G Model

IP 2546 19
Journal of Insect Physiology xxx (2010) xxxxxx

Contents lists available at ScienceDirect

Journal of Insect Physiology

journal homepage: www.elsevier.com/locate/jinsphys 1 2 3 4 5 6 7 8 9 10 11 12 13

Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y 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 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
b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 March 2010 Received in revised form 19 July 2010 Accepted 21 July 2010 Keywords: Bactrocera cucurbitae Isotope 15N Methoprene Hydrolyzed yeast SIT Total body carbon Total body nitrogen

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments known to enhance mating success in the male melon y Bactrocera cucurbitae Coquillett (Diptera: Tephritidae), supporting their use in mass rearing protocols for sterile males in the context of sterile 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 turnover of body lipids and protein to conrm the feasibility of their application in melon y SIT massrearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males were exposed to one of the following treatments: (1) topical application of methoprene and access to diet including protein (M+P+); (2) only diet including protein (MP+); (3) only methoprene (M+P) and (4) untreated, only sugar-fed, control males (MP). Total body carbon (TBC) and total body nitrogen (TBN) of ies were measured at regular intervals from emergence to 35 days of age for each of the different treatments. Nitrogen assimilation and turnover in the ies were measured using stable isotope (15N) dilution techniques. Hydrolyzed yeast incorporation into the diet signicantly increased male body weight, TBC and TBN as compared to sugar-fed males. Females had signicantly 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 signicantly 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. 2010 Elsevier Ltd. All rights reserved.

13 14 1. Introduction 15 16 17 18 19 Many insects in their adult stage are anautogenous, requiring carbohydrates, proteins and lipids to perform biological activities necessary for survival and reproduction (Chapman, 1982). The rst study on the complete nutritional requirements of adult tephritids was by Hagen (1953), who found that both sexes of Bactrocera cucurbitae, Bactrocera dorsalis and Ceratitis capitata required carbohydrates, protein in the form of free amino acids, minerals, B-complex vitamins, and water. Sucrose is needed to fuel daily foraging, ight and courtship activities and is essential for survival, but alone it does not satisfy the nutritional requirements of the ies, and protein ingestion is crucial for egg production in females (Christenson and Foote, 1960; Bateman, 1972; Sharp and Chambers, 1984; Hendrichs et al., 1991; Cangussu and Zucoloto, 1995; Teal et al., 2004). The role of dietary protein in modulating male mating success is well

* Corresponding author at: Insect Pest Control Laboratory, Joint FAO/IAEA Agriculture and Biotechnology Laboratories, A-2444 Seibersdorf, Austria. E-mail addresses: I.Haq@iaea.org, imihsan@yahoo.com (I.u. Haq). 0022-1910/$ see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.07.011

19 20 21 22 23 24 25 26 27 28 29 30

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
2 I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96

documented in tephritids (Blay and Yuval, 1997; Field and Yuval, 1999; McInnis et al., 2004; Perez-Staples et al., 2007; Shelly et al., 2007; Pereira et al., 2009, 2010). In the Mediterranean fruit y, C. capitata post-teneral feeding on protein can contribute to male gonadal and accessory gland development (Yuval et al., 2002). The increased mating success due to dietary protein may, however, have a cost for longevity (Cordts and Partridge, 1996). In C. capitata, while continuous access to dietary protein increased survival, males starved after 4 days of feeding on protein were short-lived as compared to males that had no access to protein (Kaspi and Yuval, 2000; Maor et al., 2004). However, Shelly and Kennelly (2002) reported no adverse effect of protein diet on starvation survival. Protein feeding in C. capitata males can also affect the re-mating behaviour: females mated with protein-fed males in their rst mating had less tendency to re-mate when compared to females mated with protein-deprived males in rst mating (Blay and Yuval, 1997). All these behavioural attributes are important parameters for quality of sterile males being used in sterile insect release programmes (Hendrichs et al., 2002). Protein is scarce in nature (Burroughs, 1970; Hansen, 1970; Baker and Baker, 1983), and both female and male tephritids actively forage to nd nitrogenous foods in the form of bird faeces, decomposing fruits and microbes on leaf surfaces, etc. (Drew et al., 1983; Sharp and Chambers, 1984; Hendrichs et al., 1991, 1993; Prokopy et al., 1993; Drew and Yuval, 2000). Lipid is another limiting resource not available in the natural diet of ies, but important for certain biological activities like oogenesis, pheromone production and precursors of juvenile hormone (Schooley and Baker, 1985; Jones, 1989; Williamson, 1989). Lipids are not frequently available in the adult diet of phytophagous insects, but adults are able to synthesize them in the fat body (lipogenesis) from ingested food (Chapman, 1982). Adults are unable to synthesize lipids from sucrose, and the lipid reserves in teneral adults are only a carryover from pre adult stages (Langley et al., 1972; Municio et al., 1973; Garca et al., 1980; Pagani et al., 1980). Studies on nutritionally stressed C. capitata reported a decrease in stored lipids with age (Nestel et al., 1985). However sugar-fed ies retain teneral lipid levels when tested 8 days after emergence (Nestel et al., 1986). In another study, Nestel et al. (2004) demonstrated that despite variation in the quantity of lipids in pupating larvae due to their having previously fed on different concentrations of sucrose, the emerging adults have a similar load of lipids; it was suggested that the lipid content of emerging adults may be regulated. However, further studies have now provided evidence that lipogenesis does after all take place in adult ies of C. capitata (Warburg and Yuval, 1996), Anastrepha serpentina (Jacome et al., 1995) and Anastrepha suspensa (Pereira, 2005). The melon y, B. cucurbitae is an economically important pest of fruits and vegetables (White and Elson-Harris, 1992). Relying on 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 Technique (SIT), applied as a component of an area-wide integrated pest management approach, is a well established environment-friendly technique for suppression (Vargas et al., 2004; Jang et al., 2008) or eradication (Kakinohana et al., 1990; Koyama et al., 2004). Despite these examples of successful adoption of SIT against B. cucurbitae, there still is a demand to improve the cost-effectiveness of the SIT for this species. Certain areas of importance are mating competitiveness, which is adversely affected by long-term mass rearing (Iwahashi et al., 1983; Hibino and Iwahashi, 1989; Cayol, 2000), and the long pre-copulatory period of this species. In previous studies (Haq et al., 2010b) we reported that application of the juvenile

hormone (JH) analogue methoprene and access to diet including hydrolyzed yeast reduced the pre-copulatory period and enhanced the mating success of B. cucurbitae. Application of methoprene and access to diet including hydrolyzed yeast also increased male participation in leks and pheromone calling (Haq et al., 2010a). These behavioural attributes are energetically costly and have adverse consequences for energy reserves over the life time of the y. In addition to modulating mating behaviour, application of methoprene is known to affect nutritional status and to alter resource allocation in other insects. For example, JH analogue treatment altered lipid metabolism and increased the mass of ovaries in female Gryllus rmus (Zera and Zhao, 2004), and increased the size of male accessory glands in Tribolium castaneum (Parthasarathy et al., 2009). In clinical nutrition studies it is well documented that body protein can be quantied from total body nitrogen (TBN) (Varttsky et al., 1979) since 99% of the bodys nitrogen is in the form of proteins (Kehayias et al., 1991). The two main sources of total body carbon (TBC) are fats and proteins, while the contributions from body ash and carbohydrates are typically low (<3%) (Hawk et al., 1954; Biltz and Pellegrino, 1969). Thus TBC measurement can be associated directly with body fat if a reasonable adjustment can be made for the contribution of carbon due to body protein. The evaluation of body fat through measurement of carbon was rst introduced by Kyere et al. (1982). Kehayias et al. (1991) postulated that TBC contains (>70%) carbon from fats and thus body fats can be estimated precisely from TBC. The advantages of using stable isotope dilution techniques include the possibility to determine the rate of turnover of a pool irrespective of whether there is net gain or loss in the pool of interest (IAEA, 2009). The principle of the method is that the pool of interest is labelled with the relevant stable isotope, in this case 15 N, and that the dilution of the isotope in the pool can then be measured as the organism is switched to an unlabelled diet. This then allows accurate measurement of the increase in the pool size and loss from the pool simultaneously. These techniques have been used in some entomological studies to study carbon turnover (Hood-Nowotny et al., 2006; Hood-Nowotny and Knols, 2007), but have been extensively used in soil science to measure gross N mineralization (IAEA, 2000). The elegance of the method is that it can easily be levered into entomological nutrition studies. The objective of this study was to investigate the effect of exposure to the juvenile hormone analogue methoprene and access to N sources in the diet on lipid and protein turnover during the life of adult ies from emergence to 35 days of age by estimating total body carbon (TBC), total body nitrogen (TBN), nitrogen uptake and turnover using isotope dilution techniques. Such information may provide insights into the physiological conditions that underlie male sexual performance and ultimately improve the quality of released sterile males in SIT programmes. 2. Methods

96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147

148 2.1. Strain and rearing A genetic sexing strain of B. cucurbitae, developed by USDA ARS, Hawaii (McInnis et al., 2004), and in its $59th generation, was used for all experiments. The colony was maintained on wheat based diet modied from the standard Seibersdorf diet (Hooper, 1987) at the FAO/IAEA Agriculture and Biotechnology Laboratories, Seibersdorf, Austria. The ies were maintained under low stress condition (four larvae/g of diet and $100 ies in 20 cm 20 cm 20 cm adult cages). Following emergence, the ies were maintained in the laboratory with a photoperiod of 14L:10D at 24 1 8C and 60 5% RH. 149 150 151 152 153 154 155 156 157 158 159

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx 3

159 2.2. Treatments 160 Adult male ies were subjected to one of four treatments: 163 162 161 164 163 165 164 166 165 167 166 168 167 169 168 171 170 169 172 170 174 173 171 175 172 177 176 173 178 174 179 175 180 176 181 177 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 (1) Topical application of methoprene (M), and sugar (commercial sugar; sucrose) and hydrolyzed yeast (protein source (P)) as adult food (M+P+). Hydrolyzed yeast (MP Biomedicals Inc.; www.mpbio.com) contained 60% protein, nitrogen 8.8%, alpha amino nitrogen 4.2%, moisture 3.67%, ash 11.96%, minerals 4.57%, salt 0.56% enriched with vitamins and only traces of vegetable oil (0.5%); (2) No methoprene application, but sugar and hydrolyzed yeast (protein source) as adult food (MP+); (3) Topical application of methoprene and only sugar as adult food (M+P); (4) No methoprene application and only sugar as adult food (MP). Methoprene was applied topically 34 h after adult emergence at a rate of 5 mg in 1 ml acetone solution per male by immobilizing males in a net bag (FAO/IAEA/USDA, 2003) and applying the solution via a pipette through the net onto the dorsal surface of the thorax; anaesthesia was not used to immobilize the ies. Males from each treatment were maintained in separate 30 cm 20 cm diameter cylindrical screen cages with a maximum male density of 200 ies/cage and with the type of food assigned for each treatment. In treatments without protein feeding (P) only water and sugar ad libitum were supplied to the ies. In the treatments with protein (P+), the hydrolyzed yeast was mixed with the sugar in a proportion of 3:1, sugar:hydrolyzed yeast, and supplied with water ad libitum. Females were also held in separate cages and provided with an adult diet 3:1 sugar:hydrolyzed yeast and water ad libitum. For female:male comparisons, females were compared with males of treatment MP+, since they did not receive methoprene application, but had access to protein in their adult food. 2.3. Isotope labelling and sampling 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 The ies were labelled with N during the larval stage. NGlycine at 0.1 g per 1 kg of larval diet was dissolved in water and added to the larval diet. Males and females were sexed and 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 previous work (Haq et al., 2010b) where we showed that M+P+ and MP+ males began sexual activity on day 5, that M+P and MP started sexual activities at day 8 after emergence, and that all males were sexually mature by day 15. Three individual ies (replicates) from each treatment group at each sampling date were taken and immediately stored at 20 8C. Three newly emerged unfed ies, were also collected and stored. In C. capitata, lipid contents have been found to vary according to the time of the day related to sexual activities (Warburg and Yuval, 1996). Thus ies were sampled at the same time of day (1 h after darkness). Prior to homogenization for TBC and TBN determination, fresh and dry weights of the ies were noted. 2.4. Isotope analysis 217 218 219 220 221 222 223 224 Flies were dried (60 8C for 24 h), weighed and analyzed for total N, C, 15N and 13C. Whole y samples were sealed into 8 mm 5 mm tin cups and analyzed using a Carlo Erba (Milan, Italy) carbon nitrogen (CN) analyzer, linked to an Isoprime automated isotope ratio mass spectrometer (IRMS) (GV Instruments, Manchester, UK). Samples were combusted in an atmosphere of oxygen at 1700 8C, and the resultant gas carried in a
15 15

stream of helium through a series of scrubbers to remove sulfurous impurities and residual water, as well as over hot copper to reduce oxides of nitrogen to elemental nitrogen (N2). Carbon dioxide (CO2) and N2 peaks were separated on a 3 m Porapak Q gas chromatography column. The CO2 and N2 peaks were then bled into the mass spectrometer to determine the isotopic ratio. The measurement of isotopic composition for a particular element is commonly based on the ratio of the less abundant isotope of interest to the more abundant isotope. In natural abundance studies values are conventionally reported as ratios of the lighter to the heavier isotope referenced against international standards in delta (d) units parts per thousand %. A lower-case delta value is dened as the isotopic ratio of a sample standardized to the isotopic ratio of a dened reference:   Rx Rs 1000 d Rs where Rx is the isotopic ratio of the sample and Rs is the isotopic ratio of the reference standard. The dened reference standard for carbon was Vienna PeeDee Belemnite (VPDB). The reference standard used for N was atmospheric nitrogen (Groning, 2004). 2.5. Data analysis The data were analyzed by multivariate analysis of variance using a GLM procedure, considering methoprene, protein and age as factors. The signicance value used in tests was 95% (a = 0.05). The data were analyzed using Statistica software (StatSoft, 2000). The rate of 15N loss was measured by multiple regressions. A correlation for N uptake and N excretion rates between treatments was analyzed. The relationship between TBN and d 13C in males was also measured by correlation analysis. Nitrogen (%) taken up during larval feeding and carried to the teneral stage was calculated by using simple robust equations adopted from soil fertilizer research (IAEA, 2000) by the formula
15

224 225 226 227 228 229 230 231 232 233 234 235 236 237 238

240 239 241 242 243 244 245

246 247 248 249 250 251 252 253 254 255 256

%N in adult retained from larval stage 15 TBN N retained from larval stage N derived from post-teneral diet

Nadult : Nteneral

(1) 258 257

(2)

Using isotope dilution equations it was possible to simultaneously estimate the N uptake and losses from an insect whether it be due to excretion or egg-laying. N uptake Nt N0 ln N0 = Nt lnNt =N0 Dt (3)

260 259 261 262

where N0 and Nt are initial and nal TBN in mg values, respectively and *N0 and *Nt are initial and nal atom % 15N excess values, t is time of the measure. For the sake of simplicity, average values of 3 replicate ies were taken to run the models, simple propagation of error equations were used to calculate the uncertainty, although this may have led to overestimation of the uncertainty, as the ies were destructively sampled and are thus single point measurements. The percentage standard error of each replicate set of measurements was generally far less than 10% and the compounded uncertainty was generally less than 20%, even though the data was not cleaned up to exclude outliers. It was also possible to determine the N uptake using the isotope mass balance. This method assumes that the isotopes are conserved, and any loss of isotope from one time point to the other is due to excretion or egg-laying. It was thus possible to calculate the mean isotopic enrichment of the loss and from this

264 263 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
4 I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx

280 281 282 283 284 285 286 287

value to calculate the amount of N lost; using this data and the net 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 semiindependent techniques used to calculate N uptake are mathematically robust. 3. Results

288 3.1. Body dry weight 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 Females had an average body weight of 12.2 mg (Fig. 1), which was signicantly higher (F1,53 = 59.45, P < 0.001) than that of MP+ males (9.63 mg). Female age had a signicant 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 signicant increase in body weight (F1,107 = 125.05, P < 0.001), while methoprene alone or interacting with diet including hydrolyzed yeast had no effect on body weight (F1,107 = 3.4, P = 0.06). Male age also had a signicant effect (F8,107 = 25.93, P < 0.001) and its interaction with methoprene alone, or methoprene plus diet including hydrolyzed yeast, had no signicant effect. However the interaction between male age and diet including hydrolyzed yeast had a signicant effect on body weight (F8,107 = 4.04, P < 0.001). 3.2. Total body carbon and nitrogen 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 There were signicant differences in the total body carbon (TBC) of the males compared to females (F1,53 = 53.7, P < 0.001) (Fig. 2). There were also differences in total body nitrogen (TBN) between the sexes, with females accumulating signicantly greater quantities of N (F1,53 = 157.35, P < 0.001) (Fig. 3). C:N ratios tracked these differences, with females exhibiting lower C:N ratios than the males, even though in teneral ies at emergence the C:N ratios started higher in females than in males. There was an increase in C:N ratios in the rst seven feeding days and then C:N ratios of both sexes stabilised with a signicant drop in C:N ratios in the females at 35 days (Table 1). Access to diet including hydrolyzed yeast (P+) had a signicant impact (F1,107 = 215.89, P < 0.001) on the total body carbon (TBC) in males causing almost a doubling of TBC over the rst seven days

Fig. 2. Mean total body carbon (TBC) (SD, N = 27) of Bactrocera cucurbitae females with access to a diet including hydrolyzed yeast (protein source) and of males treated 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 and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar (MP). Females received no methoprene, but had access to protein source in their diet (MP+).

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 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 signicant effects of application of methoprene on TBC observed in males with access to diet including hydrolyzed yeast (P+) (F1,107 = 0.64, P = 0.42) and sugar only (P) (F1,107 = 2.59, P = 0.11). Although the inclusion of methoprene in the M+P treatment appeared to lead to a slight decrease in TBC, this was found to be not signicantly different from the M-P- treatment. Age had signicant effects on male TBC (F8,107 = 21.44, P < 0.001). As expected there were signicant differences (F1,107 = 358.81, P < 0.001) between the total body nitrogen (TBN) of males with access to diet including hydrolyzed yeast (P+) and sugar only (P). TBN in the males with access to diet including hydrolyzed yeast (P+) increased to approximately twice that of males with access to sugar only (P), which did not decrease signicantly over the 35 days (Fig. 3). However, there was no signicant impact of the

318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336

318

Fig. 1. Mean dry weight (SD, N = 27) of Bactrocera cucurbitae females with access to a diet including hydrolyzed yeast (protein source) and of males treated 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 and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar (MP). Females received no methoprene, but had access to protein source in their diet (MP+).

Fig. 3. Mean total body nitrogen (TBN) (SD, N = 27) of Bactrocera cucurbitae females with access to a diet including hydrolyzed yeast (protein source) and of males treated 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 and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar (MP). Females received no methoprene, but had access to protein source in their diet (MP+).

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx 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 Females MP+ MP+ MP+ MP+ MP+ MP+ MP+ MP+ Males M+P+ M+P+ M+P+ M+P+ M+P+ M+P+ M+P+ M+P+ MP+ MP+ MP+ MP+ MP+ MP+ MP+ MP+ M+P M+P M+P M+P M+P M+P M+P M+P MP MP MP MP MP MP MP MP Age 0 1 5 7 8 15 20 25 35 0 1 5 7 8 15 20 25 35 1 5 7 8 15 20 25 35 1 5 7 8 15 20 25 35 1 5 7 8 15 20 25 35 %15N exc. 0.349 0.339 0.135 0.150 0.126 0.120 0.111 0.098 0.080 0.331 0.273 0.174 0.151 0.153 0.115 0.147 0.131 0.151 0.267 0.164 0.158 0.165 0.128 0.135 0.125 0.134 0.336 0.308 0.304 0.333 0.309 0.310 0.312 0.330 0.325 0.349 0.308 0.320 0.307 0.313 0.301 0.338 SD 0.032 0.017 0.010 0.040 0.006 0.028 0.025 0.012 0.002 0.043 0.023 0.010 0.011 0.031 0.019 0.007 0.007 0.046 0.020 0.007 0.003 0.019 0.003 0.010 0.007 0.013 0.023 0.030 0.015 0.039 0.023 0.035 0.031 0.039 0.025 0.017 0.035 0.010 0.018 0.024 0.011 0.011 5

d 13C
25.2 25.2 24.4 24.6 24.6 24.4 24.3 24.3 24.3 25.2 24.6 24.6 24.6 24.9 24.6 24.8 24.9 24.8 24.6 24.5 25.0 24.9 24.9 25.0 24.9 24.7 25.3 25.2 25.7 25.7 25.7 25.4 25.3 25.4 25.4 25.3 25.7 25.6 25.6 25.6 25.5 25.3

SD 0.4 0.2 0.1 0.1 0.2 0.3 0.1 0.1 0.1 0.1 0.3 0.0 0.0 0.1 0.3 0.1 0.1 0.1 0.1 0.2 0.3 0.2 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.2 0.2 0.2 0.1 0.0

C:N ratio 3.4 4.3 5.2 5.0 5.0 4.9 4.6 4.8 4.8 3.1 4.7 5.3 5.6 5.1 4.8 5.1 5.4 5.1 4.6 5.4 5.9 5.6 5.1 5.6 5.3 4.9 4.3 6.6 6.6 6.1 5.5 5.1 5.2 4.5 6.1 7.0 6.9 6.1 5.6 5.3 5.8 5.1

SD 0.2 0.6 0.2 0.3 0.1 0.5 0.0 0.2 0.0 0.1 0.4 0.2 0.3 0.2 0.2 0.4 0.1 0.2 0.3 0.2 0.4 0.5 0.4 0.5 0.4 0.3 0.3 0.7 1.4 0.8 0.3 0.5 0.5 0.1 0.3 0.5 0.4 0.6 0.4 0.8 0.2 0.5

Fig. 4. Mean (SD) nitrogen (15N) loss rate through excretion in Bactrocera cucurbitae males treated with or without methoprene and with or without access to hydrolyzed yeast (protein source) in their diet. Males were treated with methoprene and protein source (M+P+), no methoprene and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar (MP).

a Males were treated with methoprene and protein source (M+P+), no methoprene and protein source (MP+), methoprene and only sugar (M+P), or no methoprene and only sugar (MP). Females received no methoprene, but had access to protein source in their diet (MP+).

336 337 338 339 340 341

methoprene on TBN in sugar-fed (P) (F1,107 = 0.05, P = 0.81) or hydolyzed yeast-diet-fed males (F1,107 = 0.26, P = 0.6). Age also had signicant effects on TBN (F8,107 = 4.64, P < 0.001). C:N ratios of ies after the rst 7 days were not signicantly inuenced by the diet including hydrolyzed yeast (P+) (Table 1). 3.3. Isotope data

342 343 344 345 346 347 348 349 350 351 352 353 354 355

The 15N enrichment of males fed sugar only (P) remained static throughout the experiment as there was no dilution of the insects nitrogen from unlabelled N, although there was loss of nitrogen via excretion. There were no signicant differences of 15N enrichment (x2 = 0.00, P = 01) between M+P and MP males. Average 15N enrichement of males fed hydrolyzed yeast in their post-teneral diet decreased in a logarithmiccurvilinear fashion and at a signicantly higher rate (x2 = 109.15, P < 0.001) compared to sugar-fed males (Fig. 4), reecting the turnover of the metabolically active N pool which is eventually replaced by an unlabelled N pool. 15N enrichment fell to a low plateau level after about 15 days, due to a background of metabolically inert labelled tissue. There were no signicant differences of 15N enrichment

(x2 = 0.00, P = 3.00E+30) between MP+ and M+P+ males. It was only possible to accurately calculate the uptake and loss of N rates using isotope dilution equations in the initial logarithmic phase of 15 N enrichment decline, as at the moment of linearization, the incoming N has the same value as the outgoing N, therefore it was not possible to trace the dynamics of the N pool from that moment onward. The percentage of total body 15N, derived from the larval diet, of the ies in the sugar only treatments (M+P, MP) was not signicantly different from 100%, showing their obvious reliance on larval N reserves (Fig. 4). In males that had access to hydrolyzed yeast in their diet, the proportion of TBN derived from the postteneral diet, calculated by Eqs. (1) and (2), increased from 20 to 50% N from day 1 to day 5, and stabilised at around 60% at day 15. This reects the linear phase described above, where the 15N of the incoming pool is the same as the 15N of the outgoing pool. At this point, the percent of N derived from the larval diet equates to the proportion of the y which is structural N. Thus, approximately 40% (192 mg N) of the 35-day-old adult male hydrolyzed yeastdiet-fed y is structural N, however, around 60% of the teneral male and 40% (146 mg N) of the teneral female appears to be structural N. Nitrogen uptake rates, calculated by isotope dilution techniques, showed that uptake patterns appeared to vary throughout the life cycle. In males having access to diet including hydrolyzed yeast, there was rapid N uptake in the rst few days after emergence, ca. 4050 mg per day, which then stabilised to around 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 rate of the metabolically active N pool which appears to have turned over completely after 15 days. There appeared to be no signicant difference in the N uptake rates between the M+P+ and M-P+ treatments, suggesting that methoprene has little inuence on the cumulative N acquisition of ies. In males having access to hydrolyzed yeast in their diet there was a signicant linear correlation (F = 16.61, P = 0.006) between daily N uptake and N excretion rates in between 0 and 8 days of age for the average of M+P+ and MP+ treatments. In the males having access to sugar only (P), N uptake rates calculated ranged between 4 and +4 mg N per day, which reects the uncertainty range of the measurements rather than N uptake, as there was no N available in the diet. Nitrogen uptake by females during the rst days after emergence was almost double that of males (Fig. 6), with uptake rates in the

355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
6 I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx

Fig. 5. Mean (SD) nitrogen uptake rate in Bactrocera cucurbitae males treated with or without methoprene and with access to hydrolyzed yeast (protein source) in their diet. There was no nitrogen uptake in males with no access to hydrolyzed yeast in the diet. Males were treated with methoprene and protein source (M+P+) or no methoprene and protein source (MP+).

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 with or without methoprene and with access to hydrolyzed yeast in their diet. There was no nitrogen uptake in males with no access to hydrolyzed yeast in the diet. Males were treated with methoprene and protein source (M+P+) or no methoprene and protein source (MP+). Females received no methoprene, but had access to protein source in their diet (MP+).

399 400 401 402 403 404

region of 90 mg N per day between days 1 and 5 of age, which fell to around 60 mg N per day between days 5 and 8. There was no difference in N uptake between M+P+ and MP+ males (Fig. 6). A strong correlation (F = 117.6, df = 98, P = 2.04 1018) between TBN and d 13C in the male ies was also observed. 4. Discussion

405 406 407 408 409 410 411 412 413 414 415 416 417 418 419

In the present study we have measured total body nitrogen (TBN) and total body carbon (TBC) as indicators of body protein and body lipids respectively in Bactrocera cucurbitae. There was a clear effect of post-teneral diet supplementation with hydrolyzed yeast on body weight, TBC and TBN during the rst 35 days after emergence. Methoprene application to males had no effect on body weight, TBC, and TBN, regardless of whether diet was supplemented or unsupplemented. Females with access to hydrolyzed yeast in their diet had higher body weights, TBN, and TBC than similarly fed males. Males fed on diet including hydrolyzed yeast had signicantly higher body weight than sugarfed males. In C. capitata, Yuval et al. (1998) reported that there was no signicant size difference between lekking and resting males. However, lekking males were signicantly heavier and contained

signicantly more sugars and protein than resting males, but there 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 (Jacome et al., 1995; Warburg and Yuval, 1996; Pereira, 2005). The inclusion of hydrolyzed yeast in the diet had a signicant 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 TBC until the males reached sexual maturity. Slight differences were also observed, as in A. suspensa there was a decrease in lipid content after sexual maturity age, and males could never reattain the teneral lipid level (Pereira, 2005), while in B. cucurbitae we 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, inuenced the TBC in adult ies. The age of the ies was the main factor responsible for signicant differences in TBC between days. However both sex and diet interacted with age, and they inuenced the behavioural activities of the ies. In males having access to only sugar, TBC increased until males became sexually mature and then decreased a little. Pereira (2005) argued the decline in lipids in sexually mature males may be due to energy expenditures in pheromone production or malemale agonistic 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 methoprene increased pheromone production in A. suspensa (Teal et al., 2000) and its application regulates pheromone calling in B. cucurbitae (Haq et al., 2010b). In this study a slight decline in TBC in M+P males compared to MP males after sexual maturity may also have been due to higher rates of pheromone calling. However, the lack of difference in TBC between M+P+ and MP+ suggests that energy expenditures due to enhanced pheromone calling are compensated by diet including hydrolyzed yeast. TBN was twice as high in males with access to diet supplemented with hydrolyzed yeast compared to sugar-fed males and there was clearly no acquisition of N in sugar-fed males (except possibly through some access to y faeces), which leads to their reliance on N reserves accumulated during the larval stage. Previous studies on the feeding behaviour have shown that despite the scarcity of protein in nature, tephritid ies manage to harness nitrogenous compounds by feeding on a variety of compounds including leaf exudates, bird faeces, bacteria found on leaf surfaces or decomposing fruit (Drew et al., 1983; Hendrichs and Hendrichs, 1990; Prokopy et al., 1993) and also through nitrogen xation by bacteria in the gut (Behar et al., 2005). In our excperiments, however, there was no signicant increase in TBN in the sugar-fed males suggesting that symbiotic nitrogen xation by gut ora was minimal. These ndings contrast with those of

419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx 7

485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551

Pereira (2005), who reported a decrease in body protein in sugarfed males during the rst 15 days of sexual maturation after emergence and then an increase afterwards; however this increase was much higher in males with access to diet including hydrolyzed yeast. Similar to the decrease in TBC in M+P males, TBN also decreased a little after 7 days in M+P males as compared to MP males, suggesting a cost for enhanced sexual activity due to methoprene application. For both sexes there was an increase in C:N ratio during the rst seven feeding days and then stabilization. However, the signicant drop in C:N ratio in the females at 35 days may possibly be due to egg production and oviposition. From an ecological perspective, C:N ratios of ies after the rst 7 days were not signicantly inuenced by the diet including hydrolyzed yeast. This suggests lipogenensis may have been driven by the physiological requirement to maintain a distinct stoichiometry. It is evident that B. cucurbitae adults exhibited substantial variation in their elemental stoichiometry (TBC, TBN) resulting from different feeding protocols. This variation was sex specic and age related, and behavioural sex and age differences in activities likely drive these variations. However, there seemed to be a homeostasis in the stoichiometry showing that females and males of all treatments underwent physiological changes to reach certain developmental thresholds. Once the ies attained that threshold, little stoichiometric variations were observed. Interestingly there was a strong correlation between TBN and d 13C in the male ies, probably reecting the fractionation processes associated with lipogenesis. Lipid synthesis discriminates against 13 C in favour of 12C due to slight isotopic discrimination in enzymatic and kinetic pathways associated with lipogenesis (Deniro and Epstein, 1977). As increased TBN was shown to be associated with increased TBC or lipogensis, this correlation was not surprising. We have calculated uptake and loss of N by using isotope dilution and mass balance techniques. Using these techniques it was established that there were no obvious differences in the N uptake patterns between the males in the methoprene and no methoprene treatments. However, there were differences in uptake patterns between the sexes and a higher content of structural N was found in males as compared to females. This likely reects physiological differences due to resource allocation for egg laying. Estimation of N uptake by isotope dilution equations demonstrated that this is a very sensitive method for estimating N uptake allowing less than 50 mg daily uptake rates to be estimated with acceptable uncertainty. Higher N uptake during initial days and subsequent decreases in uptake compared to females may possibly be explained by the fact that after males reach sexual maturity they may not need additional N uptake. There was no acquisition of N in sugar-fed males. Interestingly there appeared to be N retention mechanisms in place as the N excretion rate in these males, similar to the males with access to diet including hydrolyzed yeast after day 1, dropped to around 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 with the loss of larval fat body tissue which contains a high proportion of protein (Maynard Smith et al., 1970). In the females the high N uptake reects the well documented large requirement of N for egg production in females. It was higher during the rst 5 days after emergence (90 mg N per day) and stable thereafter (ca. 60 mg N per day), even though egg-laying only started around 15 days of age and continued till day 35 with daily N loss rates of 1015 mg. This suggests a low N efciency, as only around 15% of the N taken up is converted to protein for egg production. This could reect the form in which the N was available in the diet, which could contain complex nutritionally

unavailable forms of N. After the maturation period there was a stable relationship between N uptake and N loss (excretion + egglaying), probably reecting the daily food foraging and oviposition activities of mature females in anautogenous tephritids (Hendrichs et al., 1991; Hendrichs and Prokopy, 1994) which do not go through the feeding and egg-laying cycles of periodic feeders such as blood feeding insects (Chapman, 1982). In this study we presented the sugar and the hydrolyzed yeast as a mixture, even though insects in natural environments regulate food ingestion by dietary self-selection behaviour that involves ingesting combinations of two or more foods in different ratios to reach a favourable nutrient balance through non-random choices (Waldbauer et al., 1984; Waldbauer and Friedman, 1991). Wild Anastrepha obliqua (Macquart) self-selecting females ingested less food and showed better performance (longevity and fecundity) when fed on sugar and yeast separately as compared to feeding on these nutrients in mixture (Cresoni-Pereira and Zucoloto, 2001; Medeiros and Zucoloto, 2006). However, continuous laboratory rearing for many generations is also reported to affect feeding behaviour, and female C. capitata showed increased fecundity when fed on a diet combining yeast and sugar in a mixture as compared to feeding on these nutrients separately (Cangussu and Zucoloto, 1995). A mixture of hydrolyzed yeast and sugar is used as a standard diet to maintain adult colonies in the mass rearing of B. cucurbitae and has been found to be a better adult food for increased fecundity as compared to these components provided separately (Sugimoto, 1978; Nakamori and Kuba, 1990). On the other hand, at y emergence and release facility, B. cucurbitae ies are fed only sugar and water for 34 days before release (Nakamori and Kuba, 1990), but recent ndings on signicant positive effect of a diet including hydrolyzed yeast on male mating competitiveness (Haq et al., 2010b) suggests that this feeding protocol could be incorporated into future SIT programmes. The ndings of this study have direct implications for B. cucurbitae SIT programmes. We showed that the incorporation of a N source into the diet of teneral sterile males at y emergence and release facilities has a positive effect on adult weight, which can directly inuence malemale interactions (Sivinski, 1993). The effects of lipid and protein reserves of males also play an important role in male mating success (Yuval et al., 1998). Since males with access to diet including hydrolyzed yeast have higher TBC and TBN levels, and application of methoprene accelerates sexual maturation without adverse effects on the acquisition of TBC, TBN and N uptake, there is good reason to incorporate methoprene and hydrolyzed yeast into the adult diet for SIT programmes. This can contribute to more nutritionally stable males, and consequently increase their effectiveness. However, it is suggested that the number of days of feeding of teneral sterile males on a diet that includes hydrolyzed yeast, and then switching them to a sugar only diet, should be evaluated as this could reduce the cost of protein feeding for the entire time that maturing sterile males are held prior to release. References
Baker, H.B., Baker, I., 1983. Floral nectar sugar constituents in relation to pollinator type. In: Jones, C.E., Little, R.J. (Eds.), Handbook of Experimental Pollination Biology. Van Nostrand Reinhold, New York, USA, pp. 117141. Bateman, M.A., 1972. The ecology of fruit ies. Annual Review of Entomology 17, 493518. Behar, A., Yuval, B., Jurkevitch, E., 2005. Enterobacteria mediated nitrogen xation in natural populations of the fruit y Ceratitis capitata. Molecular Ecology 14, 26372643. Biltz, R.M., Pellegrino, E.D., 1969. The chemical anatomy of bone. Journal of Bone and Joint Surgery (American) 51A, 456466. Blay, S., Yuval, B., 1997. Nutritional correlates to reproductive success of male Mediterranean fruit ies. Animal Behaviour 54, 5966. Burroughs, L.F., 1970. Amino acids. In: Hume, A.C. (Ed.), The Biochemistry of Fruits and Their Products. Academic Press, London, UK, pp. 119146.

551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603

604 605 606 607 608 609 610 611 612 613 614 615 616 617 618

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
8 I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx , Iwahashi, O., Ito Y., Shiyomi, M., 1983. A eld evaluation of sexual competitiveness of sterile melon ies, Dacus (Zeugodacus) cucurbitae. Ecological Entomology 8, 4348. Jacome, I., Aluja, M., Liedo, P., Nestel, D., 1995. The inuence of adult diet and age on lipid reserves in the tropical fruit y Anastrepha serpentina (Diptera: Tephritidae). Journal of Insect Physiology 41, 10791086. Jang, E.B., McQuate, G.T., Mcinnis, D.O., Harris, E.J., Vargas, R.I., Bautista, R.C., Mau, R.F., 2008. Targeted trapping, bait spray, sanitation, sterile-male, and parasitoid releases in an areawide integrated melon y (Diptera: Tephritidae) control program in Hawaii. American Entomologist 54, 240250. Jones, O.T., 1989. Mating pheromones; Ceratitis capitata. In: Robinson, A.S., Hooper, G. (Eds.), World Crop Pests: Fruit Flies Their Biology, Natural Enemies and Control, vol. 3A. Elsevier, Netherland, pp. 179183. Kakinohana, H., Kuba, H., Yamagishi, M., Kohama, T., Kiniyo, K., Tanahara, A., Sokei, Y., Kirihara, S., 1990. The eradication of the melon y from the Okinawa Islands, Japan. II. Actual control program. In: Aluja, M., Liedo, P. (Eds.), Proceedings of the International Symposium on Fruit Flies of Economic Importance. Springer, New York, NY, USA, pp. 465469. Kaspi, R., Yuval, B., 2000. Post-teneral protein feeding improves sexual competitiveness but reduces longevity of mass-reared sterile male Mediterranean Fruit Flies (Diptera: Tephritidae). Annals of the Entomological Society of America 93, 949955. Kehayias, J.J., Heymseld, S.B., LoMonte, A.F., Wang, J., Pierson Jr., R.N., 1991. In vivo determination of body fat by measuring total body carbon. American Journal of Clinical Nutrition 53, 13391344. Koyama, J., Kakinohana, H., Miyatake, T., 2004. Eradication of the melon y, Bactrocera cucurbitae, in Japan: importance of behaviour, ecology, genetics, and evolution. Annual Review of Entomology 49, 331349. Kyere, K., Oldroyd, B., Oxby, C.B., Burkinshaw, L., Ellis, R.E., Hill, G.L., 1982. The feasibility of measuring total body carbon by counting neutron in elastic scatter gamma rays. Physics in Medicine and Biology 27, 805817. Langley, P.A., Maly, H., Ruhm, F., 1972. Application of the sterility principle for the control of the Mediterranean fruit y (Ceratitis capitata): pupal metabolism in relation to mass rearing techniques. Entomologia Experimentalis et Applicata 15, 2334. Maor, M., Kamensky, B., Shloush, S., Yuval, B., 2004. Effects of post-teneral diet on foraging success of sterile male Mediterranean fruit ies. Entomologia Experimentalis et Applicata 110, 225230. Maynard Smith, J., Bozcuka, A.N., Tebbutta, S., 1970. Protein turnover in adult Drosophila. Journal of Insect Physiology 16, 601613. McInnis, D.O., Tam, S., Lim, R., Komatsu, J., Kurashima, R., Albrecht, C., 2004. Development of a pupal color-based genetic sexing strain of the melon y, Bactrocera cucurbitae Coquillett (Diptera: Tephritidae). Annals of the Entomological Society of America 97, 10261033. Medeiros, L., Zucoloto, F.S., 2006. Nutritional balancing in fruit ies: Performance of wild adult females of Anastrepha obliqua (Diptera: Tephritidae) fed on single-food or food-pair treatments. Journal of Insect Physiology 52, 1121 1127. Municio, A.M., Odnozola, J.M., Pineiro, A., Ribera, A., 1973. In vitro and in vivo [I4C] acetate incorporation during development of insects. Insect Biochemistry 3, 19 29. Nakamori, H., Kuba, H., 1990. Aerial distribution of sterile melon les, Dacus cucurbitae Coquillett, anesthetized by chilling. Japan Agricultural Research Quarterly 24, 3136. Nestel, D., Galun, R., Friedman, S., 1985. Long-term regulation of sucrose intake by the adult Mediterranean fruit y, Ceratitis capitata (Wiedemann). Journal of Insect Physiology 31, 533536. Nestel, D., Galun, R., Friedman, S., 1986. Balance energetico on el adulto irradiado de Ceratitis capitata (Wied.) (Diptera: Tephritidae). Folie Entomologica Mexicana 70, 7585. Nestel, D., Nemny-Lavy, E., Chang, C.L., 2004. Lipid and protein loads in pupating larvae and emerging adults as affected by the composition of Mediterranean fruit y (Ceratitis capitata) meridic larval diets. Archives of Insect Biochemistry and Physiology 56, 97109. Pagani, R., Suarez, A., Municio, A.M., 1980. Fatty acid patterns of the major lipid classes during development of Ceratitis capitata. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 67, 511518. Parthasarathy, R., Tan, A., Sun, Z., Chen, Z., Rankin, M., Palli, S.R., 2009. Juvenile hormone regulation of male accessory gland activity in the red our beetle. Tribolium castaneum. Mechanisms of Development 126, 563579. Pereira, R., 2005. Inuence of a juvenile hormone analog and dietary protein on male Caribbean fruit y, Anastrepha suspensa (Diptera: Tephritidae), sexual behaviour. PhD Dissertation, University of Florida, Gainesville, FL, USA. Pereira, R., Sivinski, J., Teal, P.E.A., 2009. Inuence of methoprene and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) mating aggregations. Journal of Insect Physiology 55, 328335. Pereira, R., Sivinski, J., Teal, P.E.A., 2010. Inuence of a juvenile hormone analog and dietary protein on male Anastrepha suspensa (Diptera: Tephritidae) sexual success. Journal of Economic Entomology 103, 4046. Perez-Staples, D., Prabhu, V., Taylor, P.W., 2007. Post-teneral protein feeding enhances sexual performance of Queensland fruit ies. Physiological Entomology 32, 225232. Prokopy, R.J., Hsu, C.L., Vargas, R.I., 1993. Effect of source and condition of animal excrement on attractiveness to adults of Ceratitis capitata (Diptera: Tephritidae). Environmental Entomology 22, 453458.

618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704

Cangussu, J.A., Zucoloto, F.S., 1995. Self-selection and perception threshold in adult females of Ceratitis capitata (Diptera, Tephritidae). Journal of Insect Physiology 41, 223227. Cayol, J.P., 2000. Changes in sexual behavior and in some life history traits of tephritid species caused by mass-rearing processes. In: Aluja, M., Norrbom, A. (Eds.), Fruit Flies (Tephritidae): Phylogeny and Evolution of Behavior. CRC Press, Boca Raton, FL, USA, pp. 843860. Chapman, R.F., 1982. The Insects, Structure and Function. Harvard University Press, Cambridge, MA, USA. Christenson, L.D., Foote, R.H., 1960. Biology of fruit ies. Annual Review of Entomology 15, 171192. Cordts, R., Partridge, L., 1996. Courtship reduces longevity of male Drosophila melanogaster. Animal Behaviour 52, 269278. Cresoni-Pereira, C., Zucoloto, F.S., 2001. Dietary self-selection and discrimination threshold in wild Anastrepha obliqua females (Diptera, Tephritidae). Journal of Insect Physiology 47, 11271132. Deniro, M.J., Epstein, S., 1977. Mechanism of carbon fractionation associated with lipid synthesis. Science 197, 261263. Drew, R.A.I., Yuval, B., 2000. The evolution of fruit y feeding behavior. In: Aluja, M., Norrbom, A.L. (Eds.), Fruit Flies (Tephritidae): Phylogeny and Evolution of Behavior. CRC Press, Boca Raton, FL, USA, pp. 731749. Drew, R.A.I., Courtice, A.C., Teakle, D.S., 1983. Bacteria as a natural source of food for adult fruit ies (Diptera: Tephritidae). Oecologia 60, 279284. FAO/IAEA/USDA, 2003. Manual for Product Quality Control and Shipping Procedures for Sterile Mass-reared Tephritid fruit ies, Version 5.0. International Atomic Energy Agency, Vienna, Austria, p. 85. Field, S.A., Yuval, B., 1999. Nutritional status affects copula duration in the Mediterranean fruit y, Ceratitis capitata (Insecta Tephritidae). Ethology Ecology & Evolution 11, 6170. Garca, R., Municio, A.M., Viloria, D., 1980. Glycerol phosphate and monoacylglycerol pathways of triacylglycerol biosynthesis in microsomes of Ceratitis capitata. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 66, 435437. Groning, M., 2004. International stable isotope reference materials. In: De Groot, P.A. (Ed.), Handbook of Stable Isotope Analytical Techniques, vol. I. Elsevier, pp. 874906. Hagen, K.S., 1953. Inuence of adult nutrition upon the reproduction of three fruit y species. In: Third Special Report on the Control of the Oriental fruit y (Dacus dorsalis) in the Hawaiian Islands, Senate of the State of California, USA, pp. 72 76. Hansen, E., 1970. Proteins. In: Hume, A.C. (Ed.), The Biochemistry of Fruits and Their Products. Academic Press, London, UK, pp. 147158. Haq, I., Caceres, C., Hendrichs, J., Teal, P.E.A., Stauffer, C., Robinson, A.S., 2010a. Methoprene modulates the effect of diet on male melon y Bactrocera cucurbitae (Coquillett) (Diptera: Tephritidae) performance at mating aggregations. Entomologia Experimentalis et Applicata 136, 2130. Haq, I., Caceres, C., Hendrichs, J., Teal, P.E.A., Wornoayporn, V., Stauffer, C., Robinson, A.S., 2010b. Effects of the juvenile hormone analogue methoprene and dietary protein on male melon y Bactrocera cucurbitae (Diptera: Tephritidae) mating success. Journal of Insect Physiology, doi:10.1016/j.insphys.2010.04.018. Hawk, P.B., Oser, B.L., Summerson, W.H., 1954. Practical Physiological Chemistry, 13th ed. McGraw-Hill, New York, USA. Hendrichs, J., Hendrichs, M.A., 1990. Mediterranean fruit y (Diptera: Tephritidae) in nature: location and diel pattern of feeding and other activities on fruiting and nonfruiting hosts and nonhosts. Annals of the Entomological Society of America 83, 632641. Hendrichs, J., Prokopy, R.J., 1994. Food foraging behavior of frugivorous fruit ies. In: Calkins, C.O., Klassen, W., Liedo, P. (Eds.), Fruit Flies and the Sterile Insect Technique. CRC Press, Boca Raton, FL, USA, pp. 3756. Hendrichs, J., Katsoyannos, B.I., Papaj, D.R., Prokopy, R.J., 1991. Sex differences in movement between natural feeding and mating sites and tradeoffs between food consumption, mating success and predator evasion in Mediterranean fruit ies (Diptera: Tephritidae). Oecologia 86, 223231. Hendrichs, J., Lauzon, C.R., Cooley, S.S., Prokopy, R.J., 1993. Contribution of natural food sources to adult longevity and fecundity of Rhagoletis pomonella (Diptera: Tephritidae). Ecology and Population Biology 86, 250264. Hendrichs, J., Robinson, A.S., Cayol, J.P., Enkerlin, W., 2002. Medy areawide sterile insect technique programmes for prevention, suppression or eradication: the importance of mating behavior studies. Florida Entomologist 85, 113. Hibino, Y., Iwahashi, O., 1989. Mating receptivity of wild type females for wild type males and mass-reared masles in the melon y, Dacus cucurbitae Coquillet (Diptera: Tephritidae). Applied Entomology and Zoology 24, 152154. Hood-Nowotny, R., Knols, B.G.J., 2007. Stable isotope methods in biological and ecological studies of arthropods. Entomologia Experimentalis et Applicata 124, 316. Hood-Nowotny, R., Mayr, L., Knols, B.G., 2006. Use of carbon-13 as a population marker for Anopheles arabiensis in a sterile insect technique (SIT) context. Malaria Journal 5, 6. Hooper, G.H.S., 1987. Application of quality control procedures to large scale rearing of the Mediterranean fruit y. Entomologia Experimentalis et Applicata 44, 161167. IAEA, 2000. Use of Isotope and Radiation Methods in Soil and Water Management and Crop Nutrition. Training course series No. 14, International Atomic Energy Agency, IAEA, Vienna, Austria. IAEA, 2009. Manual for the Use of Stable Isotopes in Entomology. International Atomic Energy Agency, IAEA, Vienna, Austria.

704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789 790

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011

G Model

IP 2546 19
I. Haq et al. / Journal of Insect Physiology xxx (2010) xxxxxx 9

791 790 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819

Roessler, Y., 1989. Insecticidal bait and cover sprays. In: Robinson, A.S., Hooper, G. (Eds.), World Crop Pests: Fruit Flies, Their Biology, Natural Enemies and Control, vol. 3B. Elsevier, Amsterdam, The Netherlands, pp. 329336. Schooley, D.A., Baker, F.C., 1985. Juvenile hormone biosynthesis. In: Gilbert, L.I. (Ed.), Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon, Oxford, pp. 363389. Sharp, J.L., Chambers, D.L., 1984. Consumption of carbohydrates, proteins, and amino acids by Anastrepha suspensa (Loew) (Diptera: Tephritidae) in the laboratory. Environmental Entomology 13, 768773. Shelly, T.E., Kennelly, S., 2002. Inuence of male diet on male mating success and longevity and female remating in the Mediterranean fruit y (Diptera: Tephritidae) under laboratory conditions. Florida Entomologist 85, 572579. Shelly, T.E., Edu, J., Pahio, E., 2007. Condition-dependent mating success in male fruit ies: ingestion of a pheromone precursor compensates for a low-quality diet. Journal of Insect Behavior 20, 347365. Sivinski, J., 1993. Longevity and fecundity in the Caribbean fruit y (Diptera: Tephritidae): effects of mating, strain and body size. Florida Entomologist 76, 635644. StatSoft, 2000. STATISTICA for Windows (Computer Program Manual). StatSoft, Tulsa, OK, USA. Sugimoto, A., 1978. Egg collection method in mass rearing of the melon y, Dacus cucurbitae Coquillett (Diptera: Tephritidae). Japanese Journal of Applied Entomology and Zoology 22, 6067. Teal, P.E.A., Gomez-Simuta, Y., Proveaux, A.T., 2000. Mating experience and juvenile hormone enhance sexual signaling and mating in male Caribbean fruit ies. Proceedings of the National Academy of Sciences of the United States of America 97, 37083712. Teal, P.E.A., Gavilanez-Slone, J.M., Dueben, B.D., 2004. Effects of sucrose in adult diet on mortality of males of Anastrepha suspensa (Diptera: Tephritidae). Florida Entomologist 87, 487491.

Vargas, R., Long, J., Miller, N.W., Delate, K., Jackson, C.G., Uchida, G.K., Bautista, R.C., Harris, E.J., 2004. Releases of Psyttalia etcheri (Hymenoptera: Braconidae) and sterile ies to suppress melon y (Diptera: Tephritidae) in Hawaii. Journal of Economic Entomology 97, 15311539. Varttsky, D., Ellis, K.J., Cohn, S.H., 1979. In vivo quantication of body nitrogen by neutron capture prompt gamma-ray analysis. The Journal of Nuclear Medicine 20, 11581165. Waldbauer, G.P., Friedman, S., 1991. Self-selection of optimal diets by insects. Annual Review of Entomology 36, 4363. Waldbauer, G.P., Cohen, R.W., Friedman, S., 1984. Self-selection of an optimal nutrient mix from dened diets by larvae of the corn earwrom, Heliothis zea (Boddie). Physiological Zoology 57, 590597. Warburg, M.S., Yuval, B., 1996. Effects of diet and activity on lipid levels of adult Mediterranean fruit ies. Physiological Entomology 21, 151158. White, I.M., Elson-Harris, M.M., 1992. Fruit Flies of Economic Signicance: Their Identication and Bionomics. CAB International, London, UK. Williamson, D.L., 1989. Oogenesis and spermatogenesis. In: Robinson, A.S., Hooper, G. (Eds.), World Crop Pests: Fruit Flies, Their Biology, Natural Enemies and Control, vol. 3A. Elsevier, The Netherlands, pp. 141151. Yuval, B., Kaspi, R., Shloush, S., Warburg, M.S., 1998. Nutritional reserves regulate male participation in Mediterranean fruit y leks. Ecological Entomology 23, 211215. Yuval, B., Kaspi, R., Field, S.A., Blay, S., Taylor, P.W., 2002. Effects of post-teneral nutrition on reproductive success of male Mediterranean fruit ies (Diptera: Tephritidae). Florida Entomologist 85, 165170. Zera, A.J., Zhao, Z., 2004. Effect of a juvenile hormone analogue on lipid metabolism in a wing-polymorphic cricket: implications for the endocrine-biochemical bases of life-history trade-offs. Physiological and Biochemical Zoology 77, 255266.

819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841 842 843 844 845 846 847 848

Please cite this article in press as: Haq, I., et al., Total body nitrogen and total body carbon as indicators of body protein and body lipids in the melon y Bactrocera cucurbitae: Effects of methoprene, a juvenile hormone analogue, and of diet supplementation with hydrolyzed yeast. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2010.07.011