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Journal of Insect Physiology 56 (2010) 1807–1815

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Journal of Insect Physiology

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Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon ﬂy Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast

Ihsan ul Haq a,b,*, Leopold Mayr c, P.E.A. Teal d, Jorge Hendrichs e, Alan S. Robinson a, Christian Stauffer f, Rebecca Hood-Nowotny a a Insect Pest Control Laboratory, Joint FAO/IAEA Agriculture and Biotechnology Laboratories, A-2444 Seibersdorf, Austria b National Agricultural Research Centre, Park Road, Islamabad 4500, Pakistan c Soil and Water Management and Crop Nutrition Laboratory, Joint FAO/IAEA Agriculture and Biotechnology Laboratories, A-2444 Seibersdorf, Austria d Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, Gainesville, FL 32604, USA e Insect Pest Control Section, Joint FAO/IAEA Division, IAEA, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria f Institute of Forest Entomology, Forest Pathology & Forest Protection, BOKU, Vienna, Austria

ARTICLE INFO ABSTRACT

Article history: The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments Received 18 March 2010 known to enhance mating success in the male melon fly Bactrocera cucurbitae Coquillett (Diptera: Received in revised form 19 July 2010 Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile Accepted 21 July 2010 insect technique (SIT) programmes. The objective of the present laboratory study was to investigate the effect of methoprene application and diet supplementation with hydrolyzed yeast (protein) on the Keywords: turnover of body lipids and protein to confirm the feasibility of their application in melon fly SIT mass- Bactrocera cucurbitae rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males Isotope 15N were exposed to one of the following treatments: (1) topical application of methoprene and access to diet Methoprene Hydrolyzed yeast including protein (M+P+); (2) only diet including protein (MP+); (3) only methoprene (M+P) and (4) SIT untreated, only sugar-fed, control males (MP). Total body carbon (TBC) and total body nitrogen (TBN) Total body carbon of flies were measured at regular intervals from emergence to 35 days of age for each of the different Total body nitrogen treatments. Nitrogen assimilation and turnover in the flies were measured using stable isotope (15N) dilution techniques. Hydrolyzed yeast incorporation into the diet significantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had significantly higher body weight, TBC and TBN as compared to all males. TBC and TBN showed age-dependent changes, increasing until the age of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly affect TBC or TBN. The progressive increase with age of TBC suggests that lipogenesis occurs in adult male B. cucurbitae, as is the case in other tephritids. Stable isotope dilution was shown to be an effective method for determining N uptake in B. cucurbitae. This technique was used to show that sugar-fed males rely solely on larval N reserves and that the N uptake rate in males with access to diet including hydrolyzed yeast was higher shortly after emergence and then stabilized. The implications of the results for SIT applications are discussed. ß 2010 Elsevier Ltd. All rights reserved.

1. Introduction adult tephritids was by Hagen (1953), who found that both sexes of Bactrocera cucurbitae, Bactrocera dorsalis and Ceratitis Many insects in their adult stage are anautogenous, requiring capitata required carbohydrates, protein in the form of free carbohydrates, proteins and lipids to perform biological activi- amino acids, minerals, B-complex vitamins, and water. Sucrose ties necessary for survival and reproduction (Chapman, 1982). is needed to fuel daily foraging, flight and courtship activities The first study on the complete nutritional requirements of and is essential for survival, but alone it does not satisfy the nutritional requirements of the flies, and protein ingestion is crucial for egg production in females (Christenson and Foote,

* Corresponding author at: Insect Pest Control Laboratory, Joint FAO/IAEA 1960; Bateman, 1972; Sharp and Chambers, 1984; Hendrichs Agriculture and Biotechnology Laboratories, A-2444 Seibersdorf, Austria. et al., 1991; Cangussu and Zucoloto, 1995; Teal et al., 2004). The E-mail addresses: [email protected], [email protected] (I.u. Haq). role of dietary protein in modulating male mating success is well

1808 I. Haq et al. / Journal of Insect Physiology 56 (2010) 1807–1815 documented in tephritids (Blay and Yuval, 1997; Field and yeast reduced the pre-copulatory period and enhanced the mating Yuval, 1999; McInnis et al., 2004; Pe´ rez-Staples et al., 2007; success of B. cucurbitae. Application of methoprene and access to Shelly et al., 2007; Pereira et al., 2009, 2010). In the diet including hydrolyzed yeast also increased male participation Mediterranean fruit fly, C. capitata post-teneral feeding on in leks and pheromone calling (Haq et al., 2010a). These protein can contribute to male gonadal and accessory gland behavioural attributes are energetically costly and have adverse development (Yuval et al., 2002). The increased mating success consequences for energy reserves over the life time of the fly. In due to dietary protein may, however, have a cost for longevity addition to modulating mating behaviour, application of metho- (Cordts and Partridge, 1996). In C. capitata, while continuous prene is known to affect nutritional status and to alter resource access to dietary protein increased survival, males starved after allocation in other insects. For example, JH analogue treatment 4 days of feeding on protein were short-lived as compared to altered lipid metabolism and increased the mass of ovaries in males that had no access to protein (Kaspi and Yuval, 2000; female Gryllus firmus (Zera and Zhao, 2004), and increased the size Maor et al., 2004). However, Shelly and Kennelly (2002) of male accessory glands in Tribolium castaneum (Parthasarathy reported no adverse effect of protein diet on starvation survival. et al., 2009). Protein feeding in C. capitata males can also affect the re-mating In clinical nutrition studies it is well documented that body behaviour: females mated with protein-fed males in their first protein can be quantified from total body nitrogen (TBN) (Varttsky mating had less tendency to re-mate when compared to females et al., 1979) since 99% of the body’s nitrogen is in the form of mated with protein-deprived males in first mating (Blay and proteins (Kehayias et al., 1991). The two main sources of total body Yuval, 1997). All these behavioural attributes are important carbon (TBC) are fats and proteins, while the contributions from parameters for quality of sterile males being used in sterile body ash and carbohydrates are typically low (<3%) (Hawk et al., insect release programmes (Hendrichs et al., 2002). 1954; Biltz and Pellegrino, 1969). Thus TBC measurement can be Protein is scarce in nature (Burroughs, 1970; Hansen, 1970; associated directly with body fat if a reasonable adjustment can be Baker and Baker, 1983), and both female and male tephritids made for the contribution of carbon due to body protein. The actively forage to find nitrogenous foods in the form of bird faeces, evaluation of body fat through measurement of carbon was first decomposing fruits and microbes on leaf surfaces, etc. (Drew et al., introduced by Kyere et al. (1982). Kehayias et al. (1991) postulated 1983; Sharp and Chambers, 1984; Hendrichs et al., 1991, 1993; that TBC contains (>70%) carbon from fats and thus body fats can Prokopy et al., 1993; Drew and Yuval, 2000). Lipid is another be estimated precisely from TBC. limiting resource not available in the natural diet of flies, but The advantages of using stable isotope dilution techniques important for certain biological activities like oogenesis, phero- include the possibility to determine the rate of turnover of a pool mone production and precursors of juvenile hormone (Schooley irrespective of whether there is net gain or loss in the pool of and Baker, 1985; Jones, 1989; Williamson, 1989). Lipids are not interest (IAEA, 2009). The principle of the method is that the pool of frequently available in the adult diet of phytophagous insects, but interest is labelled with the relevant stable isotope, in this case 15N, adults are able to synthesize them in the fat body (lipogenesis) and that the dilution of the isotope in the pool can then be from ingested food (Chapman, 1982). Adults are unable to measured as the organism is switched to an unlabelled diet. This synthesize lipids from sucrose, and the lipid reserves in teneral then allows accurate measurement of the increase in the pool size adults are only a carryover from pre-adult stages (Langley et al., and loss from the pool simultaneously. These techniques have been 1972; Municio et al., 1973; Garcıá et al., 1980; Pagani et al., 1980). used in some entomological studies to study carbon turnover Studies on nutritionally stressed C. capitata reported a decrease in (Hood-Nowotny et al., 2006; Hood-Nowotny and Knols, 2007), but stored lipids with age (Nestel et al., 1985). However sugar-fed flies have been extensively used in soil science to measure gross N retain teneral lipid levels when tested 8 days after emergence mineralization (IAEA, 2000). The elegance of the method is that it (Nestel et al., 1986). In another study, Nestel et al. (2004) can easily be levered into entomological nutrition studies. demonstrated that despite variation in the quantity of lipids in The objective of this study was to investigate the effect of pupating larvae due to their having previously fed on different exposure to the juvenile hormone analogue methoprene and concentrations of sucrose, the emerging adults have a similar load access to N sources in the diet on lipid and protein turnover of lipids; it was suggested that the lipid content of emerging adults during the life of adult flies from emergence to 35 days of age by may be regulated. However, further studies have now provided estimating total body carbon (TBC), total body nitrogen (TBN), evidence that lipogenesis does after all take place in adult flies of C. nitrogen uptake and turnover using isotope dilution techniques. capitata (Warburg and Yuval, 1996), Anastrepha serpentina (Jaćome Such information may provide insights into the physiological et al., 1995) and Anastrepha suspensa (Pereira, 2005). conditions that underlie male sexual performance and ulti- The melon fly, B. cucurbitae is an economically important pest of mately improve the quality of released sterile males in SIT fruits and vegetables (White and Elson-Harris, 1992). Relying on programmes. conventional chemical control to manage tephritid pests (Roessler, 1989) has led to increasing environmental concerns and thus alternative strategies have been sought. The Sterile Insect 2. Methods Technique (SIT), applied as a component of an area-wide integrated pest management approach, is a well established 2.1. Strain and rearing environment-friendly technique for suppression (Vargas et al., 2004; Jang et al., 2008) or eradication (Kakinohana et al., 1990; A genetic sexing strain of B. cucurbitae, developed by USDA ARS, Koyama et al., 2004). Despite these examples of successful Hawaii (McInnis et al., 2004), and in its 59th generation, was used adoption of SIT against B. cucurbitae, there still is a demand to for all experiments. The colony was maintained on wheat based improve the cost-effectiveness of the SIT for this species. Certain diet modified from the standard Seibersdorf diet (Hooper, 1987)at areas of importance are mating competitiveness, which is the FAO/IAEA Agriculture and Biotechnology Laboratories, Sei- adversely affected by long-term mass rearing (Iwahashi et al., bersdorf, Austria. The flies were maintained under low stress 1983; Hibino and Iwahashi, 1989; Cayol, 2000), and the long pre- condition (four larvae/g of diet and 100 flies in 20 cm copulatory period of this species. In previous studies (Haq et al., 20 cm 20 cm adult cages). Following emergence, the flies 2010b) we reported that application of the juvenile hormone (JH) were maintained in the laboratory with a photoperiod of 14L:10D analogue methoprene and access to diet including hydrolyzed at 24 1 8C and 60 5% RH. Author's personal copy

I. Haq et al. / Journal of Insect Physiology 56 (2010) 1807–1815 1809

2.2. Treatments stream of helium through a series of scrubbers to remove sulfurous impurities and residual water, as well as over hot copper to reduce

Adult male flies were subjected to one of four treatments: oxides of nitrogen to elemental nitrogen (N2). Carbon dioxide (CO2) and N2 peaks were separated on a 3 m Porapak Q gas (1) Topical application of methoprene (M), and sugar (commercial chromatography column. The CO2 and N2 peaks were then bled sugar; sucrose) and hydrolyzed yeast (protein source (P)) as into the mass spectrometer to determine the isotopic ratio. adult food (M+P+). Hydrolyzed yeast (MP Biomedicals Inc.; The measurement of isotopic composition for a particular www.mpbio.com) contained 60% protein, nitrogen 8.8%, alpha element is commonly based on the ratio of the less abundant amino nitrogen 4.2%, moisture 3.67%, ash 11.96%, minerals isotope of interest to the more abundant isotope. In natural 4.57%, salt 0.56% enriched with vitamins and only traces of abundance studies values are conventionally reported as ratios of vegetable oil (0.5%); the lighter to the heavier isotope referenced against international (2) No methoprene application, but sugar and hydrolyzed yeast standards in delta (d) units parts per thousand %. A lower-case (protein source) as adult food (MP+); delta value is defined as the isotopic ratio of a sample standardized (3) Topical application of methoprene and only sugar as adult food to the isotopic ratio of a defined reference: (M+P); (4) No methoprene application and only sugar as adult food ðR R Þ (MP). x s 1000 ¼ d Rs Methoprene was applied topically 3–4 h after adult emergence where Rx is the isotopic ratio of the sample and Rs is the isotopic at a rate of 5 mgin1ml acetone solution per male by immobilizing ratio of the reference standard. The defined reference standard for males in a net bag (FAO/IAEA/USDA, 2003) and applying the carbon was Vienna PeeDee Belemnite (VPDB). The reference solution via a pipette through the net onto the dorsal surface of the standard used for N was atmospheric nitrogen (Gro¨ning, 2004). thorax; anaesthesia was not used to immobilize the flies. Males from each treatment were maintained in separate 30 cm 20 cm 2.5. Data analysis diameter cylindrical screen cages with a maximum male density of 200 flies/cage and with the type of food assigned for each The data were analyzed by multivariate analysis of variance treatment. In treatments without protein feeding (P) only water using a GLM procedure, considering methoprene, protein and age and sugar ad libitum were supplied to the flies. In the treatments as factors. The significance value used in tests was 95% (a = 0.05). with protein (P+), the hydrolyzed yeast was mixed with the sugar The data were analyzed using Statistica software (StatSoft, 2000). in a proportion of 3:1, sugar:hydrolyzed yeast, and supplied with The rate of 15N loss was measured by multiple regressions. A water ad libitum. correlation for N uptake and N excretion rates between treatments Females were also held in separate cages and provided with an was analyzed. The relationship between TBN and d 13C in males adult diet 3:1 sugar:hydrolyzed yeast and water ad libitum. For was also measured by correlation analysis. Nitrogen (%) taken up female:male comparisons, females were compared with males of during larval feeding and carried to the teneral stage was treatment MP+, since they did not receive methoprene applica- calculated by using simple robust equations adopted from soil tion, but had access to protein in their adult food. fertilizer research (IAEA, 2000) by the formula

15 2.3. Isotope labelling and sampling Nadult %N in adult retained from larval stage ¼ 15 : (1) Nteneral The flies were labelled with 15N during the larval stage. 15N- Glycine at 0.1 g per 1 kg of larval diet was dissolved in water and TBN ¼ N retained from larval stage added to the larval diet. Males and females were sexed and þ N derived from post-teneral diet (2) maintained separately from emergence until 1, 5, 7, 8, 15, 20, 25 and 35 days of age. We selected these ages for sampling based on Using isotope dilution equations it was possible to simultaneously previous work (Haq et al., 2010b) where we showed that M+P+ and estimate the N uptake and losses from an insect whether it be due MP+ males began sexual activity on day 5, that M+P and MP to excretion or egg-laying. started sexual activities at day 8 after emergence, and that all Nt N lnð N = NtÞ males were sexually mature by day 15. N uptake ¼ 0 0 (3) Dt lnðNt=N Þ Three individual flies (replicates) from each treatment group at 0 each sampling date were taken and immediately stored at 20 8C. where N0 and Nt are initial and final TBN in mg values, respectively * * 15 Three newly emerged unfed flies, were also collected and stored. In and N0 and Nt are initial and final atom % N excess values, t is C. capitata, lipid contents have been found to vary according to the time of the measure. For the sake of simplicity, average values of 3 time of the day related to sexual activities (Warburg and Yuval, replicate flies were taken to run the models, simple propagation of 1996). Thus flies were sampled at the same time of day (1 h after error equations were used to calculate the uncertainty, although darkness). Prior to homogenization for TBC and TBN determina- this may have led to overestimation of the uncertainty, as the flies tion, fresh and dry weights of the flies were noted. were destructively sampled and are thus single point measurements. The percentage standard error of each replicate set of 2.4. Isotope analysis measurements was generally far less than 10% and the com- pounded uncertainty was generally less than 20%, even though the Flies were dried (60 8C for 24 h), weighed and analyzed for total data was not cleaned up to exclude outliers. N, C, 15N and 13C. Whole fly samples were sealed into It was also possible to determine the N uptake using the isotope 8mm 5 mm tin cups and analyzed using a Carlo Erba (Milan, mass balance. This method assumes that the isotopes are Italy) carbon nitrogen (CN) analyzer, linked to an Isoprime conserved, and any loss of isotope from one time point to the automated isotope ratio mass spectrometer (IRMS) (GV Instru- other is due to excretion or egg-laying. It was thus possible to ments, Manchester, UK). Samples were combusted in an atmo- calculate the mean isotopic enrichment of the loss and from this sphere of oxygen at 1700 8C, and the resultant gas carried in a value to calculate the amount of N lost; using this data and the net Author's personal copy

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N gain data it was then possible to calculate the gross N uptake of the insect. This method was compared with the isotope dilution method and was found to give almost identical results, with r2 greater than 0.95 for each treatment, suggesting these semi- independent techniques used to calculate N uptake are mathe- matically robust.

3. Results

3.1. Body dry weight

Females had an average body weight of 12.2 mg (Fig. 1), which was significantly higher (F1,53 = 59.45, P < 0.001) than that of MP+ males (9.63 mg). Female age had a significant effect on body weight (F8,53 = 20.41, P < 0.001). The average weight of males varied between 6.22 and 11.29 mg (Fig. 1) and access to diet including hydrolyzed yeast led to a significant increase in body Fig. 2. Mean total body carbon (TBC) (SD, N = 27) of Bactrocera cucurbitae females weight (F1,107 = 125.05, P < 0.001), while methoprene alone or with access to a diet including hydrolyzed yeast (protein source) and of males treated interacting with diet including hydrolyzed yeast had no effect on with or without methoprene and with or without access to hydrolyzed yeast in their diet. Males were treated with methoprene and protein source (M+P+), no methoprene body weight (F1,107 = 3.4, P = 0.06). Male age also had a significant and protein source (M P+), methoprene and only sugar (M+P ), or no methoprene and effect (F = 25.93, P < 0.001) and its interaction with metho- 8,107 only sugar (MP). Females received no methoprene, but had access to protein source prene alone, or methoprene plus diet including hydrolyzed yeast, in their diet (MP+). had no significant effect. However the interaction between male age and diet including hydrolyzed yeast had a significant effect on body weight (F8,107 = 4.04, P < 0.001). which stabilised to around 2.5 mg C at 10 days, compared to the treatments with access to sugar only (P), which increased to a 3.2. Total body carbon and nitrogen maximum of around 1.5 mg C and decreased to 1.2 mg C by the end of the experiment (Fig. 2). There were no significant effects of There were significant differences in the total body carbon application of methoprene on TBC observed in males with access to

(TBC) of the males compared to females (F1,53 = 53.7, P < 0.001) diet including hydrolyzed yeast (P+) (F1,107 = 0.64, P=0.42) and (Fig. 2). There were also differences in total body nitrogen (TBN) sugar only (P)(F1,107 = 2.59, P=0.11). Although the inclusion of between the sexes, with females accumulating significantly methoprene in the M+P treatment appeared to lead to a slight greater quantities of N (F1,53 = 157.35, P < 0.001) (Fig. 3). C:N decrease in TBC, this was found to be not significantly different ratios tracked these differences, with females exhibiting lower C:N from the M-P- treatment. Age had significant effects on male TBC ratios than the males, even though in teneral flies at emergence the (F8,107 = 21.44, P < 0.001). C:N ratios started higher in females than in males. There was an As expected there were significant differences (F1,107 = 358.81, increase in C:N ratios in the first seven feeding days and then C:N P < 0.001) between the total body nitrogen (TBN) of males with ratios of both sexes stabilised with a significant drop in C:N ratios access to diet including hydrolyzed yeast (P+) and sugar only (P). in the females at 35 days (Table 1). TBN in the males with access to diet including hydrolyzed yeast Access to diet including hydrolyzed yeast (P+) had a significant (P+) increased to approximately twice that of males with access to impact (F1,107 = 215.89, P < 0.001) on the total body carbon (TBC) sugar only (P), which did not decrease significantly over the 35 in males causing almost a doubling of TBC over the first seven days days[(Fig._3)TD$IG] (Fig. 3). However, there was no significant impact of the [(Fig._1)TD$IG]

Fig. 1. Mean dry weight (SD, N = 27) of Bactrocera cucurbitae females with access to a Fig. 3. Mean total body nitrogen (TBN) (SD, N = 27) of Bactrocera cucurbitae females diet including hydrolyzed yeast (protein source) and of males treated with or without with access to a diet including hydrolyzed yeast (protein source) and of males treated methoprene and with or without access to hydrolyzed yeast in their diet. Males were with or without methoprene and with or without access to hydrolyzed yeast in their treated with methoprene and protein source (M+P+), no methoprene and protein diet. Males were treated with methoprene and protein source (M+P+), no methoprene source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and (MP). Females received no methoprene, but had access to protein source in their only sugar (MP). Females received no methoprene, but had access to protein source diet (MP+). in their diet (MP+). Author's personal copy

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Table 1 Exchange of 15N(SD), d 13C(SD) and C:N ratio (SD) in Bactrocera cucurbitae females with access to a diet including hydrolyzed yeast (protein source) and malesa treated with or without methoprene and with or without access to hydrolyzed yeast in their diet (n = 27 per treatment).

Treatments Age %15N exc. SD d 13C SD C:N ratio SD

Females 0 0.349 0.032 25.2 0.4 3.4 0.2 MP+ 1 0.339 0.017 25.2 0.2 4.3 0.6 MP+ 5 0.135 0.010 24.4 0.1 5.2 0.2 MP+ 7 0.150 0.040 24.6 0.1 5.0 0.3 MP+ 8 0.126 0.006 24.6 0.2 5.0 0.1 MP+ 15 0.120 0.028 24.4 0.3 4.9 0.5 MP+ 20 0.111 0.025 24.3 0.1 4.6 0.0 MP+ 25 0.098 0.012 24.3 0.1 4.8 0.2 MP+ 35 0.080 0.002 24.3 0.1 4.8 0.0 Males 0 0.331 0.043 25.2 0.1 3.1 0.1 M+P+ 1 0.273 0.023 24.6 0.3 4.7 0.4 M+P+ 5 0.174 0.010 24.6 0.0 5.3 0.2 Fig. 4. Mean (SD) nitrogen (15N) loss rate through excretion in Bactrocera cucurbitae M+P+ 7 0.151 0.011 24.6 0.0 5.6 0.3 males treated with or without methoprene and with or without access to hydrolyzed M+P+ 8 0.153 0.031 24.9 0.1 5.1 0.2 yeast (protein source) in their diet. Males were treated with methoprene and protein M+P+ 15 0.115 0.019 24.6 0.3 4.8 0.2 source (M+P+), no methoprene and protein source (MP+), methoprene and only sugar M+P+ 20 0.147 0.007 24.8 0.1 5.1 0.4 (M+P), or no methoprene and only sugar (MP). M+P+ 25 0.131 0.007 24.9 0.1 5.4 0.1 M+P+ 35 0.151 0.046 24.8 0.1 5.1 0.2 MP+ 1 0.267 0.020 24.6 0.1 4.6 0.3 MP+ 5 0.164 0.007 24.5 0.2 5.4 0.2 2 MP+ 7 0.158 0.003 25.0 0.3 5.9 0.4 (x = 0.00, P=3.00E+30) between MP+ and M+P+ males. It was MP+ 8 0.165 0.019 24.9 0.2 5.6 0.5 only possible to accurately calculate the uptake and loss of N rates MP+ 15 0.128 0.003 24.9 0.1 5.1 0.4 using isotope dilution equations in the initial logarithmic phase of MP+ 20 0.135 0.010 25.0 0.1 5.6 0.5 15N enrichment decline, as at the moment of linearization, the MP+ 25 0.125 0.007 24.9 0.1 5.3 0.4 MP+ 35 0.134 0.013 24.7 0.1 4.9 0.3 incoming N has the same value as the outgoing N, therefore it was M+P 1 0.336 0.023 25.3 0.2 4.3 0.3 not possible to trace the dynamics of the N pool from that moment M+P 5 0.308 0.030 25.2 0.1 6.6 0.7 onward. M+P 7 0.304 0.015 25.7 0.1 6.6 1.4 The percentage of total body 15N, derived from the larval diet, M+P 8 0.333 0.039 25.7 0.2 6.1 0.8 of the flies in the sugar only treatments (M+P ,M P ) was not M+P 15 0.309 0.023 25.7 0.1 5.5 0.3 M+P 20 0.310 0.035 25.4 0.2 5.1 0.5 significantly different from 100%, showing their obvious reliance M+P 25 0.312 0.031 25.3 0.2 5.2 0.5 on larval N reserves (Fig. 4). In males that had access to hydrolyzed M+P 35 0.330 0.039 25.4 0.1 4.5 0.1 yeast in their diet, the proportion of TBN derived from the post- MP 1 0.325 0.025 25.4 0.1 6.1 0.3 teneral diet, calculated by Eqs. (1) and (2), increased from 20 to MP 5 0.349 0.017 25.3 0.1 7.0 0.5 MP 7 0.308 0.035 25.7 0.1 6.9 0.4 50% N from day 1 to day 5, and stabilised at around 60% at day 15. 15 MP 8 0.320 0.010 25.6 0.2 6.1 0.6 This reflects the linear phase described above, where the N of the MP 15 0.307 0.018 25.6 0.2 5.6 0.4 incoming pool is the same as the 15N of the outgoing pool. At this MP 20 0.313 0.024 25.6 0.2 5.3 0.8 point, the percent of N derived from the larval diet equates to the MP 25 0.301 0.011 25.5 0.1 5.8 0.2 proportion of the fly which is structural N. Thus, approximately MP 35 0.338 0.011 25.3 0.0 5.1 0.5 40% (192 mg N) of the 35-day-old adult male hydrolyzed yeast- a Males were treated with methoprene and protein source (M+P+), no diet-fed fly is structural N, however, around 60% of the teneral methoprene and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar (MP). Females received no methoprene, but had male and 40% (146 mg N) of the teneral female appears to be access to protein source in their diet (MP+). structural N. Nitrogen uptake rates, calculated by isotope dilution techni- methoprene on TBN in sugar-fed (P)(F1,107 = 0.05, P=0.81) or ques, showed that uptake patterns appeared to vary throughout hydolyzed yeast-diet-fed males (F1,107 = 0.26, P=0.6). Age also had the life cycle. In males having access to diet including hydrolyzed significant effects on TBN (F8,107 = 4.64, P < 0.001). C:N ratios of yeast, there was rapid N uptake in the first few days after flies after the first 7 days were not significantly influenced by the emergence, ca. 40–50 mg per day, which then stabilised to around diet including hydrolyzed yeast (P+) (Table 1). 20 mg per day at around 8 days (Fig. 5). After that it was not possible to reliably determine N uptake rates due to rapid turnover 3.3. Isotope data rate of the metabolically active N pool which appears to have turned over completely after 15 days. There appeared to be no The 15N enrichment of males fed sugar only (P) remained significant difference in the N uptake rates between the M+P+ and static throughout the experiment as there was no dilution of the M-P+ treatments, suggesting that methoprene has little influence insect’s nitrogen from unlabelled N, although there was loss of on the cumulative N acquisition of flies. In males having access to nitrogen via excretion. There were no significant differences of 15N hydrolyzed yeast in their diet there was a significant linear enrichment (x2 = 0.00, P=01) between M+P and MP males. correlation (F = 16.61, P = 0.006) between daily N uptake and N Average 15N enrichement of males fed hydrolyzed yeast in their excretion rates in between 0 and 8 days of age for the average of post-teneral diet decreased in a logarithmic–curvilinear fashion M+P+ and MP+ treatments. In the males having access to sugar and at a significantly higher rate (x2 = 109.15, P < 0.001) compared only (P), N uptake rates calculated ranged between 4 and to sugar-fed males (Fig. 4), reflecting the turnover of the +4 mg N per day, which reflects the uncertainty range of the metabolically active N pool which is eventually replaced by an measurements rather than N uptake, as there was no N available in unlabelled N pool. 15N enrichment fell to a low plateau level after the diet. about 15 days, due to a background of metabolically inert labelled Nitrogen uptake by females during the first days after emergence tissue. There were no significant differences of 15N enrichment wasalmostdoublethatofmales(Fig. 6), with uptake rates in the Author's personal copy

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was no difference in lipids among both behavioural groups. A positive effect of protein in the diet on male weight was also observed in A. suspensa (Pereira, 2005). These results with B. cucurbitae substantiate the positive effect on male weight of incorporating protein in the post-teneral diet. TBC relates mainly to the lipids in the body (Kehayias et al., 1991), an increase in TBC levels from teneral stage to sexual maturity in sugar-fed males suggests that lipogenesis occurs in adults of this species as it does in other tephritids (Ja´come et al., 1995; Warburg and Yuval, 1996; Pereira, 2005). The inclusion of hydrolyzed yeast in the diet had a signiﬁcant impact on TBC, which was almost doubled compared to treatments without hydrolyzed yeast. This acquisition of TBC due to post-teneral feeding on diet including hydrolyzed yeast is similar to lipid accumulation in A. suspensa (Pereira, 2005). In both cases there was a rise in lipids and

Fig. 5. Mean (SD) nitrogen uptake rate in Bactrocera cucurbitae males treated with or TBC until the males reached sexual maturity. Slight differences without methoprene and with access to hydrolyzed yeast (protein source) in their diet. were also observed, as in A. suspensa there was a decrease in lipid There was no nitrogen uptake in males with no access to hydrolyzed yeast in the diet. content after sexual maturity age, and males could never reattain Males were treated with methoprene and protein source (M+P+) or no methoprene and the teneral lipid level (Pereira, 2005), while in B. cucurbitae we protein source (M P+). [(Fig._6)TD$IG] found that there was no decrease in TBC after males reached sexual maturity and TBC remained higher than at the teneral stage. For sugar-fed A. suspensa males there was a much steeper decline in lipids from day 1 onward as compared to males with access to diet including hydrolyzed yeast. However, our results on the B. cucurbitae showed little decline in TBC after males became sexually mature and TBC never fell below that of the teneral level, even though a small decline in TBC in M+P males was observed as compared to MP males. The differences in both studies may be due to analytical methodological differences or species biology. Our results suggest that daily, age related, and behavioural activity differences, and depletion of reserves associated with these activity levels, influenced the TBC in adult flies. The age of the flies was the main factor responsible for significant differences in TBC between days. However both sex and diet interacted with age, Fig. 6. Mean (SD) cumulative nitrogen uptake rate in Bactrocera cucurbitae females with access to a diet including hydrolyzed yeast (protein source) and in males treated and they influenced the behavioural activities of the flies. In males with or without methoprene and with access to hydrolyzed yeast in their diet. There having access to only sugar, TBC increased until males became was no nitrogen uptake in males with no access to hydrolyzed yeast in the diet. Males sexually mature and then decreased a little. Pereira (2005) argued were treated with methoprene and protein source (M+P+) or no methoprene and the decline in lipids in sexually mature males may be due to energy protein source (MP+). Females received no methoprene, but had access to protein expenditures in pheromone production or male–male agonistic source in their diet (MP+). interactions. Nestel et al. (1985) suggested that lipid reserves in C. capitata may play a role in regulation and production of pheromone. This may, indeed, be the case as application of region of 90 mg N per day between days 1 and 5 of age, which fell to methoprene increased pheromone production in A. suspensa (Teal around 60 mg N per day between days 5 and 8. There was no et al., 2000) and its application regulates pheromone calling in B. difference in N uptake between M+P+ and MP+ males (Fig. 6). cucurbitae (Haq et al., 2010b). In this study a slight decline in TBC in 18 A strong correlation (F = 117.6, df = 98, P = 2.04 10 ) M+P males compared to M P males after sexual maturity may 13 between TBN and d C in the male flies was also observed. also have been due to higher rates of pheromone calling. However, the lack of difference in TBC between M+P+ and MP+ suggests 4. Discussion that energy expenditures due to enhanced pheromone calling are compensated by diet including hydrolyzed yeast. In the present study we have measured total body nitrogen TBN was twice as high in males with access to diet (TBN) and total body carbon (TBC) as indicators of body protein supplemented with hydrolyzed yeast compared to sugar-fed and body lipids respectively in Bactrocera cucurbitae. There was a males and there was clearly no acquisition of N in sugar-fed clear effect of post-teneral diet supplementation with hydrolyzed males (except possibly through some access to fly faeces), which yeast on body weight, TBC and TBN during the first 35 days after leads to their reliance on N reserves accumulated during the larval emergence. Methoprene application to males had no effect on stage. Previous studies on the feeding behaviour have shown that body weight, TBC, and TBN, regardless of whether diet was despite the scarcity of protein in nature, tephritid flies manage to supplemented or unsupplemented. Females with access to harness nitrogenous compounds by feeding on a variety of hydrolyzed yeast in their diet had higher body weights, TBN, compounds including leaf exudates, bird faeces, bacteria found and TBC than similarly fed males. Males fed on diet including on leaf surfaces or decomposing fruit (Drew et al., 1983; Hendrichs hydrolyzed yeast had significantly higher body weight than sugar- and Hendrichs, 1990; Prokopy et al., 1993) and also through fed males. In C. capitata, Yuval et al. (1998) reported that there was nitrogen fixation by bacteria in the gut (Behar et al., 2005). In our no significant size difference between lekking and resting males. excperiments, however, there was no significant increase in TBN in However, lekking males were significantly heavier and contained the sugar-fed males suggesting that symbiotic nitrogen fixation by significantly more sugars and protein than resting males, but there gut flora was minimal. These findings contrast with those of Author's personal copy

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Pereira (2005), who reported a decrease in body protein in sugar- stable relationship between N uptake and N loss (excretion + egg- fed males during the first 15 days of sexual maturation after laying), probably reflecting the daily food foraging and oviposition emergence and then an increase afterwards; however this increase activities of mature females in anautogenous tephritids (Hendrichs was much higher in males with access to diet including hydrolyzed et al., 1991; Hendrichs and Prokopy, 1994) which do not go yeast. Similar to the decrease in TBC in M+P males, TBN also through the feeding and egg-laying cycles of periodic feeders such decreased a little after 7 days in M+P males as compared to as blood feeding insects (Chapman, 1982). MP males, suggesting a cost for enhanced sexual activity due to In this study we presented the sugar and the hydrolyzed yeast methoprene application. as a mixture, even though insects in natural environments regulate For both sexes there was an increase in C:N ratio during the first food ingestion by dietary self-selection behaviour that involves seven feeding days and then stabilization. However, the significant ingesting combinations of two or more foods in different ratios to drop in C:N ratio in the females at 35 days may possibly be due to reach a favourable nutrient balance through non-random choices egg production and oviposition. From an ecological perspective, (Waldbauer et al., 1984; Waldbauer and Friedman, 1991). Wild C:N ratios of flies after the first 7 days were not significantly Anastrepha obliqua (Macquart) self-selecting females ingested less influenced by the diet including hydrolyzed yeast. This suggests food and showed better performance (longevity and fecundity) lipogenensis may have been driven by the physiological require- when fed on sugar and yeast separately as compared to feeding on ment to maintain a distinct stoichiometry. It is evident that B. these nutrients in mixture (Cresoni-Pereira and Zucoloto, 2001; cucurbitae adults exhibited substantial variation in their elemental Medeiros and Zucoloto, 2006). However, continuous laboratory stoichiometry (TBC, TBN) resulting from different feeding proto- rearing for many generations is also reported to affect feeding cols. This variation was sex specific and age related, and behaviour, and female C. capitata showed increased fecundity behavioural sex and age differences in activities likely drive these when fed on a diet combining yeast and sugar in a mixture as variations. However, there seemed to be a homeostasis in the compared to feeding on these nutrients separately (Cangussu and stoichiometry showing that females and males of all treatments Zucoloto, 1995). A mixture of hydrolyzed yeast and sugar is used as underwent physiological changes to reach certain developmental a standard diet to maintain adult colonies in the mass rearing of B. thresholds. Once the flies attained that threshold, little stoichio- cucurbitae and has been found to be a better adult food for metric variations were observed. Interestingly there was a strong increased fecundity as compared to these components provided correlation between TBN and d 13C in the male flies, probably separately (Sugimoto, 1978; Nakamori and Kuba, 1990). On the reflecting the fractionation processes associated with lipogenesis. other hand, at fly emergence and release facility, B. cucurbitae flies Lipid synthesis discriminates against 13C in favour of 12C due to are fed only sugar and water for 3–4 days before release (Nakamori slight isotopic discrimination in enzymatic and kinetic pathways and Kuba, 1990), but recent findings on significant positive effect of associated with lipogenesis (Deniro and Epstein, 1977). As a diet including hydrolyzed yeast on male mating competitiveness increased TBN was shown to be associated with increased TBC (Haq et al., 2010b) suggests that this feeding protocol could be or lipogensis, this correlation was not surprising. incorporated into future SIT programmes. We have calculated uptake and loss of N by using isotope The findings of this study have direct implications for B. dilution and mass balance techniques. Using these techniques it cucurbitae SIT programmes. We showed that the incorporation of was established that there were no obvious differences in the N a N source into the diet of teneral sterile males at fly emergence uptake patterns between the males in the methoprene and no and release facilities has a positive effect on adult weight, which methoprene treatments. However, there were differences in can directly influence male–male interactions (Sivinski, 1993). uptake patterns between the sexes and a higher content of The effects of lipid and protein reserves of males also play an structural N was found in males as compared to females. This likely important role in male mating success (Yuval et al., 1998). Since reflects physiological differences due to resource allocation for egg males with access to diet including hydrolyzed yeast have higher laying. Estimation of N uptake by isotope dilution equations TBC and TBN levels, and application of methoprene accelerates demonstrated that this is a very sensitive method for estimating N sexual maturation without adverse effects on the acquisition of uptake allowing less than 50 mg daily uptake rates to be estimated TBC, TBN and N uptake, there is good reason to incorporate with acceptable uncertainty. Higher N uptake during initial days methoprene and hydrolyzed yeast into the adult diet for SIT and subsequent decreases in uptake compared to females may programmes. This can contribute to more nutritionally stable possibly be explained by the fact that after males reach sexual males, and consequently increase their effectiveness. However, it maturity they may not need additional N uptake. is suggested that the number of days of feeding of teneral sterile There was no acquisition of N in sugar-fed males. Interestingly males on a diet that includes hydrolyzed yeast, and then switching there appeared to be N retention mechanisms in place as the N them to a sugar only diet, should be evaluated as this could excretion rate in these males, similar to the males with access to reduce the cost of protein feeding for the entire time that diet including hydrolyzed yeast after day 1, dropped to around maturing sterile males are held prior to release. 6 mg N per day at day 8, a third of that of the males fed on hydrolyzed yeast-diet, and continued to fall to less than 1 mg N per day at 35 days. The initial high loss of N may have been associated References with the loss of larval fat body tissue which contains a high proportion of protein (Maynard Smith et al., 1970). Baker, H.B., Baker, I., 1983. Floral nectar sugar constituents in relation to pollinator In the females the high N uptake reflects the well documented type. In: Jones, C.E., Little, R.J. 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