Which of the following combinations of flies showed the highest number of mating instances?

Journal Article

Todd E Shelly,

USDA-APHIS

41-650 Ahiki Street, Waimanalo, HI 96795

USA

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Nicholas C Manoukis

USDA-ARS, Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center

Hilo, HI 96720

USA

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Received:

02 November 2021

Editorial decision:

09 February 2022

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Todd E Shelly, Nicholas C Manoukis, Mating Competitiveness of Bactrocera dorsalis (Diptera: Tephritidae) Males From a Genetic Sexing Strain: Effects of Overflooding Ratio and Released Females, Journal of Economic Entomology, Volume 115, Issue 3, June 2022, Pages 799–807, //doi.org/10.1093/jee/toac027
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Abstract

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a global pest that infests a range of fruit and vegetables. Males are attracted to methyl eugenol, and control is often achieved by the Male Annihilation Technique, where methyl eugenol + insecticide dispensers are deployed to eliminate males, preclude matings, and reduce population growth. The Sterile Insect Technique (SIT) has also been used to control B. dorsalis. The SIT involves the release of mass-reared, sterilized males to achieve matings with wild females, who then produce inviable eggs. Two key elements of SIT include the overflooding ratio achieved (sterile: wild males) and the strain type utilized, namely bisexual or genetically sexed (allowing male-only releases). Here, we describe the effects of these two factors on the mating competitiveness of a males from a genetic sexing strain of B. dorsalis, termed DTWP. Mating success was scored for DTWP versus wild males in field cages at ratios of 1:2, 1:1, 2:1, and 10:1 both when DTWP females were or were not concurrently released with DTWP males. Close correspondence was found between observed numbers of matings of particular male–female combinations and expected numbers based on the numbers of flies released of each sex and each strain. As a result, the proportion of total matings achieved by the DTWP across the eight treatments showed a corresponding increase with overflooding ratio. At a given ratio, DTWP males had a higher relative mating success when DTWP females were absent rather than present, although the reason for this was unclear.

The Sterile Insect Technique (SIT) is an environmentally benign, species-specific approach to suppress or eradicate populations of pestiferous insects (Pérez-Staples et al. 2021). SIT is widely used against agriculturally important fruit flies (Diptera: Tephritidae), most notably the Mediterranean fruit fly (medfly), Ceratitis capitata (Wiedemann) (Barnes et al. 2004, Plá et al. 2021), the melon fly, Zeugodacus cucurbitae (Coquillett) (Koyama et al. 2004), the South American fruit fly, Anastrepha fraterculus (Wiedemann) (Krüger et al. 2021), and the Mexican fruit fly, A. ludens (Loew) (Ramírez-Santos et al. 2021). SIT involves the production and sterilization of large numbers of the target pest and the subsequent release of these insects in impacted areas. The goal is to procure sterile male-by-wild female matings, which result in the production of inviable eggs and reduction of the population growth rate of the pest species (Pérez-Staples et al. 2021). SIT is generally used with other control measures, such as protein bait sprays, parasitoids, sanitary measures, and male annihilation, that first lower pest abundance, thus increasing the efficacy of the released sterile insects (Barclay 2005).

With respect to fruit flies, SIT is a complex process whose effectiveness is influenced by many factors. Perhaps most importantly, the released, sterile males must possess sufficient biological quality or vigor to allow location and copulation with wild females (Parker et al. 2021). Released males must be capable of flight and dispersal, survival under field conditions, and production of sexual signals (including pheromones and courtship displays) that are ‘acceptable’ to wild females. Conditions of mass-rearing, the sterilization procedure, and artificial selection in long-established, domestic colonies may negatively impact these performance parameters, and accordingly these influences have received considerable study (e.g., Cayol 2000, Hernández et al. 2007, Duarte et al. 2020). In addition, the release strategy adopted will greatly impact SIT’s effectiveness, where key decisions include selection of the mass-reared strain for release (e.g., males-only vs. bisexual strains, discussed in detail below), choice of release mode (e.g., ground vs. aerial), and determination of the frequency, spacing, and size (number of insects) of releases. The latter point, in particular, is considered critical as the effectiveness of SIT is likely dependent on the overflooding ratio (sterile:wild males) achieved through releases (Barclay 2005, Flores et al. 2017, Mastrangelo et al. 2018). For the medfly, in particular, program response to the inferior sexual competitiveness of mass-reared males has been to produce extremely large numbers of males for release (Parker et al. 2021).

The present study focuses on release strategy and in particular measures the impact of two elements—overflooding ratio and use of male-only versus bisexual strains—under semi-natural field cage conditions for the oriental fruit fly, Bactrocera dorsalis (Hendel). This species is a serious, worldwide agricultural pest, and females oviposit, and larvae subsequently develop, in over 400 plant species (USDA 2020), including many commercially important crops, such as mango (Mangifera indica L.), papaya (Carica papaya L.), and guava (Psidium guajava L.). The species is also highly invasive, owing to its polyphagy, high dispersal (Froerer et al. 2010), high fecundity (Yang et al. 1994), and competitive superiority over resident tephritid species (Duyck et al. 2004). While the Male Annihilation Technique (MAT; an attract-and-kill approach based on strong male attraction to the lure methyl eugenol, Vargas et al. 2014) is often implemented as a stand-alone approach to control B. dorsalis, SIT has occasionally been employed after MAT, and this MAT-then-SIT strategy has resulted in successful eradication (Steiner et al. 1970, Habu et al. 1984, Shiga 1989) or effective suppression (Orankanok et al. 2007). More recently, combinations of MAT and SIT have received increased attention for the control and eradication of Bactrocera fruit flies, increasing the relevance of SIT research for these species (Barclay et al. 2014, Khan et al. 2017).

The experimental approach adopted here followed earlier studies by Robinson et al. (1986), McInnis et al. (1986), and Hendrichs et al. (1996) by establishing different overflooding ratios in outdoor field cages and scored matings by laboratory (i.e., mass-reared) and wild males under these different ratios. Additionally, for each overflooding ratio tested, we either released laboratory males only or laboratory females along with the laboratory males. While the laboratory strain used here was a genetic sexing strain developed to allow male-only releases (after optical sorting and removal of female [white] pupae, see below), releases of laboratory females were included to assess their impact on the mating frequency of laboratory males with wild females and hence estimate the presumed benefit (in terms of heightened mating frequency with wild females) derived from male-only releases. Finally, as initial observations suggested laboratory males mated earlier in the day than wild males, we recorded times of observed matings between (1) wild females and laboratory or wild males in an outdoor cage and (2) females and males of both strains in individual male–female pairings established in Petri dishes.

Materials and Methods

Insects

Wild B. dorsalis derived from a laboratory colony started with ≈500 adults reared from fruits of common guava (Psidium guajava L.) collected at the University of Hawaii’s Urban Garden, Pearl City, Oahu, during June 2020. Fruits were placed in opaque plastic boxes that rested on wire-mesh screening over a layer of vermiculite for pupation. Pupae were sifted and divided evenly between two screen cages (60 × 40 × 30 cm, l:w:h). Adults were provided a food mixture of sugar and yeast hydrolysate (3:1 v/v) and water ad libitum. After 14 d, Nalgene (Nalge Nunc International Corporation, Rochester, NY) plastic bottles, containing guava juice-soaked sponges, were introduced into the cages periodically and served as oviposition devices. Eggs were rinsed from the bottles and placed on an artificial medium (Tanaka et al. 1969). As above, containers with larval diet were placed over vermiculite, and pupae were collected via sifting. Emerged adults were then either used to initiate the next generation of the colony or were used in the mating trials. To obtain adults for the mating trials, pupae were placed in plexiglass cages (40 × 30 × 30 cm), and adults were separated by sex within 3 d of emergence, well before reaching sexual maturity at 14–16 d of age (Shelly, unpublished data), and placed in cubical (30 cm per side) screen cages (approximately 250 flies per cage) with food and water as noted above. When used in the mating trials, wild flies were 18–23 d old and were 2–4 generations removed from the wild. All life stages were held at 23–27°C and 50–80% relative humidity under natural photoperiod (≈12:12 [L:D] hr).

The genetic (or pupal color) sexing strain, termed DTWP (dorsalis translocation white pupae), was developed by linking—via translocation—alleles for pupal color with the Y chromosome, such that males express the wild-type brown phenotype and females the mutant white phenotype (McCombs and Saul 1995). This strain has been reared continuously since 1995 (approximately 290 generations, Fezza et al. 2018) by the USDA-ARS, Hilo, HI, following the procedures given by Vargas (1989). The DTWP colony used in the present study was held and maintained in the same manner described above for wild flies. When used in the mating trials, DTWP flies were 12–16 d old, reflecting their shorter prematuration period (Shelly et al. 2000).

Note that, aside from preliminary mating trials, the DTWP flies used in this study were not irradiated, i.e., were fertile. This approach eliminated the logistical issues related to irradiating and shipping pupae from the main DTWP colony on the Big Island to Oahu and thus facilitated the completion of a relatively large number of mating trials. Importantly, an earlier study showed that irradiation (150 Gy) had no effect on the mating competitiveness of DTWP males (Shelly et al. 2000). In addition, two recent studies (Shelly 2020, Fezza et al. 2021), while not directly comparing irradiated and nonirradiated males, found that irradiated DTWP males (100 Gy) had similar mating success as (fertile) wild males in competition for wild females. Thus, we were confident that the data obtained using nonirradiated DTWP flies would apply to irradiated flies. Nonetheless, as a precaution and for reconfirmation, the present study included preliminary trials (following the procedures outlined below) that compared the mating success of irradiated and nonirradiated DTWP males relative to wild males in competition for wild females.

To distinguish wild and DTWP flies in the mating trials, flies from one strain were chilled on ice for 5–10 min and marked by placing a small dot of enamel paint on the thorax. The same color (silver) was used for marking in all trials. This marking method is widely used in tephritid fruit fly research and has no apparent effect on fly vigor or behavior (e.g., Poramarcom and Boake 1991, Lance et al. 2000). Flies were marked 1–2 d before use in an experiment. The identity of the marked strain (i.e., DTWP or wild) was alternated between successive test days for a given treatment (see following section).

Mating Trials

Mating trials were conducted in two nylon-screen field cages (3 m diam., 2.5 m height) that contained three potted guava trees (2–2.5 m high) to provide shaded resting sites for the flies (see FAO/IAEA/USDA 2019 for the photo of standard walk-in, field cage) On a given test day, flies were released into both cages approximately 2 hr before sunset (the period of peak sexual activity, Arakaki et al. 1984). Cages were checked at 5–10 min intervals from 1 hr before sunset to 0.5 hr past sunset; a flashlight was used to locate mating pairs at low light levels. Once coupled, pairs remain in copula until the following morning (Arakaki et al. 1984). Mating pairs were collected by gently coaxing them into plastic vials and placed in a freezer at the end of a trial. Collected flies were checked the next morning for the presence or absence of marking.

Before assessing the impact of overflooding ratios and the presence of DTWP females on the mating success of DTWP males, we conducted preliminary mating trials to confirm the lack of an irradiation effect on the mating competitiveness of DTWP males. Following standard practice (FAO/IAEA/USDA 2019), flies were irradiated as pupae, under hypoxia, 2 d before emergence at 100 Gy using a CXR-105 X-ray tube (Comet AG, Flamatt, Switzerland) at the USDA-ARS facility on Hawaii Island and then express mailed to Oahu, where the adults emerged. Trials were performed on eight different nights, with 70 wild males and 70 wild females placed in both tents along with 70 irradiated DTWP males in one tent and 70 nonirradiated DTWP males in the other tent.

The experimental mating trials included four overflooding ratios (defined as the ratio of DTWP:wild males released per field cage) each of which was evaluated with and without the simultaneous release of DTWP females (Table 1). When both sexes of DTWP flies were released, a 1:1 sex ratio was always employed. As shown in Table 1, the numbers of flies released were adjusted to maintain an overall total number of released flies (i.e., sexes and strains combined) of approximately 200. This uniformity in total numbers was deemed important to reduce possible density effects that might have confounded data interpretation. Eight trials (replicates) were conducted for each of the eight treatments listed in Table 1 (treatments hereafter refer to the different combinations of overflooding ratio and presence/absence of DTWP females). On a given day, the same treatment was established in each of the two field cages, meaning a given treatment was examined over four different days (4 d × 2 cages/d = 8 replicates). The treatments to be tested on a given day were selected randomly.

Table 1.

Overflooding ratios tested and corresponding numbers of DTWP and wild individuals released for each overflooding ratio

Overflooding ratioMalesFemalesTotalDTWPWildDTWP Wild

1:2 f+	34	68	34	68	204
1.2 f-	40	80	0	80	200
1:1 f+	50	50	50	50	200
1:1 f-	67	67	0	67	201
2:1 f+	68	34	68	34	204
2:1 f-	100	50	0	50	200
10:1 f+	90	9	90	9	198
10:1 f-	170	17	0	17	204

Overflooding ratioMalesFemalesTotalDTWPWildDTWP Wild

1:2 f+	34	68	34	68	204
1.2 f-	40	80	0	80	200
1:1 f+	50	50	50	50	200
1:1 f-	67	67	0	67	201
2:1 f+	68	34	68	34	204
2:1 f-	100	50	0	50	200
10:1 f+	90	9	90	9	198
10:1 f-	170	17	0	17	204

Overflooding ratio equals DTWP:wild males released in a given field cage. Note that the 1:2 ratio represents ‘underflooding’, but for brevity it is labeled an overflooding ratio. Superscripts f+ and f- designate the presence or absence of released DTWP females, respectively.

Table 1.

Overflooding ratios tested and corresponding numbers of DTWP and wild individuals released for each overflooding ratio

Overflooding ratioMalesFemalesTotalDTWPWildDTWP Wild

1:2 f+	34	68	34	68	204
1.2 f-	40	80	0	80	200
1:1 f+	50	50	50	50	200
1:1 f-	67	67	0	67	201
2:1 f+	68	34	68	34	204
2:1 f-	100	50	0	50	200
10:1 f+	90	9	90	9	198
10:1 f-	170	17	0	17	204

Overflooding ratioMalesFemalesTotalDTWPWildDTWP Wild

1:2 f+	34	68	34	68	204
1.2 f-	40	80	0	80	200
1:1 f+	50	50	50	50	200
1:1 f-	67	67	0	67	201
2:1 f+	68	34	68	34	204
2:1 f-	100	50	0	50	200
10:1 f+	90	9	90	9	198
10:1 f-	170	17	0	17	204

In addition to the primary experiment, an ancillary test was conducted to assess the impact of prerelease exposure of DTWP males to the compound methyl eugenol (4-allyl-1,2-dimethoxy benzene). Several studies have demonstrated that male feeding on this chemical, which occurs in a wide diversity of plants (Tan and Nishida 2012), enhances their mating success, owing to an increased level of sexual signaling (pheromone calling) and the production of a more attractive pheromone to females (Shelly and Dewire 1994, Tan and Nishida 1996). DTWP males (12–14 d old) were provided with methyl eugenol for 30 min (1000–1030 hr) 2 d before testing. The chemical (0.5 ml) was applied to a cotton wick resting in a Petri dish, which was then placed into a holding cage. These cages were isolated from other flies to avoid inadvertent exposure to the odor of methyl eugenol. Chemically treated DTWP males were tested only at the 1:2 overflooding ratio as the effect of methyl eugenol feeding would presumably be most evident when DTWP males were at a numerical disadvantage. Six replicates were conducted with or without the release of DTWP females, and thus treated DTWP males were exposed to methyl eugenol in groups of 34 (females present) or 40 (females absent) individuals per cage (Table 1).

Timing of Matings

In the mating trials described above, pairs were collected without recording the time of collection. However, it appeared that matings involving DTWP males were occurring earlier than those involving wild males. To investigate this possibility more closely, we conducted two different sets of observations. First, using only one of the field cages, times were recorded when pairs were first observed and collected, with continuous observations beginning 1.5 hr before sunset. Equal numbers of DTWP males and wild males and females were released (67 individuals/category, 201 flies total). Second, 20 pairs each of DTWP♂ × DTWP♀, DTWP♂ × wild♀, wild♂ × DTWP♀, and wild♂ × wild♀ were placed in Petri dishes (8.5 cm diam.), and mating times were recorded. Flies were chilled in ice approximately 2 hr before sunset to allow transfer to the Petri dishes; lids contained a screened opening (2 cm diam.) to allow air exchange. Individual Petri dishes (80 total) were labeled according to the fly pair contained and placed randomly on a table in a shaded location outside the laboratory. Starting 1.5 hr before sunset, the pairs were observed continuously and times were recorded when flies coupled. Data on mating times from the field cage or the Petri dishes were made on clear days in November-December 2020, with air temperatures between 25–27°C and sunset between 1748–1751 hr. In addition, the ambient light level was measured (in Lux using an ennoLogic eL200K Light Meter, ennoLogic, Eugene, OR) at 5 min intervals with the sensor held horizontally in the canopy of guava trees in the field cage or placed on the same table as the Petri dishes holding the paired flies.

Statistical Analysis: Mating Trials

For the preliminary trials, comparisons of mating numbers between DTWP (irradiated or nonirradiated) and wild males were made using t-tests as data were normally distributed. A minimum of 20% of the females mated in all field cages and thus, based on standard guidelines (FAO/IAEA/USDA 2019), data from all cages were included in the analysis.

For the main experiment, a goodness-of-fit analysis was undertaken to determine whether observed numbers of matings obtained by DTWP and wild males differed from those expected by chance (i.e., those based solely on the relative numbers of males of each strain released in the field cages). The chi-square test compared average numbers of matings observed per replicate for each possible mating combination against the expected numbers computed as the product of the relative abundance of the 2 male types and the average numbers of females of a given strain that mated per replicate. Expected proportions of matings by DTWP males for the 1:2, 1:1, 2:1, and 10: 1 overflooding ratios were 33, 50, 67, and 91%, respectively. To provide additional confirmation of the results of these aggregate chi-square tests, we conducted chi-square analysis for the individual replicates and briefly summarize these results. Over all tests, the Yates correction factor was employed when df = 1. In all field cages, at least 20% of females from each strain mated, and data from all cages were included in the analyses.

Because the numbers of flies released in the field cages varied among treatments, comparisons of the mating success of DTWP males in the different treatments were made on a relative, and not absolute, basis. For all replicates, we calculated the proportion of matings by wild females that involved DTWP males. In SIT studies, this value is termed the Relative Sterility Index (RSI; McInnis et al. 1996), but since the DTWP flies used in our study were fertile, we used the acronym RMI (Relative Mating Index) to refer to this proportion. Variation in RMIs was analyzed with a 2-way ANOVA (overflooding ratio and DTWP female presence/absence as the main factors) using arcsine transformed proportions, which met parametric assumptions, with the Tukey test used to identify pair wise differences among the treatments. In the ancillary experiment, t-tests were used to compare RMI values obtained for methyl eugenol-fed DTWP males with RMI values measured for unfed DTWP males in the main experiment at a 1:2 overflooding ratio (arcsine transformed proportions met parametric assumptions).

Statistical Analysis: Timing of Matings

Data on the timing of matings from both the field tent and the Petri dishes were analyzed in the same manner. For a particular day (n = 4 in both cases), the time interval between the first and last matings was determined, and individual matings were then assigned a relative mating time (RMT) based on their occurrence in this interval. If, for example, the first and last matings occurred at 1700 and 1750 hr, respectively, a mating that occurred at 1725 hr was assigned an RMT of 0.50 (25/50) as it occurred at the midpoint of the interval encompassing all matings on that day. Arcsine transformed RTMs were not normally distributed for field cage or Petri dish observations, consequently nonparametric tests were used to analyze data from both the field cage (Mann–Whitney test, test statistic Z using normal approximation) and the Petri dishes (Kruskal–Wallis test, test statistic H). Dunn’s test was performed to identify pairwise difference following the Kruskal–Wallis test.

Statistical analyses were conducted using SigmaPlot 11.0 (Systat Software, San Jose, CA). Means ± 1SD are given.

Results

Mating Success of Irradiated and Nonirradiated DTWP Males

Results of the preliminary trials confirmed that, as shown by earlier studies, irradiation had no effect on the mating ability of DTWP males. Nonirradiated DTWP males obtained 22.9 ± 5.3 matings per replicate compared to 20.1 ± 2.9 matings by wild males (t = 1.3, df = 7, P = 0.22). Similarly, irradiated DTWP males achieved 26.0 ± 7.0 matings per replicate compared to 21.6 ± 3.61 for wild males (t = 1.8, df = 7, P = 0.10). The number of matings obtained per replicate by irradiated and nonirradiated males did not differ significantly (t = 1.3, df = 7, P = 0.20).

Mating Frequencies: DTWP Females Absent

Based on an aggregate chi-square test, observed numbers of matings for DTWP and wild males did not differ significantly from expected values based on their relative abundances for any of the overflooding ratios tested (Table 2). This same result was found for the great majority of the individual replicates. For the 1:2 and 10:1 overflooding ratios, none of the replicates (n = 8 per ratio) showed significant deviation from random mating. Significant deviation from random was detected for one replicate for the 1:1 overflooding ratio and two replicates for the 2:1 ratio. Thus, over all overflooding ratios, only three of 32 replicates (four treatments × eight replicates each) indicated nonrandom mating frequencies. In these three instances, DTWP males mated more often, and wild males less often, than expected by chance.

Table 2.

Numbers of matings observed in field cage trials over four overflooding ratios with DTWP females absent or present

DTWP femalesOverflooding ratio (D:W males)Matings per replicateDDDWWDWWχ2P

Absent	1:2	–	18.2 ± 1.9 (14.9)	–	27.1 ± 5.7 (30.4)	0.79	0.34
1:1	–	24.4 ± 5.7 (20.2)	–	16.0 ± 3.0 (20.2)	1.36	0.28
2:1	–	22.1 ± 5.2 (21.1)	–	9.4 ± 3.0 (10.4)	0.03	0.87
10:1	–	9.9 ± 2.1 (9.4)	–	0.4 ± 0.5 (0.9)	0.00	1.00
Present	1:2	10.7 ± 2.1 (7.1)	8.9 ± 1.6 (11.3)	10.9 ± 3.3 (14.5)	25.2 ± 3.8 (22.8)	3.48	0.49
1:1	22.1 ± 6.5 (16.0)	13.1 ± 1.4 (13.5)	9.9 ± 2.1 (16.0)	13.9 ± 2.0 (13.5)	4.66	0.21
2:1	37.7 ± 5.7 (33.4)	13.1 ± 1.7 (13.4)	12.2 ± 2.1 (16.5)	6.9 ± 2.0 (6.6)	1.69	0.65
10:1	47.7 ± 3.6 (49.1)	4.5 ± 0.9 (4.7)	6.3 ± 2.5 (4.9)	0.7 ± 0.7 (0.5)	0.56	0.91

DTWP femalesOverflooding ratio (D:W males)Matings per replicateDDDWWDWWχ2P

Absent	1:2	–	18.2 ± 1.9 (14.9)	–	27.1 ± 5.7 (30.4)	0.79	0.34
1:1	–	24.4 ± 5.7 (20.2)	–	16.0 ± 3.0 (20.2)	1.36	0.28
2:1	–	22.1 ± 5.2 (21.1)	–	9.4 ± 3.0 (10.4)	0.03	0.87
10:1	–	9.9 ± 2.1 (9.4)	–	0.4 ± 0.5 (0.9)	0.00	1.00
Present	1:2	10.7 ± 2.1 (7.1)	8.9 ± 1.6 (11.3)	10.9 ± 3.3 (14.5)	25.2 ± 3.8 (22.8)	3.48	0.49
1:1	22.1 ± 6.5 (16.0)	13.1 ± 1.4 (13.5)	9.9 ± 2.1 (16.0)	13.9 ± 2.0 (13.5)	4.66	0.21
2:1	37.7 ± 5.7 (33.4)	13.1 ± 1.7 (13.4)	12.2 ± 2.1 (16.5)	6.9 ± 2.0 (6.6)	1.69	0.65
10:1	47.7 ± 3.6 (49.1)	4.5 ± 0.9 (4.7)	6.3 ± 2.5 (4.9)	0.7 ± 0.7 (0.5)	0.56	0.91

Values represent means ± 1 SD, with n = 8 in all cases. Numbers in parentheses are expected values based on random mating. DTWP flies were designated as D, and wild flies were designated as W. In the four mating combinations, the first letter indicates male strain and the second indicates female strain. In chi-square tests, df = 1 when DTWP females were absent, and df = 3 when these females were present. Table 1 provides the numbers of flies released for each strain and each sex in the different treatments.

Table 2.

Numbers of matings observed in field cage trials over four overflooding ratios with DTWP females absent or present

DTWP femalesOverflooding ratio (D:W males)Matings per replicateDDDWWDWWχ2P

Absent	1:2	–	18.2 ± 1.9 (14.9)	–	27.1 ± 5.7 (30.4)	0.79	0.34
1:1	–	24.4 ± 5.7 (20.2)	–	16.0 ± 3.0 (20.2)	1.36	0.28
2:1	–	22.1 ± 5.2 (21.1)	–	9.4 ± 3.0 (10.4)	0.03	0.87
10:1	–	9.9 ± 2.1 (9.4)	–	0.4 ± 0.5 (0.9)	0.00	1.00
Present	1:2	10.7 ± 2.1 (7.1)	8.9 ± 1.6 (11.3)	10.9 ± 3.3 (14.5)	25.2 ± 3.8 (22.8)	3.48	0.49
1:1	22.1 ± 6.5 (16.0)	13.1 ± 1.4 (13.5)	9.9 ± 2.1 (16.0)	13.9 ± 2.0 (13.5)	4.66	0.21
2:1	37.7 ± 5.7 (33.4)	13.1 ± 1.7 (13.4)	12.2 ± 2.1 (16.5)	6.9 ± 2.0 (6.6)	1.69	0.65
10:1	47.7 ± 3.6 (49.1)	4.5 ± 0.9 (4.7)	6.3 ± 2.5 (4.9)	0.7 ± 0.7 (0.5)	0.56	0.91

DTWP femalesOverflooding ratio (D:W males)Matings per replicateDDDWWDWWχ2P

Absent	1:2	–	18.2 ± 1.9 (14.9)	–	27.1 ± 5.7 (30.4)	0.79	0.34
1:1	–	24.4 ± 5.7 (20.2)	–	16.0 ± 3.0 (20.2)	1.36	0.28
2:1	–	22.1 ± 5.2 (21.1)	–	9.4 ± 3.0 (10.4)	0.03	0.87
10:1	–	9.9 ± 2.1 (9.4)	–	0.4 ± 0.5 (0.9)	0.00	1.00
Present	1:2	10.7 ± 2.1 (7.1)	8.9 ± 1.6 (11.3)	10.9 ± 3.3 (14.5)	25.2 ± 3.8 (22.8)	3.48	0.49
1:1	22.1 ± 6.5 (16.0)	13.1 ± 1.4 (13.5)	9.9 ± 2.1 (16.0)	13.9 ± 2.0 (13.5)	4.66	0.21
2:1	37.7 ± 5.7 (33.4)	13.1 ± 1.7 (13.4)	12.2 ± 2.1 (16.5)	6.9 ± 2.0 (6.6)	1.69	0.65
10:1	47.7 ± 3.6 (49.1)	4.5 ± 0.9 (4.7)	6.3 ± 2.5 (4.9)	0.7 ± 0.7 (0.5)	0.56	0.91

Mating Frequencies: DTWP Females Present

Based on an aggregate chi-square test, observed numbers of the four different mating combinations did not differ significantly from expected values based on the relative abundances of the DTWP and wild males for any of the overflooding ratios tested (Table 2). As above, most of the replicates likewise indicated random mating frequencies. For the 2:1 overflooding ratio, none of the eight replicates showed significant deviation from random mating. Significant deviation from random was detected for three replicates for the 1:2 overflooding ratio, one replicate for the 1:1 ratio, and three replicates for the 10:1 ratio. Thus, over all overflooding ratios, seven of 32 replicates (four treatments × eight replicates each) indicated nonrandom mating frequencies. In most of these seven cases, nonrandom mating frequencies resulted from higher than expected numbers of matings between DTWP flies and lower numbers of matings between wild males and DTWP females.

RMI Values

Both overflooding ratio (F3, 56 = 99.9, P < 0.001) and presence/absence of DTWP females (F1, 56 = 15.7, P < 0.001) had significant effects on RMI values (Fig. 1). The interaction between these 2 factors was not significant (F3, 56 = 0.96, P = 0.42). The multiple comparisons Tukey test revealed that, independent of overflooding ratio, RMI values differed significantly between the females present versus absent conditions. Likewise, independent of female presence or absence, RMI values differed significantly in all pair wise comparisons between the different overflooding ratios. The effect of DTWP females on RMI generally declined as overflooding ratio increased. At 1:2 and 1:1, RMI values measured when females were absent were, respectively, 55 and 23% greater than those realized when females were present. At 2:1 and 10:1, however, RMI values measured when females were absent were, respectively, only 5 and 12% greater than those observed when females were present.

Fig. 1.

RMI values (% total matings achieved by DTWP males) at different overflooding ratios and with or without the concurrent release of DTWP females. Symbols represent means ± 1 SD. Triangles (filled and unfilled) represent trials in which DTWP males fed on methyl eugenol before testing. Eight replicates were performed for each of the eight treatments in the main experiment (represented by circles), and six replicates were conducted with or without DTWP females, respectively, in the ancillary experiment involving methyl eugenol.

RMI Values and Methyl Eugenol Feeding

In trials conducted at 1:2 overflooding ratio, prerelease feeding on methyl eugenol by DTWP males enhanced their mating performance (Fig. 1). In the absence of females, the RMI of methyl eugenol-treated DTWP males was 47.4 ± 4.3% compared to 40.5 ± 3.7 for untreated males (t = 2.25, df = 12, P = 0.04). When DTWP females were released, the RMI of methyl eugenol-treated DTWP males was 36.2 ± 3.5% compared to 26.1 ± 4.3 for untreated males (t = 4.69, df = 12, P < 0.001).

Timing of Matings

Data from the field cage did not reveal a pronounced temporal difference in mating times between DTWP and wild males in competition for copulations with wild females (Fig. 2A). The proportion of matings occurring before 1715 hr was slightly higher for DTWP (21%) than wild (16%) males, and conversely, the proportion of matings occurring after 1745 hr was slightly higher for wild (14%) than DTWP (8%) males. Nonetheless, the mating times of DTWP and wild males showed broad overlap, and as a result, RMTs did not differ significantly between the male types (Z = 0.96, P = 0.34; Fig. 2B). Mean RMTs were 0.53 and 0.57 for matings with DTWP and wild males, respectively.

Fig. 2.

Timing of matings recorded in a field tent that, for a given replicate, contained 67 DTWP males, 67 wild males, and 67 wild females. DTWP flies were designated as D, and wild flies were designated as W. In the two mating combinations, the first letter indicates male strain and the second indicates female strain. The top graph (A) presents the proportions of total matings of DW and WW pairs that were collected during a given time interval. The bottom graph (B) presents the relative mating times for DW and WW pairs as defined in the text. For both graphs, data were pooled over four days when sunset occurred between 175.48–5.50 pm; lux values are averages of measurements taken on the 4 test days. Total numbers of matings observed were: DW—103, WW—77.

In contrast, data from the Petri dishes showed an obvious temporal trend in the occurrence of the different mating combinations (Fig. 3A). Specifically, the 4 male–female combinations occurred in rough succession, with DTWP♂ × DTWP♀ occurring earliest (mean RTM = 0.42), followed by DTWP♂ × wild♀ (mean RMT = 0.51), then wild♂ × DTWP♀ (mean RMT = 0.64), and wild♂ × wild♀ (mean RMT = 0.69) occurring latest (Fig. 3B).

Fig. 3.

Timing of matings for paired flies in Petri dishes. DTWP flies were designated as D, and wild flies were designated as W. In the four mating combinations, the first letter indicates male strain and the second indicates female strain. Ordinate represents the proportion of total matings observed for a particular male–female combination that occurred within a given time interval. Data were pooled over four days on which sunset occurred between 1748–1750 hr; lux values are averages of measurements taken on the four test days. Total numbers of matings observed were: DD—77, DW—68, WD—59, WW—63.

The timing of matings varied noticeably between the field cage and the Petri dishes (Figs. 2A and 3A, respectively). Despite higher light levels, matings occurred earlier in the field cage than in the Petri dishes. Combined over both DTWP and wild males, 36% (62/180) of all matings occurred before 1725 hr in the field cage when Lux readings ranged from approximately 400–650 Lux. By contrast, only 3% (4/131) of all DTWP♂ × wild♀ and wild♂ × wild♀ matings recorded in the Petri dishes occurred before 1725 hr even though light levels were lower (543 Lux at 1700 hr [data not shown in Fig. 3] and 330 Lux at 1725 hr). Similarly, peak mating activity in the field cage occurred roughly between 1726–1745 hr when 53% (95/180) of all matings were collected, whereas in the Petri dishes the peak numbers of matings involving wild females (83/140 = 59%) were observed in the time intervals 1741–1755 hr.

Discussion

The most notable result of the present study is that, based on data collected in semi-natural habitats afforded by field cages, males from a pupal-color genetic sexing strain of B. dorsalis are equivalent to wild males in mating competitiveness. Goodness-of-fit tests for both pooled (i.e., averaged across replicates) and individual replicates showed close correspondence between observed numbers of matings of particular male–female combinations (with respect to strain identity, i.e., DTWP or wild) and expected numbers expected based solely on the numbers of flies released of each sex and each strain. In short, the mating frequencies of DTWP and wild males across all eight treatments indicated that females mated randomly between DTWP and wild males. In those specific replicates where nonrandom mating was observed, DTWP females mated significantly more frequently with DTWP males and mated less frequently with wild males than expected by chance. Whether this finding reflects an evolved preference of DTWP females for DTWP males or a possible inter-strain difference in the timing of mating activity (see below) is unknown.

The apparent randomness in female mate selection differs dramatically from that commonly reported for the medfly, where mass-reared males are, in nearly all studies, inferior sexual competitors relative to their wild counterparts (Shelly and McInnis 2016 and references therein). However, the finding is consistent with previous studies on B. dorsalis and Z. cucurbitae: a survey of published data (field cage studies with 1:1 ratio of mass-reared and wild males) found that, averaged over these two species, mass-reared males obtained 49% (n = 5) of all matings (Shelly and McInnis 2016). Pérez-Staples et al. (2009) likewise found that, when fed a protein-rich diet, mass-reared males of the Queensland fruit fly, Bactrocera tryoni (Froggatt), achieved similar numbers of matings with wild females as did wild males. Interestingly, the mass-reared males used in these previous studies were all irradiated, indicating that the sterilization process did not reduce male sexual competitiveness, a finding consistent with the present study.

Why mass-rearing negatively affects mating success in medfly but not in the oriental fruit is unknown, although it may reflect interspecific differences in the complexity of male courtship (Lance and McInnis 2021). Both species appear to exhibit lek behavior, and receptive females seek male aggregations generally located on host trees (e.g., Shelly and Kaneshiro 1991, Whittier et al. 1992). Moreover, in both species, perching males signal their location via emission of pheromones, intense wing-fanning, and accompanying sound production (Shelly 2018). However, upon detection of an approaching female, males of the two species behave quite differently. Males of C. capitata males produce a suite of close-range signals, including rapid forward-backward wing flicks, continued pheromone emission but with the abdomen curved downward and forward, and oscillatory head movements, all of which are performed while directly facing the female (Arita and Kaneshiro 1989). After a few seconds, and if the female remains still, the male mounts the female and attempts intromission. In contrast, B. dorsalis males cease wing-fanning and abruptly attempt to mount the female; specific courtship behaviors or signals are not apparent (Arakaki et al. 1984, Shelly and Kaneshiro 1991). Under prolonged mass-rearing, artificial selection might be expected to generate greater changes in complex courtship than in mating attempts that follow directly from the mere occurrence of intersexual encounters. In fact, Briceño and Eberhard (1998, 2000) have documented changes in the duration and timing of courtship elements of mass-reared C. capitata males, which they attributed to crowded holding conditions that favor accelerated courtship. They further proposed that modified courtship is responsible for the low mating competitiveness of mass-reared males relative to their wild counterparts.

Whatever the underlying reason, the equivalence in mating success generated increases in RMI that corresponded directly with increasing overflooding ratio. RMI values obtained in the presence/absence of DTWP females bracketed 33% at 1:2, 50% at 1:1, 67% at 2:1, and 91% at 10:1. Within this overall trend, RMI values were significantly greater for male-only releases than bisexual releases at each overflooding ratio tested, with the greatest differences observed at the lower overflooding ratios (1:2 ad 1:1). It is not obvious what factor(s) was responsible for this result, particularly given the apparent similarity in mating ability between DTWP and wild males. Although the numbers of matings observed did not, in general, differ from those expected, the RMI values for male-only releases were consistently above expected values, while those for bisexual releases were consistently below expected frequencies, which, in turn, generated the significant difference in RMIs observed for each overflooding ratio.

The production and release of male-only genetic sexing strains in SIT programs have several benefits, two of which are paramount (Hendrichs et al. 1995). First, culling of females before release dramatically reduces rearing and holding costs for released flies. This benefit is most fully realized in species, like C. capitata, where females are eliminated at the egg stage (via thermal stress), as resources required for larval and pupal rearing can be allocated to males exclusively. Still, even where female removal occurs after pupation (via visual sorting based on pupal color), as in B. dorsalis, costs associated with adult eclosion, prerelease holding, and field release would be lowered substantially. Second, male-only releases may increase the effectiveness of SIT, because matings between released flies are eliminated, and the mating activities of released males will be directed at wild females exclusively.

While reduced rearing and handling costs are demonstrable, documenting enhanced efficacy of SIT via male-only releases is more difficult. Moreover, all such studies have focused exclusively on the medfly. For example, field cage studies have either not included comparisons between male-only and bisexual releases at the same overflooding ratio (Robinson et al. 1986, Hendrichs et al. 1996) or used nonirradiated, mass-reared flies in lieu of wild flies (McInnis et al. 1986). Field data are more compelling, as studies in coffee (Coffea arabica L.) plantations in Hawaii (McInnis et al. 1994) and Guatemala (Rendón et al. 2004) show that egg hatch was 3–7 times lower in areas receiving male-only releases compared to areas receiving bisexual releases. However, a study (Shelly and Whittier 1996) based on observed matings in a mixed Hawaiian fruit orchard indicated that release of males only did not increase the frequency of sterile male by wild female matings above that recorded in bisexual releases. To our knowledge, the present study constitutes the first explicit test regarding the efficacy of male-only versus bisexual releases for any Bactrocera species, but field studies on this genus are clearly needed to supplement the field cage tests described here and better assess the effectiveness of male-only releases.

Mass-reared flies have been found to display earlier mating times than their wild counterparts in the melon fly (Suzuki and Koyama 1980, Kuba and Koyama 1982; but see Matsuyama and Kuba [2009] for contrasting data) and B. tryoni (Weldon 2005), and preliminary observations suggested the same for B. dorsalis. However, data gathered in two separate experiments were inconsistent. Results from the field cage showed no significant difference in the timing of matings between wild females and DTWP or wild males. In the Petri dishes, on the other hand, a well-defined, temporal sequence of mating was observed: crosses of DTWP♂ x DTWP♀ generally occurred earliest, followed by DTWP♂ × wild♀, wild♂ × DTWP♀, and wild♂ × wild♀. This pattern indicates that (1) DTWP flies mated earlier than wild flies, because intrastrain matings occurred earlier for DTWP flies than for wild flies and (2) DTWP males may gain an advantage over wild males in competition for wild females, because DTWP♂ × wild♀ matings generally occurred before wild♂ × wild♀ pairings.

Our failure to detect a similar trend in the field cage may have reflected inaccuracy in the scoring of mating times arising from the complex nature of the guava tree foliage, which made it difficult to locate copulating pairs and thus obtain reliable copulation start times. To our knowledge, no other studies have compared the timing of matings using the same strain(s) of flies in Petri dishes (or other small cages) and field cages. In future tests, multiple observers should be employed to collect pairs and record times. Independent of this issue, comparing data from the field cage and the Petri dishes is confounded by different time-dependent changes in ambient light levels. In the field cage, which was placed in a relatively bright location, the initial light level (at 1700 hr) was approximately 700 Lux, and the majority of matings occurred when light levels ranged from 125 to 500 Lux. In contrast, the Petri dishes were placed in a shadier location, with the initial light intensity being approximately only 350 Lux, and maximum mating activity was recorded at light levels of approximately 25–150 Lux. The difference observed in the relationship between light level and mating activity for the field cage and the Petri dishes was unexpected and remains unexplained. Observing Petri dishes inside the same field cage used to collect mating pairs would presumably standardize light conditions, and, if detected, any difference in mating times between tree canopy and Petri dishes would reflect, not light level, but differing effects of the comparative density of the flies in the two experimental environments.

In conclusion, the present study indicates that the SIT is potentially a powerful control measure for B. dorsalis that would be effective in conjunction with male annihilation or alone in circumstances where male annihilation is not feasible (e.g., protected natural areas). Mass-reared DTWP males competed equally with wild males for copulations with wild females, and, if appropriate methodology is developed (Tan and Tan 2013), the incorporation of prerelease methyl eugenol feeding would further enhance the sexual performance of mass-reared males. The high, endogenous competitiveness, plus the potential for methyl eugenol-mediated enhancement, suggest that suppression or even eradication of invasive populations could be achieved with much low overflooding ratios than those used against C. capitata. Reduction in production demand would, in turn, reduce the rearing and release costs associated with SIT and increase the cost-effectiveness of this tactic against B. dorsalis.

Acknowledgments

We are grateful to Song So (California Department of Food and Agriculture) for supplying rearing materials and Rick Kurashima (USDA APHIS) for assistance in maintaining the fly colonies.

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Published by Oxford University Press on behalf of Entomological Society of America 2022.

This work is written by (a) US Government employee(s) and is in the public domain in the US.

Published by Oxford University Press on behalf of Entomological Society of America 2022.

Subject Editor: Hannah Burrack

Hannah Burrack

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Which of the following combinations of flies showed the highest number of mating instances?

Cite

Abstract

Materials and Methods

Insects

Mating Trials

Timing of Matings

Statistical Analysis: Mating Trials

Statistical Analysis: Timing of Matings

Results

Mating Success of Irradiated and Nonirradiated DTWP Males

Mating Frequencies: DTWP Females Absent

Mating Frequencies: DTWP Females Present

RMI Values

RMI Values and Methyl Eugenol Feeding

Timing of Matings

Discussion

Acknowledgments

References Cited

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