The relevance of age and nutritional status on the mating competitiveness of medfly males ( Diptera : Teprhitidae )

Results of previous investigations trying to ascertain which physiological factors are more important to the mating success of medfly males are controversial. In part, this controversy owes to the fact that each factor was evaluated by an independent study using different experimental designs and populations. In the present study we compare the roles of age and nutritional status (immature and adult phases) on the mating competitiveness of Ceratitis capitata (Wiedemann, 1824) males. Three parameters were used to evaluate the male mating success: calling behavior (pheromone emission), lek participation and copulation (ability to be chosen by a female). Females gave preference to the males that were given a high protein diet in the larval phase. By contrast, females did not give preference to males that had been well-nourished in the adult phase only. The other parameters evaluated followed the same pattern: young males and males that had been fed a high protein diet during their immature phase had a greater participation in leks and called more often than older males and males that had been fed a diet poor in protein during their larval phase. Therefore, we conclude that the mating success of C. capitata males is determined both by age and nourishment during the immature stage.

The medfly, Ceratitis capitata (Wiedemann, 1824), is an important crop pest.These flies attack many species of fruits and vegetables of economic importance and cause enormous damage to fruit crops throughout the world (MALAVASI & ZUCCHI 2000).One important topic when studying economically important species such as the medfly is reproductive behavior.Ceratitis capitata mates in a lek (PROKOPY & HENDRICHS 1979, WHITTIER et al. 1992), defined as a polygyny system in which males aggregate and enclose a territory for the attraction of females without defending resources (ALCOCK 2011).
Leks of C. capitata contain from 2 to 10 males and generally form on the leaves of trees, where males call from, while releasing of a sex pheromone by raising the back of their abdomen and everting the membranous portion of their rectal epithelium.Calling is usually accompanied by rhythmic wing beats (FERON 1962, ARITA & KANESHIRO 1989, SIVINSKY et al. 2001), which are essential to attract the females to the leks (ALCOCK 2011).Inside the lek, males dispute preferential sites through agonistic behavior (EBERHARD 1999).When a female arrives, the male initiates a courtship display and then jumps on her in an attempt to copulate.Females evaluates males participating in a lek, and reject most of the courtship displays, leaving before or even after an undesirable male has jumped on her (WHITTIER et al. 1994, WHITTIER & KANESHIRO 1995).It is possible that there are some variables that influence the female's decision, for instance the distance and angle between male and female (BRICEÑO et al. 2007), and tactile stimulation from the male's arista (BRICEÑO & EBERHARD 2002).It is assumed that the variability of male mating success is determined by female choices (WHITTIER et al. 1994).Inside the lek, males that perform better in territory disputes and/or courtship behavior are more successful attracting females (NORRY et al. 1999).The better performance of some males with respect to others may reflect the conditions they were subjected to during the immature or adult phases.
Protein intake by C. capitata immatures is essential for larval development, emergence of large adults and egg production (CANGUSSU & ZUCOLOTO 1993, 1997, ANJOS-DUARTE et al. 2011a), because protein is an essential component of all body structures (SLANSKY & SCRIBER 1985).The effect of protein intake during the immature phase on the sexual success of males has not been directly studied.However several studies have linked the size of the adult to its reproductive success (BLAY & YUVAL 1997, TAYLOR & YUVAL 1999).
In order to determine which factors are involved in the mating success of males, it is fundamental to investigate why some males are successful in courting and others are not.Different studies have associated male mating success with protein ingestion (BLAY & YUVAL 1997, PAPADOPOULOS et al. 1998, YUVAL et al. 2002, MAOR et al. 2004), morphometric traits of the body (RODRIGUERO et al. 2002), youth (PAPADOPOULOS et al. 1998) and The relevance of age and nutritional status on the mating competitiveness of medfly males (Diptera: Teprhitidae) ABSTRACT.Results of previous investigations trying to ascertain which physiological factors are more important to the mating success of medfly males are controversial.In part, this controversy owes to the fact that each factor was evaluated by an independent study using different experimental designs and populations.In the present study we compare the roles of age and nutritional status (immature and adult phases) on the mating competitiveness of Ceratitis capitata (Wiedemann, 1824) males.Three parameters were used to evaluate the male mating success: calling behavior (pheromone emission), lek participation and copulation (ability to be chosen by a female).Females gave preference to the males that were given a high protein diet in the larval phase.By contrast, females did not give preference to males that had been well-nourished in the adult phase only.The other parameters evaluated followed the same pattern: young males and males that had been fed a high protein diet during their immature phase had a greater participation in leks and called more often than older males and males that had been fed a diet poor in protein during their larval phase.with certain mating activities such as pheromone emission, lek formation and courtship behavior (NORRY et al. 1999).However, other studies have failed to successfully associate any of these factors with male mating success (ARITA & KANESHIRO 1989, WHITTIER et al. 1994, KASPI & YUVAL 2000, SHELLY & KENNELLY 2002, SHELLY & MCINNIS 2003).It is possible that the conflicting results among various studies are due to the fact that different methodologies and and/or fly strains have been used in each, as pointed out by YUVAL et al. (2007).
Evaluating the variables that import on the mating success of C. capitata males is relevant to the success of the sterile insect technique (SIT) -a pest control method based on the release of large numbers of sterile males in the field to compete for females with wild type males (BRICEÑO et al. 2007).Large quantities of these insects are reared in bio-production facilities (HENDRICHS et al. 1995) and then released.In order to complete with wild males to be chosen by wild females, they must have desirable and adequate physiological and behavioral characteristics.
The present study used a standardized methodology to analyze the influence of age and nutrition of immatures and adults on the mating competitiveness of C. capitata males reared in the same laboratory.

MATERIAL AND METHODS
Flies employed in the experiments were brought from a laboratory lineage with frequent introductions of wild flies (hybrid lineage).This colony had been reared for 12 years at the Laboratório de Ecologia Nutricional de Insetos (LENI) of the Departamento de Biologia Geral do Instituto de Biologia at Universidade Federal da Bahia according to the methodology described by ZUCOLOTO (1987).Males in the immature and adult phases were divided into distinct categories according age and nutritional status for the different experiments (Table I).
In the experiment testing the role of age, we used flies fed a high-protein diet (with 6.5 g of brewer's yeast as protein source) during the immature and adult phases.In the tests on the effects of nutrition during the immature stage, larvae received diets contained 6.5 g (high protein) or 3.0 g (low protein) of brewer's yeast (protein source).To evaluate the influence of adult nutrition, newly-emerged flies received diets contained 6.5 g of brewer's yeast (protein source), or a diet without this component for 4-5 days.The diet given to all larvae contained sucrose (11.0 g), citric acid (1.0 g), nipagin (1.0 mL), and distilled water (100.0mL).All adults also received water and a saturated sugar solution on a daily basis.Adults used in nutritional status experiments had 4-5 days old.All males and females were kept separately in plastic cages (16x11x10 cm) from emergence to the age required for the studies.
The mate choice tests were performed at the Ondina campus of the Universidade Federal da Bahia, Salvador, state of Bahia, Brazil.Fifty "young" females (4-5 days old), which had been well-nourished in the immature and adult phases (diets with high protein levels), were placed into a screened field cage (2.30x 1.50x2.30m) with 50 males from each age or nutritional groups confronted: (YM) young males, (OM) old males, (HI) high protein diet in immature phase, (LI) low protein diet in immature phase, (PA) protein diet in adult phase, (WA) without protein diet in adult phase.The cages contained a "pitanga" tree (Eugenia uniflora Linnaeus, 107 cm tall and with crown diameters of 40 cm) on a wooden, 60 cm high table.Pieces of apple (Malus domestica Linnaeus) were placed on the extremities of some of the tree branches to simulate food resources and oviposition sites.The males from each age or nutritional groups were marked (on their thorax) with different colors (green or blue) using non-toxic paint for identification.The colors were alternated with each experimental repetition.In each experiment, behavioral observations were performed in the morning for a period of 3 hours (starting time 08:00), quantifying copulations and identifying the mated males.After the removal of the copulating pair, a male of the opposite group was also removed from the cage to maintain equal proportions of males from the different categories inside the cage during the experiment (SILVA- NETO et al. 2009).
The copulating couples (as well the males and the females that did not copulate) were captured in test-tubes.Five replicates of each experiment were made.The average temperature during the field experiments was 30 ± 1°C and relative humidity was 80-85%.
The parameters, participation in lek formation and calling behavior, were evaluated in a laboratory cage (0.68x0.68x 0.90 m) containing a small potted "pitanga" (Eugenia uniflora Linneus) tree, 60 cm tall and 40 cm radius.Pieces of apple (Malus domestica Borkhausen) were placed on the tree branches.The experiments were performed in the morning hours (starting time 08:00 h) under controlled laboratory conditions (temperature 25 ± 1°C, and 70% relative humidity).
In the tests of lek formation, 20 HI and 20 LI males, previously marked (green or blue), were released into the laboratory cage and observed for 90 minutes, indentifying the lek formations.The same methodology was applied to evaluate the variables age and adult nutrition.When a lek (a grouping of at least two males on the abaxial face of neighboring leaves, according with ARITA & KANESHIRO (1989) formed, we observed it for one entire minute and then the males involved were collected for later identification.Each experiment had five replicates.
The calling behavior tests followed the same general methodology described above.After the males were released into the cage, we made continuous observations on their pheromone emission for one hour.When pheromone emission was observed (eversion of anal epithelium with the liberation of drop of the hormone) that male was collected for identification.Five replicates were conducted for each test.
We took biometric measurements of half of the males in each experimental procedure, in order to determine body size (according to the methodology described by ZUCOLOTO 1987) and to compare it among males from different test groups.The non-parametric T test was employed to analyze the data, using the GraphPad InStat version 3.00 (GraphPad Software, San Diego California USA).The data was tested for normality using the Kolmogorov and Smirnov test and analyzed for variance homogeneity.When homogeneity of variance was not detected in the groups being compared, we applied the Welch correction to the data; ␣ = 5% was assumed in all analyses.

RESULTS
General observations during mate choice tests indicated that most males position themselves on the underside of leaves to release pheromones and to perform their courtship rituals.Most of the copulations occurred on the abaxial surface of a leaf, where lek formation was observed.Only a single male in the lek occupied the leaf at a given time, and when another male landed there, both engaged in territorial disputes.
An assessment of the size of males showed that all males that were given a high protein diet in the immature stage, and which were used to test the effects of age and nutritional status on the adult had a similar size: age group (µ = 2.7, = 0.13, p = 0.55) and adult nutrition group (µ = 2.86, = 0.68, p = 0.49).However, males given distinct protein concentrations in the immature stage (HI and LI) differed in size: the HI were larger (µ = 2.9, = 0.74) than the LI ( = 2.47, = 0.10) (T test, ␣ = 0.05, p <0.001).

DISCUSSION
Our results indicate that, in C. capitata, young adults and adults that had access to more protein in the immature stage have a greater chance of mating.
Viability indicator models of age-related mate choice predict that females prefer older males because they have already proven that they are able to survive and are able to convey that information in their sexual display (PROULX et al. 2002).However, in the present study, the number of old males that emitted pheromone, participated in lek formation and succeeded in mating was lower than the number of young males engaged in those activities.Thus, the hypothesis that females prefer older males despite their reduced fertilization success or viability of their offspring (JONES 2000, JONES & ELGAR 2004) does not seem to apply to C. capitata, at least when there is a choice between young and old males.The data obtained by us is in agreement with other recent studies on different populations of C. capitata (SILVA-NETO et al. 2009, SHELLY et al. 2011, PAPANASTASIOU et al. 2011).Working with the same population used in this study, ANJOS-DUARTE et al. (2011b) had found that The relevance of age and nutritional status on the mating competitiveness of medfly males ZOOLOGIA 30 (5): 506-512, October, 2013 females did not distinguish between males of 4 and 12 days of age and accepted to mate equally with both; however, they preferred four or 12 days-old males than 20 days-old ones.Male ageing may affect their courtship ability and/or pheromone production and females apparently are capable of detecting it.For example, longer courtship, as found in 4 day-old males compared to a 20 day-old male in the study of ANJOS-DUARTE et al. (2011b), are energetically more costly and may signal to the female that the male is vigorous.
The results of studies on the influence of body size on sexual performance have not been consistent.Different studies have demonstrated that larger males have advantages in terms of sexual performances (CHURCHILL-STANLAND et al. 1986, BLAY & YUVAL 1997, TAYLOR & YUVAL 1999), whereas others have failed to do so (WHITTIER et al. 1994, SHELLY & KENNELLY 2002).
According KASPI et al. (2002) the use of diets with different concentrations of protein and sucrose during the larval stage import on the emission of pheromone by males, but the authors did not investigate the relationship between diet and size.In order to participate in leks, males must insert themselves into them and emit pheromones to attract females.The greater participation of larger males (HI) when compared to smaller (LI) males in these aggregations may be due to the fact that smaller individuals are less competent emitting pheromones and/or disputing territories.Territorial defense is essential to the mating success of medfly males (ARITA & KANESHIRO 1985).Males dispute territories through agonistic behaviors, using their heads to butt one another until one of them gives up and leaves the site (EBERHARD 1999); larger males (HI) presumably have an advantage during these territorial disputes.Even when large males were put in smaller proportions than the small ones inside a cage, females preferred to mate with the largest males (SILVA-NETO et al. 2012).In addition, females of different sizes (small and large) also preferred to mate with larger males (ANJOS-DUARTE et al. 2011a).
Protein ingestion by C. capitata males at the beginning of the adult phase has been positively correlated with their repro-ductive behavior in a number of studies (BLAY & YUVAL 1997, PAPADOPOULOS et al. 1998, TAYLOR & YUVAL 1999, KASPI & YUVAL 2000, SHELLY et al. 2002).PEREZ STAPLES et al. (2008) studied the addition of a protein source to the diet of Bactrocera tryoni (Froggatt, 1897) males 24 or 48 hours after emergence, finding that it was sufficient to increase the probability that the adult would mate, increase the number of sperm stored, the longevity of the flies and decrease mating latency and female re-mating.Furthermore, eating protein at the beginning of the adult phase has been associated with an improvement in the mating performance of sterile males of B. tryoni (PEREZ STAPLES et al. 2009).However, in the present study on C. capitata, we did not find any evidence that the adult diet influences male pheromone emission, lek formation or mating success, corroborating other studies (SHELLY & KENNELLY 2002, SHELLY & MCINNIS 2003, JOACHIM-BRAVO et al. 2009).The observed similarities in the behavior of males that ingested brewer's yeast in the initial phase of their adult lives and those that did not have access to this protein source may be explained by the fact that they were both already metabolizing protein reserves acquired in their larval phase.
Many holometabolic insects use food resources derived from their larval diets to supplement nutritional deficiencies encountered in their adult phase (BOGGS 1981, KARLSSON 1995).JOACHIM-BRAVO et al. (2009) demonstrated that protein intake by C. capitata in the first four days of life did not contribute to increase the mating success of males when compared with those fed on a no-protein diet.Ceratitis capitata males with access to protein resources also emitted more sexual signals and copulated more than males that did not consume proteins (KASPI et al. 2002), which suggests that the proteins may be indispensable to the synthesis of sexual pheromones (BLAY & YUVAL 1997).In the study of COSTA et al. (2012), males in the adult phase which were fed protein produced and transferred more sperm to females.In Anastrepha fraterculus (Wiedmann, 1830), the amount of sperm stored in wild females was lower than the amount stored in laboratory females, even though both mated with males fed the same amount of protein as adults (ABRAHAM Figures 1-3.Influence of factors, age (1), feeding on the immature phase (2) and feeding in adulthood (3) in the reproductive success of male Ceratitis capitata, assessed by the parameters: the calling behavior (pheromone emission), participation and success in lek mating.The data represent the means and standard deviations of five replicates (experimental cages) for each independent factor and parameter evaluated.Asterisks represent statistical differences between the bars corresponding to the same parameter (T test with Welch correction p < 0.05), (YM) young males, (OM) old males, (HI) high protein diet in immature phase, (LI) low protein diet in immature phase, (PA) protein diet in adult phase, (WA) without protein diet in adult phase.
1 2 ZOOLOGIA 30 (5): 506-512, October, 2013506-512, October, et al. 2012)).Flies originating from laboratory rearing receive, during the immature phase, a high-protein diet (PEREIRA et al. 2009(PEREIRA et al. , 2010)), and the resulting adults emerge with a high energy reserve, which is consumed throughout the flies' life (SLANSKY & SCRIBER 1985).Thus, the protein requirement in early adulthood may be greater if the larval diet is deficient in protein.In the present study, the influence of the nutritional status of the adults was tested with well-nourished flies in the immature phase, which may explain the similarity in the performance of males fed protein or no-protein diets as adults.
The synergistic effects of both nutrition and age cannot be forgotten.Lipid reserves, for example, acquired during the immature phase of C. capitata males are consumed (at least in part) during their sexual activities in the adult phase (WARBURG & YUVAL 1996).Therefore, it is logical to assume that there is a decline in the energy reserves, which is associated with aging, and that can affect male competitiveness.This decline, however, may vary according to the nutrition of the immature and the adult.Comparisons of protein intake during early and middle adult life of lab-reared C. capitata showed that, despite the fact that protein intake during the four first days of the adult life does not import on male mating success, when the period of protein intake is extended to 12 days, males given a protein diet had greater mating success with females and calling than males fed on a no-protein diet at the same period (SILVA-NETO et al. 2009).However, being a young male and having been well-nourished in the immature stage are the most important factors in the competition for mating according the present study.
Situations involving competition for females between males of distinct sizes, ages or nutritional histories, presumably occur under natural conditions with wild males.As such, the mass-rearing and release of young and sexually mature males with standardized sizes that are even larger than those encountered in field and that can compete successfully with wild-type males will improve the effectiveness of the sterile insect techniques (SIT) now in use.

Table I .
Categories and condition of males used in different experiments of mating success.