Introduction
Sterile insect technique (SIT) is an environmentally friendly pest control method, consisting of the mass-rearing of the target insect, subsequent male irradiation and release into the field (Knipling, Reference Knipling1955). Once in the field, sterile males must obtain food and water to survive until they reach sexual maturity, find and mate with a wild female, transfer an adequate ejaculate, trigger the typical behavior of a mated female and induce a refractory, or non-receptive, period in the mated female. The SIT is used around the world to control many insect pests, such as certain fruit flies of the Tephritidae family, as the Mediterranean fruit fly Ceratitis capitata (Enkerlin et al., Reference Enkerlin, Gutiérrez-Ruelas, Villaseñor Cortes, Cotoc Roldan, Midgarden, Lira, Zavala López, Hendrichs, Liedo and Trujillo Arriaga2015) and the oriental fruit fly Bactrocera dorsalis (Vargas et al., Reference Vargas, Piñero, Mau, Jang, Klungness, McInnis, Harris, Mcquate, Bautista and Wong2010).
The South American fruit fly Anastrepha fraterculus (Diptera: Tephritidae) is an important pest of many commercial crops from the Southern USA to Central Argentina (Salles, Reference Salles1995; Malavasi et al., Reference Malavasi, Zucchi, Sugayama, Malavasi and Zucchi2000). Currently, SIT has been proposed for A. fraterculus as a feasible strategy for area-wide control of this pest (Ortíz, Reference Ortíz1999), and a great effort has been made in the past two decades to gain the knowledge necessary to develop this technique for this species (Jaldo et al., Reference Jaldo, Gramajo and Willink2001; Allinghi et al., Reference Allinghi, Gramajo, Willink and Vilardi2007; Segura et al., Reference Segura, Petit-Marty, Sciurano, Vera, Calcagno, Allinghi, Gomez-Cendra, Cladera and Vilardi2007, Reference Segura, Utgés, Liendo, Rodríguez, Devescovi, Vera and Cladera2013; Vera et al., Reference Vera, Abraham, Oviedo and Willink2007, Reference Vera, Oviedo, Abraham, Ruiz, Mendoza, Chang and Willink2014; Abraham et al., Reference Abraham, Liendo, Devescovi, Peralta, Yusef, Ruiz, Cladera, Vera and Segura2013; Liendo et al., Reference Liendo, Devescovi, Bachmann, Utgés, Abraham, Vera and Teal2013).
Anastrepha and Bactrocera species have a long pre-maturation period, around 10–15 days approximately, according to species. In an SIT program, the release of sexually immature males is detrimental and reduces the efficiency of this technique as males are under predation risk, may face stressful environmental conditions (such as rain, extreme temperature, low humidity, etc.), and must forage for water and food to reach sexual maturity (Gómez-Cendra et al., Reference Gómez-Cendra, Segura, Allinghi, Cladera and Vilardi2007). On the other hand, if males are held in the facilities for days until they reach sexual maturity, crowded holding conditions for an extended period have undesirable consequences, such as wing damage and a decrease in their mating competitiveness (Gaskin et al., Reference Gaskin, Futerman and Chapman2002; Enkerlin, Reference Enkerlin2007; Díaz-Fleischer et al., Reference Díaz-Fleischer, Arredondo and Aluja2009; Andress et al., Reference Andress, War and Shelly2013). Another concern is that holding insects before release for extended periods increases economic costs and creates logistic difficulties for SIT programs. Shortening the sexual maturation period of males is a viable option to incorporate into the pre-release protocols, which can be achieved by using methoprene, a juvenile hormone (JH) analog (Teal et al., Reference Teal, Gómez-Simuta and Proveaux2000, Reference Teal, Pereira, Segura, Haq, Gómez-Simuta, Robinson and Hendrichs2013; Muñoz-Barrios et al., Reference Muñoz-Barrios, Cruz-López, Rojas, Hernández, Liedo, Gómez-Simuta and Malo2016; Gómez-Simuta et al., Reference Gómez-Simuta, Diaz-Fleischer, Arredondo, Díaz-Santiz and Pérez-Staples2017). Ideally, post teneral/pre-release treatments must have a positive effect not only on the mating behavior of the treated males (accelerating or/and increasing male mating behavior), but also on their ejaculate characteristics and their ability to induce female refractoriness (Morelli et al., Reference Morelli, Paranhos, Coelho, Castro, Garziera, Lopes and Bento2013; Akter and Taylor, Reference Akter and Taylor2018).
Physiological processes in insects, such as reproduction and metamorphosis, are regulated, in part, by JH (Gilbert et al., Reference Gilbert, Granger and Roe2000; Dubrovsky, Reference Dubrovsky2005). In tephritid fruit flies of economic importance the application of methoprene accelerates male sexual maturation in Anastrepha obliqua (Teal et al., Reference Teal, Gomez-Simuta, Dueben, Holler, Olson, Vreysen, Robinson and Hendrichs2007), Anastrepha suspensa (Teal et al., Reference Teal, Gómez-Simuta and Proveaux2000; Teal and Gómez-Simuta, Reference Teal and Gómez-Simuta2002; Pereira et al., Reference Pereira, Sivinski and Teal2009), Anastrepha ludens (Gómez-Simuta et al., Reference Gómez-Simuta, Teal and Pereira2013), Zeugodacus cucurbitae (Haq et al., Reference Haq, Caceres, Hendrichs, Teal, Wornoayporn, Stauffer and Robinson2010), and Bactrocera tryoni (Collins et al., Reference Collins, Reynolds and Taylor2014).
Efforts have recently been made to apply methoprene into the adult male diet (Muñoz-Barrios et al., Reference Muñoz-Barrios, Cruz-López, Rojas, Hernández, Liedo, Gómez-Simuta and Malo2016; Gómez-Simuta et al., Reference Gómez-Simuta, Diaz-Fleischer, Arredondo, Díaz-Santiz and Pérez-Staples2017; Adnan et al., Reference Adnan, Mendez, Morelli, Akter, Farhana and Taylor2018, Reference Adnan, Farhana, Inskeep, Rempoulakis and Taylor2020c; Pereira et al., Reference Pereira, Yuval, Liedo, Teal, Shelly, McInnis, Haq, Taylor, Hendrichs, Dick, Hendrichs and Robinson2021; Reyes-Hernández et al., Reference Reyes-Hernández, Macías-Díaz del Castillo, Abraham, Arredondo and Pérez-Staples2021), and not topically diluted in acetone, as the handling of acetone in mass-rearing facilities can be difficult. Methoprene application not only increases male mating success, but may also affect post-copulatory male ability to modulate female refractoriness. However, in A. ludens 6-day-old males fed with diet with methoprene had similar ability to inhibit female remating as mature males (Gómez-Simuta et al., Reference Gómez-Simuta, Diaz-Fleischer, Arredondo, Díaz-Santiz and Pérez-Staples2017; Reyes-Hernández et al., Reference Reyes-Hernández, Macías-Díaz del Castillo, Abraham, Arredondo and Pérez-Staples2021). Similarly, topical application of methoprene in Z. cucurbitae or incorporation of methoprene into the male adult diet in B. tryoni did not affect the ability of young males to inhibit female remating (Haq et al., Reference Haq, Vreysen, Teal and Hendrichs2014; Adnan et al., Reference Adnan, Farhana, Rempoulakis and Taylor2020b). In A. fraterculus, topical applications in adult males and pupal immersion in methoprene diluted in acetone accelerated male sexual behavior, with 6-day-old males having similar mating success compared to mature males (Liendo et al., Reference Liendo, Devescovi, Bachmann, Utgés, Abraham, Vera and Teal2013; Segura et al., Reference Segura, Utgés, Liendo, Rodríguez, Devescovi, Vera and Cladera2013). However, importantly, those males were less able to inhibit female remating than mature males, although sperm transferred and stored by females was not affected (Abraham et al., Reference Abraham, Liendo, Devescovi, Peralta, Yusef, Ruiz, Cladera, Vera and Segura2013).
The inhibition of female sexual receptivity in A. fraterculus is mediated by the whole ejaculate composed of sperm and accessory gland proteins (Abraham et al., Reference Abraham, Lara-Pérez, Rodríguez, Contreras-Navarro, Nuñez-Beverido, Ovrusky and Pérez-Staples2016). JH accelerates the maturation of male accessory glands in insects (Chen, Reference Chen1984; Couche et al., Reference Couche, Gillott, Tobe and Feyereisen1985; Regis et al., Reference Regis, Gomes and Furtado1985; Gold and Davey, Reference Gold and Davey1989) and has a central role in regulating protein synthesis in these glands (Yamamoto et al., Reference Yamamoto, Chadarevian and Pellegrini1988). For example, in Aedes aegypti mosquitoes, JH-treated males have more protein in their accessory glands compared to untreated males (Fernández and Klowden, Reference Fernández and Klowden1995). Similarly, in the beetle Tribolium castaneum, males treated with hydroprene (another JH analog), have more protein content in their accessory glands and have bigger accessory glands (Parthasarathy et al., Reference Parthasarathy, Tan, Sun, Chen, Rankin and Palli2009). The positive correlation between size of the gland and quantity of secretions was also corroborated in the nasuta subgroup of Drosophila (Ram and Ramesh, Reference Ram and Ramesh2002), and males of Drosophila melanogaster with bigger accessory glands produced and transferred a higher amount of sex peptide, a key seminal component responsible of inhibiting female sexual receptivity in this species (Wigby et al., Reference Wigby, Sirot, Linklater, Buehner, Calboli, Bretman, Wolfner and Chapman2009). However, the effects of methoprene on male reproductive tissues and its possible consequences on female post-copulatory behaviors still warrants further studies.
Within the Tephritidae family, in A. ludens the incorporation of methoprene and protein to the adult diet resulted in bigger male accessory glands (Reyes-Hernández et al., Reference Reyes-Hernández, Macías-Díaz del Castillo, Abraham, Arredondo and Pérez-Staples2021). However in B. tryoni, the addition of methoprene to the protein diet accelerated the development of testes and ejaculatory apodeme but did not increase male accessory gland size (Adnan et al., Reference Adnan, Pérez-Staples and Taylor2020a). Compared to other tephritid species studied to date, only in A. fraterculus there has been a negative effect of topically applied methoprene on female remating behavior, thus we were interested in further elucidating the effect of methoprene applied in the diet on A. fraterculus adult male reproductive organs and female remating behavior. We predicted that males treated with methoprene would have smaller accessory glands and testes than untreated males of the same nutritional status. Likewise, less ability of these treated males in suppressing female remating was expected, based on previous information in this species.
Materials and methods
Source and treatment of flies
Adults were obtained from a laboratory colony established at the LIEMEN-PROIMI, Tucumán, Argentina. Rearing followed methods described by Jaldo et al. (Reference Jaldo, Gramajo and Willink2001) and Vera et al. (Reference Vera, Abraham, Oviedo and Willink2007). On the day of emergence, laboratory flies were sorted by sex and were transferred to 12 liter plastic containers in groups of 50 adults, with water and food provided ad libitum. Females were fed with adult diet consisting of sugar (57.9%) (Ledesma S.A., Jujuy, Argentina), hydrolyzed yeast (14.5%) (Yeast Hydrolyzed Enzymatic, MP Biomedicals®), hydrolyzed corn (27.3%) (Gluten Meal, ARCOR®, Tucumán, Argentina), and vitamin E (0.3%) (Parafarm®, Buenos Aires, Argentina) (w/w) (Jaldo et al., Reference Jaldo, Gramajo and Willink2001), hereafter named ‘complete diet = CD’. Females were 12 ± 2 days old in all experiments.
Male diet and age varied according to the trials. In trial 1 (diet at 0.01%), 10 g of CD was mixed with 10 ml of methoprene solution at 0.01% (1 μl of methoprene ‘Agrisent’ in 10 ml of distilled water). For control untreated males, 10 g of CD was mixed with 10 ml of distilled water. Diet was mixed, homogenized, placed in Petri dishes and covered with a disk of absorbent paper. Treated and untreated males of 6 and 12 days old were used. Two repetitions of trial 1 were carried out. In trial 2 (diets at 0.01, 0.02, 0.05, and 0.1%), diets were prepared as in trial 1, but four doses of methoprene were evaluated: 0.01, 0.02, 0.05, and 0.1%. Six-day-old treated males were evaluated, and 6- and 12-day-old untreated males were used as control. Three repetitions of trial 2 were carried out. In trial 3, methoprene was placed directly in the diet. For example, 1 μl of methoprene was placed in 10 g of CD and homogenized with an entomological pin for 60 s to obtain a methoprene diet at 0.01%. Four doses were evaluated: 0.01, 0.1, 0.5, and 1%, using 6-day-old treated males, and 6- and 12-day-old untreated males were used as control. Two repetitions of trial 3 were carried out. Diets were prepared, placed in the cages, and not replaced until the beginning of the experiment, and methoprene males were maintained in a separate room to avoid interference with untreated control males.
Accessory gland and testes size: general procedure
At 5, 6, and 12 days after emergence, 20 virgin treated and untreated males were sacrificed in cold. For treated males, methoprene at 0.1% directly applied to the diet without dissolving in water was used. Males were dissected under a stereomicroscope (Leica EZ4D) with fine forceps to extract accessory glands and testes. Organs were measured by taking a photograph using Leica application Suite EZ software, connected to a stereomicroscope (Leica EZ4D). The two long mesodermal glands were measured and the area was calculated averaging the area of these two measures. Similarly, for testes the area was calculated averaging the area of the two testicles. Additionally, head width was measured to be used as a co-variable. Organ area was determined by taking a photograph of 1040 × 770 pixels at a 3.2× magnification. Images were analyzed with ImageJ Version 1.50 software (3/26/16). For calibration, a photograph of a microscopy rule was taken using the same specifications used for measuring glands (1040 × 770 pixels at 3.2×). The number of pixels in a millimeter was measured (result: 1 mm = 933 pixels).
Male mating success and female remating
At 8:00 am two males from each of the different treatments described above (trials 1–3) were placed in individual 500 cm3 containers containing cotton soaked with water and sugar. That is, treated males did not compete against control males (‘no choice’ scenario). One female of 12 (±2) days of age was placed in each of the containers at 8:30–9:00 am. The number of copulations was recorded for each treatment. The unsuccessful males were removed from the containers to avoid disturbing copulating pairs. Copulas were registered until approximately 11:00 am. After the mating pairs separated, the males were discarded, and the females were kept in individual containers, alone, with cotton soaked with water and sugar. The females were labeled with the type of male with which they had copulated. The cotton with water and sugar were moistened daily until the evaluation of female remating.
Two days after the copulations were obtained, two untreated males of 14 (±2) days of age were placed at 8:00 am with each female, and the number of remating females were recorded. Cotton was discarded from each vial. The number of remating females was registered until approximately 11:00 am.
Data analysis
Accessory gland and testes size were compared using a two-way analysis of variance (ANOVA) where male age (5, 6, or 12 days old), methoprene treatment (with/without methoprene), and the interaction between factors were the class variables, while head width was used as co-variable. Male mating success and female remating were compared within each trial (trial 1, 2, 3) by one-way ANOVA using male treatment as class variable. Post-hoc comparisons were carried out with Tukey's test. The normality assumption was analyzed with a Shapiro–Wilks test. A Kruskal–Wallis test was used when ANOVA assumptions were not achieved. Data were analyzed using InfoStat (2009) software (free version).
Results
Accessory gland and testes size
Male age and methoprene treatment affected testes size (male age F = 17.25; d.f. = 2113; P < 0.001, methoprene treatment F = 5.40; d.f. = 1113; P = 0.022), while the interaction between them was not statistically significant (F = 0.23; d.f. = 2113; P = 0.793). Testes size of 5 and 6 days old were similar and smaller than that of 12-day-old males. Testes size of methoprene males was bigger than that of untreated males (fig. 1). Similarly, male age and methoprene treatment affected accessory gland size (male age F = 159.66; d.f. = 2113; P < 0.001, methoprene treatment F = 6.34; d.f. = 1113; P = 0.013), while the interaction between them was not statistically significant (F = 1.6; d.f. = 2113; P = 0.206). Twelve-day-old males had the largest accessory gland size, while those of 5 days the smallest, and those of 6 days an intermediate size. Accessory gland size of methoprene-fed males was bigger than that of untreated males (fig. 2). The co-variable was not statistically significant both for testes and male accessory gland size (testes F = 0.34; d.f. = 1113; P = 0.560; male accessory glands F = 0.0001; d.f. = 1113; P = 0.991).
Figure 1. Testes size (area, mean ± SE) of virgin A. fraterculus males of different ages treated with methoprene or untreated (control). Different uppercase letters indicate significant differences between ages and different lowercase letters indicate differences between treated or untreated males (two-way ANOVA, P < 0.05).
Figure 2. Male accessory gland size (area, mean ± SE) of virgin A. fraterculus males of different ages treated with methoprene or untreated (control). Different uppercase letters indicate significant differences between ages and different lowercase letters indicate differences between treated or untreated males (two-way ANOVA, P < 0.05).
Male mating success and female remating
Trial 1 (0.01% methoprene diet applied in solution)
Male mating success was affected by male treatment (fig. 3, F = 41.99; d.f. = 3, 4; P = 0.002). Six-day-old methoprene-treated males had higher mating success than untreated males of the same age and similar mating success than 12-day-old males (treated and untreated). Female remating was not affected by the dietary treatment of the first male (fig. 3; F = 0.62; d.f. = 3, 4; P = 0.640).
Figure 3. Trial 1. Percentage of male mating success (mean + SE) and female remating (mean + SE) at 48 h of first copulation of A. fraterculus females mated with untreated control males (6/12 days old) or methoprene-treated males (6/12 days old) at 0.01% (methoprene applied in solution). Numbers inside bars represent sample sizes. Different letters indicate significant differences (ANOVA, P < 0.05).
Trial 2 (four methoprene doses applied to the diet in solution)
Neither male mating success nor female remating were affected by male treatment (fig. 4, male mating success F = 1.20; d.f. = 5, 12; P = 0.364, female remating F = 0.86; d.f. = 5, 12; P = 0.533).
Figure 4. Trial 2. Percentage of male mating success (mean + SE) and female remating (mean + SE) at 48 h of first copulation of A. fraterculus females mated with untreated control males (6/12 days old) or 6-day-old methoprene-treated males at four different doses (0.1, 0.05, 0.02, and 0.01%, methoprene applied in solution). Numbers inside bars represent sample sizes. Different letters indicate significant differences (ANOVA, P > 0.05).
Trial 3 (direct placement of methoprene in diet)
There was no significant effect of incorporation of methoprene directly into the male diet on male mating success (fig. 5, F = 0.45; d.f. = 5, 6; P = 0.801). However, female remating was affected by the identity of the first male; females initially mated with males treated with methoprene at 1% had the highest remating frequency, while female remating was the lowest in females mated with 6-day-old untreated males (fig. 5, F = 15.32, d.f. = 5, 6; P = 0.002).
Figure 5. Trial 3. Percentage of male mating success (mean + SE) and female remating (mean + SE) at 48 h of first copulation of A. fraterculus females mated with untreated control males (6/12 days old) or 6-day-old methoprene-treated males at four different doses (1, 0.5, 0.1, and 0.01%, direct placement of methoprene in diet). Numbers inside bars represent sample sizes. Different letters indicate significant differences (ANOVA, P < 0.05).
Discussion
Effect of methoprene and male age on male reproductive organ size
As expected, mature males (12 days old) had bigger accessory glands and testes than younger immature males (5–6 days old). These organs were also affected by methoprene added to the protein diet: methoprene-treated males had bigger reproductive organs compared with untreated males. This same effect was observed in A. ludens for male accessory gland size (Reyes-Hernández et al., Reference Reyes-Hernández, Macías-Díaz del Castillo, Abraham, Arredondo and Pérez-Staples2021) and in B. tryoni for testes and ejaculatory apodeme size, but not male accessory glands (Adnan et al., Reference Adnan, Pérez-Staples and Taylor2020a). Similar to the present study, T. castaneum males fed with hydroprene showed an increase in male accessory gland size and had higher protein content in their accessory glands (Parthasarathy et al., Reference Parthasarathy, Tan, Sun, Chen, Rankin and Palli2009). Likewise, in A. aegypti, JH administered to starved males increased the levels of total accessory gland proteins in their accessory glands (Fernández and Klowden, Reference Fernández and Klowden1995). The positive correlation between accessory gland size and protein content has also been observed in several species of the nasuta subgroup of Drosophila (Ram and Ramesh, Reference Ram and Ramesh2002). In D. melanogaster, males with large accessory glands had significantly increased competitive reproductive success and also produced and transferred significantly more sex peptide, which is directly involved in inhibiting females from remating (Wigby et al., Reference Wigby, Sirot, Linklater, Buehner, Calboli, Bretman, Wolfner and Chapman2009). For A. fraterculus, we found that methoprene-treated males had bigger accessory glands, but the injection of homogenates of these glands from methoprene-treated males, albeit topically treated, were less effective at inhibiting female remating (Abraham et al., Reference Abraham, Cladera, Goane and Vera2012). This result suggests that male accessory gland size and their protein content may not be positively correlated in this species.
There is evidence in the literature that development of male internal reproductive structures is associated with the time to reach sexual maturity. For example, in several Drosophila species, Pitnick et al. (Reference Pitnick, Markow and Spicer1995) showed that the time required to reach sexual maturity is positively correlated with testes size, whereas in the stalk-eyed fly Cyrtodiopsis dalmanni, on the contrary, accessory gland size seems to be the critical determinant of sexual maturity and male mating success, as the accessory gland growth was more closely associated with the time taken to reach sexual maturity than testes growth (Baker et al., Reference Baker, Denniff, Futerman, Fowler, Pomiankowski and Chapman2003; Rogers et al., Reference Rogers, Chapman, Fowler and Poniankowski2005). Likewise, in A. ludens, male accessory gland size grew with age and was more closely related to sexual maturity than testes size (Reyes-Hernández and Pérez-Staples, Reference Reyes-Hernández and Pérez-Staples2017). Also, in B. tryoni male diet affects accessory gland size but not testes size (Vijaysegaran et al., Reference Vijaysegaran, Walter and Drew2002). These studies point out that male accessory gland size may be a good indicator of male sexual maturity and indeed mating success more than testes size (see, Bangham et al., Reference Bangham, Chapman and Partridge2002; Wigby et al., Reference Wigby, Sirot, Linklater, Buehner, Calboli, Bretman, Wolfner and Chapman2009). Nevertheless, male accessory gland and testes size seems to be a plastic trait that responds to factors, such as social cues of sperm competition, male diet, and age (e.g. Pérez-Staples et al., Reference Pérez-Staples, Weldon and Taylor2011). Males raised under high level of competition may evolve larger testes and sperm production (Schärer and Vizoso, Reference Schärer and Vizoso2007) or larger accessory glands (Crudgington et al., Reference Crudgington, Fellows, Badcock and Snook2009; Lemaître et al., Reference Lemaître, Ramm, Hurst and Stockley2011) than those that experienced a low level of competition.
Effect of methoprene treatment on male mating success and female remating
The effect of methoprene on male mating success has been studied over the past decade in different tephritids. While in B. dorsalis and C. capitata the effect of the topical application of methoprene dissolved in acetone did not produce a significant acceleration in male maturation (Shelly et al., Reference Shelly, Nishimoto and Edu2009), whereas in Bactrocera cucurbitae, topical application of methoprene in protein-fed males greatly accelerated their sexual maturity, showing a higher percentage of copulations at younger ages (5 days old) compared to males without methoprene or protein, which copulated at 9 days old (Haq et al., Reference Haq, Caceres, Hendrichs, Teal, Wornoayporn, Stauffer and Robinson2010). In A. obliqua, males with access to protein and methoprene in the diet reduced the time required to reach sexual maturity in approximately 2 days (Muñoz-Barrios et al., Reference Muñoz-Barrios, Cruz-López, Rojas, Hernández, Liedo, Gómez-Simuta and Malo2016). In A. ludens, 6-day-old males with access to protein and methoprene in the adult diet had similar mating success to that of mature 13-day-old untreated males (Gómez-Simuta et al., Reference Gómez-Simuta, Diaz-Fleischer, Arredondo, Díaz-Santiz and Pérez-Staples2017). In B. tryoni young methoprene-treated males were more competitive than untreated males (Adnan et al., Reference Adnan, Mendez, Morelli, Akter, Farhana and Taylor2018), and even under field cage conditions, young treated males were more competitive than mature wild and lab males (Adnan et al., Reference Adnan, Farhana, Inskeep, Rempoulakis and Taylor2020c). The effect of methoprene treatment on male sexual maturation in A. fraterculus was previously evaluated but using methoprene dissolved in acetone and applied topically in the thorax of newly emerged males and by pupal immersion in methoprene. Using such methods, the sexual maturity period was shortened 2–3 days compared with untreated males in this species (Segura et al., Reference Segura, Utgés, Liendo, Rodríguez, Devescovi, Vera and Cladera2013). In our study we demonstrated that it is possible to reduce sexual maturation of males by feeding them with a methoprene diet, suggesting that doses as low as 0.01% accelerate sexual maturity, with males being competitive at 6 days of age. However, this method of methoprene incorporation seems to be dependent on environmental conditions, such as relative humidity, which could affect diet consistency and therefore the amount of diet ingested by males. More studies in A. fraterculus are needed to clarify the specific conditions necessary for the optimal supplementation of methoprene in the adult diet.
The use of methoprene to accelerate sexual maturation has shown that treated males are ready to perform calling behavior, emit pheromones, and obtain copulations, increasing their pre-copulatory success. However, this does not imply that the post-copulatory performance of the male is also maximized. Methoprene treatment should not only increase male sexual development and mating success but also the ability to transfer an adequate ejaculate in quality and quantity, directly impacting female remating. In A. fraterculus, females mated with 6-day-old sterile males treated with topical application of methoprene or by pupae immersion exhibited shorter copula durations, higher percentages of remating and shorter refractory periods in comparison with females mated with untreated males (Abraham et al., Reference Abraham, Liendo, Devescovi, Peralta, Yusef, Ruiz, Cladera, Vera and Segura2013). Also, when females were injected with the male accessory glands of methoprene-treated males, they again had higher remating probabilities (Abraham et al., Reference Abraham, Cladera, Goane and Vera2012). Here we show that indeed if males are fed a diet with the direct incorporation of methoprene at 1% then they have a lower ability to inhibit female remating. This suggests the need of further studies for the effects of methoprene on the male ejaculate and female behavior at higher doses. However, in the context of the SIT, incorporating methoprene into the diet at lower doses than 1% yielded young males with higher mating success and increased ability to inhibit females from remating. Furthermore, the percentage of females remating after 48 h from the first copulation are sufficiently low in this species so as not compromise the efficiency of the SIT. Other studies have also found that methoprene treatment topically or added to male adult protein diet had no detrimental effect on male ability to inhibit females remating (Haq et al., Reference Haq, Caceres, Hendrichs, Teal, Wornoayporn, Stauffer and Robinson2010; Gómez-Simuta et al., Reference Gómez-Simuta, Diaz-Fleischer, Arredondo, Díaz-Santiz and Pérez-Staples2017; Adnan et al., Reference Adnan, Farhana, Rempoulakis and Taylor2020b; Reyes-Hernández et al., Reference Reyes-Hernández, Macías-Díaz del Castillo, Abraham, Arredondo and Pérez-Staples2021).
We found that methoprene effect on the adult diet was not dose-dependent as was found previously by Segura et al. (Reference Segura, Utgés, Liendo, Rodríguez, Devescovi, Vera and Cladera2013). Methoprene-treated males were able to inhibit female remating in most cases (trials 1 and 2), except when females were mated with males treated with high doses of methoprene (trial 3). Differences in studies could be due to the methods used for methoprene incorporation into the diet, which was liquid for trials 1 and 2 and more solid for trial 3. This could have affected male capacity to ingest the contents of the diet. If the water content of the diet evaporates and dries too much, males can no longer consume it. Thus, the amount of diet ingested by the males may depend on the relative humidity of the environment. Trial 1 was carried out during the summer months, when the relative humidity is higher than in the winter months during which trials 2 and 3 were carried out. Drier winter conditions may have reduced male ability to ingest a sufficient amount of nutrients and methoprene to accelerate and/or enhance their sexual maturity. Tests where the relative humidity of the environment is controlled by placing the cages in chambers with humidity control would help to discern the effect of this variable on water evaporation of the diet, its consistency and the consequent ability of the flies to feed on such diets. Nevertheless, the positive effects of methoprene incorporation into the male diet at 0.01% on male accessory gland size, testes size, mating success and ability to inhibit female remating suggests that this method could potentially be used in an SIT program for A. fraterculus.
Acknowledgements
We acknowledge Compañía Argentina de Levaduras S.A. (CALSA®) for providing brewer's yeast and ARCOR® S.A. for providing corn protein for the diets. We thank Julio Dominguez (ECOSUR) for providing methoprene. Funding was partially provided by the International Atomic Energy Agency (Individual Research Contract 18463).
Conflict of interest
The authors declare none.
