Methoprene treatment and its effect on male reproductive organ size and female remating in a fruit fly

Abstract

Methoprene, a juvenile hormone analog, is used to accelerate sexual maturation in males of species of economic importance in support to the sterile insect technique (SIT). In the SIT, mass-reared sterile males are released into the field and need to survive until they reach sexual maturation, find a wild female, mate with her and then induce female sexual refractoriness, so she will not remate with a wild counterpart. The use of methoprene shortens the time between release and copulation. However, in South American fruit flies, Anastrepha fraterculus, the ability of methoprene-treated males to inhibit female remating has been shown to be lower than wild males, when methoprene was applied by pupal immersion or topical application. Here we evaluated the possibility of incorporating methoprene into the male diet at different doses and the ability of those males to inhibit female remating, as well as the effect of methoprene on male reproductive organ size, due to the possible correlation between male accessory gland size and their content, and the role of male accessory gland proteins in female inhibition. We found that A. fraterculus males fed with methoprene in the adult protein diet at doses as high as 1% were less likely to inhibit female remating, however, at all other lower doses males had the same ability as untreated males to inhibit female remating. Males fed with methoprene had bigger male accessory glands and testes compared to methoprene-deprived males. We demonstrate that the incorporation of methoprene in adult male diets is possible in this species and potentially useful as a post-teneral, pre-release supplement at doses as low as 0.01%. Even at higher doses, the percentage of females remating after 48 h from the first copulation is sufficiently low in this species so as not compromise the efficiency of the SIT.

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, 1955). 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., 2015) and the oriental fruit fly Bactrocera dorsalis (Vargas et al., 2010).

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, 1995; Malavasi et al., 2000). Currently, SIT has been proposed for A. fraterculus as a feasible strategy for area-wide control of this pest (Ortíz, 1999), 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., 2001; Allinghi et al., 2007; Segura et al., 2007, 2013; Vera et al., 2007, 2014; Abraham et al., 2013; Liendo et al., 2013).

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., 2007). 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., 2002; Enkerlin, 2007; Díaz-Fleischer et al., 2009; Andress et al., 2013). 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., 2000, 2013; Muñoz-Barrios et al., 2016; Gómez-Simuta et al., 2017). 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., 2013; Akter and Taylor, 2018).

Physiological processes in insects, such as reproduction and metamorphosis, are regulated, in part, by JH (Gilbert et al., 2000; Dubrovsky, 2005). In tephritid fruit flies of economic importance the application of methoprene accelerates male sexual maturation in Anastrepha obliqua (Teal et al., 2007), Anastrepha suspensa (Teal et al., 2000; Teal and Gómez-Simuta, 2002; Pereira et al., 2009), Anastrepha ludens (Gómez-Simuta et al., 2013), Zeugodacus cucurbitae (Haq et al., 2010), and Bactrocera tryoni (Collins et al., 2014).

Efforts have recently been made to apply methoprene into the adult male diet (Muñoz-Barrios et al., 2016; Gómez-Simuta et al., 2017; Adnan et al., 2018, 2020c; Pereira et al., 2021; Reyes-Hernández et al., 2021), 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., 2017; Reyes-Hernández et al., 2021). 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., 2014; Adnan et al., 2020b). 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., 2013; Segura et al., 2013). 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., 2013).

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., 2016). JH accelerates the maturation of male accessory glands in insects (Chen, 1984; Couche et al., 1985; Regis et al., 1985; Gold and Davey, 1989) and has a central role in regulating protein synthesis in these glands (Yamamoto et al., 1988). For example, in Aedes aegypti mosquitoes, JH-treated males have more protein in their accessory glands compared to untreated males (Fernández and Klowden, 1995). 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., 2009). The positive correlation between size of the gland and quantity of secretions was also corroborated in the nasuta subgroup of Drosophila (Ram and Ramesh, 2002), 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., 2009). 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., 2021). 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., 2020a). 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. (2001) and Vera et al. (2007). 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., 2001), 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., 2021) and in B. tryoni for testes and ejaculatory apodeme size, but not male accessory glands (Adnan et al., 2020a). 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., 2009). Likewise, in A. aegypti, JH administered to starved males increased the levels of total accessory gland proteins in their accessory glands (Fernández and Klowden, 1995). 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, 2002). 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., 2009). 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., 2012). 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. (1995) 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., 2003; Rogers et al., 2005). 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, 2017). Also, in B. tryoni male diet affects accessory gland size but not testes size (Vijaysegaran et al., 2002). 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., 2002; Wigby et al., 2009). 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., 2011). Males raised under high level of competition may evolve larger testes and sperm production (Schärer and Vizoso, 2007) or larger accessory glands (Crudgington et al., 2009; Lemaître et al., 2011) 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., 2009), 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., 2010). 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., 2016). 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., 2017). In B. tryoni young methoprene-treated males were more competitive than untreated males (Adnan et al., 2018), and even under field cage conditions, young treated males were more competitive than mature wild and lab males (Adnan et al., 2020c). 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., 2013). 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., 2013). Also, when females were injected with the male accessory glands of methoprene-treated males, they again had higher remating probabilities (Abraham et al., 2012). 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., 2010; Gómez-Simuta et al., 2017; Adnan et al., 2020b; Reyes-Hernández et al., 2021).

We found that methoprene effect on the adult diet was not dose-dependent as was found previously by Segura et al. (2013). 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.