10 days old), and 50 wild-like females (8–12 days old). Flies were released between 8:30–9:00 AM (males were released 15 min before females), and mating pairs were collected over the next 4 h by gently coaxing them into a vial. Pairs were then placed in a freezer, and males were identified using a dissecting microscope under black light (to determine the presence of pink dye). As the number of field cages available was limited, mating tests on a given day were conducted using males from only low- or high-density treatments exclusively. Mating trials were conducted over 12 different days for sterile males held in plastic buckets and 12 different days for sterile males held in tower trays.
As shown below, sterile males held in tower trays and chilled for 3 h had significantly lower mating success than males from the other treatments. To determine whether this reduction in mating competitiveness was temporary, we performed an additional set of mating trials in which males from tower trays were chilled 3 h and then held 3 days (instead of 1 day as above) before testing. Non-chilled males (derived from the same pupal batch but held in separate tower trays) were tested simultaneously to serve as a control. Sterile males were thus 6 days old when tested (as above, sterile males were held in plastic buckets after day 3). Two field cages (with chilled or non-chilled sterile males, respectively) were established on each of eight test days.
For flight ability, a two-way anova with chill duration (0, 1, or 3 h) and batch (1–7) as the main factors was performed separately for the two holding conditions (plastic buckets or tower trays). Data met the assumptions of normality and equal variance in both cases. Subsequent pair-wise comparisons were made with the Tukey test. For a particular chilling regime, a t-test was used to compare flight ability of sterile males held in plastic buckets vs. tower trays. For mating competitiveness, a one-way anova with chill duration as the main factor was performed separately for the two holding conditions, with data entered as the proportion of the total matings obtained by sterile males in a given replicate (values were arc sine transformed for analysis). Analyses included only those trials (field cages) in which at least 20% of the females mated. Potential between-batch differences in mating success were not examined, because only three replicates (field cages) were conducted per batch (test day) over all chill treatments. As before, for each chilling regime, a t-test was used to compare mating success of sterile males held at low vs. high density. Means are given ±1 SE.
For sterile males held in the plastic buckets, chill treatment did not have a significant effect on the proportion of flight-able sterile males (F2,84 = 2.7, P = 0.07). The average percentage of fliers was similar among chill treatments, varying only between 79% and 81% (fig. 1). However, there was significant variation in flight ability among batches (F6,84 = 87.4, P < 0.001, fig. 2). Over all chill treatments, the lowest average score for a particular batch (n = 15 readings) was 69% fliers, and the highest score was 94%. The interaction term was also significant (F12,84 = 2.8, P = 0.003). This latter result most likely reflected the fact that the rank order of flight ability observed among the chill treatments varied among batches. For example, in batch 1, the average proportion of fliers was greatest for no chill and lowest for 3 h chill, while the reverse ranking was found for batch 2 (i.e., 3 h chill highest, no chill lowest).
For sterile males held in emergence tower trays, chill treatment had a significant effect on flight ability (F2,84 = 8.7, P < 0.001, fig. 1). Flight ability for the no chill treatment was significantly higher than the 1 and 3 h chill treatments, which did not differ significantly from one another (P = 0.05, Tukey test). The average percentage of fliers was 77% for the no chill treatment compared with 73% and 72% for the 1 and 3 h chill treatments, respectively. Batch also had a significant effect on flight ability (F6,84 = 32.8, P < 0.001, fig. 2). Over all chill treatments, the lowest average score for a particular batch (n = 15 readings) was 65% fliers, and the highest score was 83%. For males held in the tower trays, the interaction term between chill treatment and batch was not significant (F12,84 = 1.3, P = 0.25). Based on average values, the flight ability scores for plastic buckets and tower trays were positively related across batches (fig. 2), although this correlation was not statistically significant (r = 0.59, P = 0.18).
For unchilled males, flight ability did not differ significantly between sterile males held in plastic buckets or tower trays (t = 1.9, P = 0.06). However, sterile males held in plastic buckets displayed significantly higher flight ability than sterile males held in the tower trays following 1 (t = 2.9, P = 0.004) or 3 h (t = 3.2, P = 0.002) of chilling (d.f. = 68 for all tests, data pooled over batches).
There was no significant variation in relative mating success among chill treatments for sterile males held in plastic buckets (F2,33 = 0.04, P = 0.96, fig. 3). However, among sterile males held in tower trays, relative mating success varied significantly among the chill treatments (F2,33 = 3.6, P = 0.04, fig. 3). Specifically, the mating success of males chilled for 3 h was significantly lower than that observed for unchilled males or males chilled for only 1 h, which did not differ significantly from one another (Tukey test, P = 0.05).
There was no significant difference in the relative mating success of sterile males from low and high-density conditions when the males were unchilled (t = 0.2, P = 0.82) or chilled for only 1 h (t = 0.6, P = 0.56). However, when chilled for 3 h, sterile males held at low density achieved a significantly higher proportion of the total matings per replicate than sterile males held at high density (t = 2.8, P = 0.01, d.f. = 22 for all tests).
While the above analyses reveal a negative effect of the 3 h chill treatment on mating success of sterile males held in tower trays, the auxiliary experiment in which mating trials were performed 3 days post-chilling indicate that this negative effect is short-lasting. In this experiment, there was no significant difference in relative mating success between 6-day-old sterile males that were unchilled (mean = 28.4 ± 3.3%, n = 8) or chilled for 3 h (mean = 30.5 ± 2.9%, n = 8) (t = 0.36, d.f. = 14, P = 0.72). As this finding suggests, for sterile males held in tower trays and chilled for 3 h, their relative mating success was significantly greater in trials conducted 3-day post-chilling than in trials conducted 1-day post-chilling (30.5% vs. 17.1%, t = 2.6, d.f. = 18, P = 0.02). This result did not appear to derive from the 2-day difference in age between sterile males used in the different post-chill treatments (i.e., 6-day-old vs. 4-day-old sterile males from tower trays), because unchilled sterile males tested when 6 days old had a similar level of mating success as unchilled sterile males tested when 4 days old (28.4 ± 3.3% vs. 29.7 ± 3.9%, t = 0.15, d.f. = 18, P = 0.88).
The present findings show that the effects of chilling on the quality of sterile Mediterranean fruit fly males were dependent on the conditions under which the males were held prior to being chilled. For males maintained in low densities in the plastic buckets, chilling had no detectable effect on flight ability or mating competitiveness. In contrast, for males held at a much higher density in the tower trays, chilling reduced flight ability below that observed for unchilled males, and males chilled for 3 h had lower mating success than unchilled males or males chilled for 1 h (at least in tests conducted 1 day after chilling). Flight ability and mating competitiveness were similar between unchilled males held at low and high density, whereas chilled males kept at low density generally displayed higher performance levels than chilled males held at high density for both of these parameters.
Male Mediterranean fruit flies kept at relatively high densities may display increased rates of aggression and decreased longevity compared with males held at lower densities (Gaskin et al. 2002; see also Carey et al. 1995). In the present study, however, density per se did not negatively affect the performance parameters measured, because there was no significant difference in either flight ability or mating competitiveness for unchilled males held at low or high density. While lacking an independent role, high density in combination with chilling resulted in reduced male quality. In other words, male quality was not affected by either high density or chilling acting independently but was reduced when both stresses were imposed.
Initial studies on chilling Mediterranean fruit flies were made as part of the larger effort to develop and improve the aerial release method and were undertaken to compare the biological effects of chilling against alternative anesthetics or procedures involving no anesthetics at all (Hooper 1970; Harris et al. 1975; see also Tanahara and Kirihara 1989 and Nakamori et al. 1989 for similar studies on the melon fly, Bactrocera cucurbitae [Coquillett]). Few published studies have specifically examined the association between chill duration and flight ability, and the results have not been uniform. Consistent with the present findings, several studies (Nakamori et al. 1989; Tanahara and Kirihara 1989) on the melon fly demonstrated a decrease in flight ability with length of time chilled (the test flies were held at high density in paper bags used for release). In contrast, Salvato et al. (2003), working with the Mediterranean fruit fly, reported no significant decrease in flight ability between unchilled flies and those chilled for 2.5 h (males used were held before release at very high density in Plastic Adult Rearing Containers [so-called PARC boxes] that are used in many fly emergence and release facilities, USDA-APHIS 2009). As Salvato et al. (2003) used the same strain of C. capitata as the present study, it is not known what factor(s) accounted for the differing results.
To our knowledge, there exists only one published study relating cold treatment to mating behavior in C. capitata (see Mangan 1996 and Barron 2000 for relevant data on Anastrepha and Drosophila, respectively). In that study, Taylor et al. (2001) found that sterile males kept at low density (maximum 300 males per 15-l cage) and chilled for 4 h on the morning of testing had similar mating success to control, unchilled sterile males. In an unpublished study, Liedo (El Colegio de la Frontera Sur, Mexico, personal communication) showed that chilling (for 30–60 min) sterile C. capitata males (from high-density conditions) did not reduce the production of four major components of the male sex pheromone. Results of these two studies appear consistent with the present study: chilling had no adverse effect on mating frequency when sterile males (i) were held at low density (Taylor et al. 2001) or (ii) were held at high density but subject to cold for only 1 h (Liedo, unpublished data). As the auxiliary experiment showed, the negative effects of chilling on mating performance appear to be temporary. At first glance, this result may suggest that the negative impact of chilling is negligible. However, if sterile males survive only a few days after release, then even 1 day of diminished sexual competitiveness could severely reduce the efficacy of an SIT program. This would appear a serious concern, as Barry et al. (2002) reported over 50% mortality of sterile males by 5 days of age even under presumably optimal conditions, i.e., in-door cages supplied with ample food and water and lacking predators.
In addition, the present results reveal significant day-to-day variation in the flight ability of sterile males independent of chill treatment. As noted above, measurements of flight ability ranged approximately 20% among different batches for both unchilled and chilled males. Although outside the scope of the present study, this finding should raise broader questions regarding the level of variability characteristic of other quality parameters (male size, emergence, and longevity) and the extent to which the different quality parameters co-vary. Note that this variation occurred independently of any ‘external’ effects associated with handling and transport of irradiated pupae. Any temporal variation in handling procedures or travel times could conceivably amplify or diminish the intrinsic variability in quality control parameters deriving from the mass-rearing process itself.
The authors would like to thank Ed Baltazar and Mamadou War (both CDFA) and Amy Young (USDA) for supplying information on operating procedures in California and Florida, respectively, Tim Holler and Mark Salvato for discussion of their paper, Pablo Liedo for sharing unpublished data, Jorge Hendrichs, Pablo Liedo, and Rui Pereira for comments on an earlier draft, and Don McInnis (USDA) for granting permission to use the field cages.