Ihsan.prot.Lipid.article

8/6/2019 Ihsan.prot.Lipid.article

1/9

Total body nitrogen and total body carbon as indicators of body protein and bodylipids in the melon fly Bactrocera cucurbitae: Effects of methoprene, a juvenilehormone 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, Austriab National Agricultural Research Centre, Park Road, Islamabad 4500, Pakistanc Soil and Water Management and Crop Nutrition Laboratory, Joint FAO/IAEA Agriculture and Biotechnology Laboratories, A-2444 Seibersdorf, Austriad Center for Medical, Agricultural and Veterinary Entomology, USDA, ARS, Gainesville, FL 32604, USAe

Insect Pest Control Section, Joint FAO/IAEA Division, IAEA, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, AustriafInstitute of Forest Entomology, Forest Pathology & Forest Protection, BOKU, Vienna, Austria

1. Introduction

Many insects in their adult stage are anautogenous, requiring

carbohydrates, proteins and lipids to perform biological activi-

ties necessary for survival and reproduction (Chapman, 1982).

The first 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, flight and courtship activities

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,

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

Journal of Insect Physiology xxx (2010) xxxxxx

11

2

2

2

2

2

2

2

2

2

2

3

A R T I C L E I N F O

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

SITTotal body carbon

Total body nitrogen

A B S T R A C T

The application of methoprene, and providing access to diet including hydrolyzed yeast, are treatments

known to enhance mating success in the male melon fly 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 confirm the feasibility of their application in melon fly SIT mass-

rearing programmes. While females had access to a diet that included hydrolyzed yeast (protein), males

wereexposed to oneof thefollowingtreatments: (1)topical application of methopreneand 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 flies were measured at regular intervals from emergence to 35 days of age for each of the different

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 TBNas compared to sugar-fed males. Females had significantly higher body weight, TBC

and TBN as compared to all males. TBCand TBN showed age-dependent changes,increasing until the age

of sexual maturity and decreasing afterwards in both sexes. Methoprene treatment did not significantly

affect TBCor TBN. Theprogressive increase with ageof TBCsuggests 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 toshow 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.

* Corresponding author at: Insect Pest Control Laboratory, Joint FAO/IAEA

Agriculture and Biotechnology Laboratories, A-2444 Seibersdorf, Austria.

E-mail addresses: [email protected], [email protected] (I.u. Haq).

G Model

IP 2546 19

Pleasecite this article in press as:Haq, I.,et al., Total body nitrogenand total body carbonas indicators of body protein and body lipidsin

the melon fly 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

Contents lists available at ScienceDirect

Journal of Insect Physiology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j i n s p h y s

0022-1910/$ see front matter 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2010.07.011
http://dx.doi.org/10.1016/j.jinsphys.2010.07.011mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://www.sciencedirect.com/science/journal/00221910http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://www.sciencedirect.com/science/journal/00221910http://dx.doi.org/10.1016/j.jinsphys.2010.07.011mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.jinsphys.2010.07.011


2/9

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 fly, 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 first

mating had less tendency to re-mate when compared to females

mated with protein-deprived males in first 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 find 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

flies, but important for certain biological activities like oogene-

sis, 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 (Nestelet al., 1985). However sugar-fed flies 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 flies of C. capitata

(Warburg and Yuval, 1996), Anastrepha serpentina (Jacome et al.,

1995) and Anastrepha suspensa (Pereira, 2005).

The melon fly, 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 fly. 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

firmus (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

proteincan be quantified fromtotalbodynitrogen (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 (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 case15N, 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 sourcesin thediet on lipid andprotein turnover during

the life of adult flies 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

2.1. Strain and rearing

A genetic sexing strain ofB. 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 modified from the standard Seibersdorf diet (Hooper,

1987) at the FAO/IAEA Agriculture and Biotechnology Laboratories,

Seibersdorf, Austria. The flies were maintained under low stress

condition (four larvae/g of diet and $100 flies in

20 cm 20 cm 20 cm adult cages). Following emergence, the

flies were maintained in the laboratory with a photoperiod of

14L:10D at 24

18

C and 60

5% RH.

9

9

9

9

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

11

1

1

1

1

1

1

1

1

1

1

1

1

11

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

I. Haq et al./ Journal of Insect Physiology xxx (2010) xxxxxx2

G Model

IP 2546 19

Pleasecite this article in press as:Haq, I.,et al., Total body nitrogen and total body carbon as indicators of body protein andbodylipids in


http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://dx.doi.org/10.1016/j.jinsphys.2010.07.011


3/9

2.2. Treatments

Adult male flies were subjected to one of four treatments:

(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 immobilizingmales 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 flies. Males

from each treatment were maintained in separate 30 cm 20 cmdiameter cylindrical screencageswith a maximum male density of

200 flies/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 flies. 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 applica-

tion, but had access to protein in their adult food.

2.3. Isotope labelling and sampling

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

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) wherewe showedthat 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 flies (replicates) from each treatment group at

each sampling date were taken and immediately stored at 20 8C.

Threenewly emerged unfedflies, were also collected and stored. In

C. capitata, lipid contents have been found to vary according to thetime of the day related to sexual activities (Warburg and Yuval,

1996). Thus flies were sampled at the same time of day (1 h after

darkness). Prior to homogenization for TBC and TBN determina-

tion, fresh and dry weights of the flies were noted.

2.4. Isotope analysis

Flies were dried (60 8C for 24 h), weighed and analyzed for total

N, C, 15N and 13C. Whole fly 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 Instru-

ments, Manchester, UK). Samples were combusted in an atmo-

sphere of oxygen at 17008

C, and the resultant gas carried in a

streamof heliumthrough a series of scrubbersto remove sulfurous

impurities and residual water, as well as over hot copper to reduce

oxides of nitrogento elemental nitrogen(N2). Carbondioxide(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 definedas the isotopic ratio of a sample standardized

to the isotopic ratio of a defined reference:

Rx Rs

Rs

1000 d

where Rx is the isotopic ratio of the sample and Rs is the

isotopic ratio of the reference standard. The defined 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 significance 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

%N inadultretained from larval stage 15Nadult

15Nteneral: (1)

TBN N retained fromlarval stage

N derived from post-teneral diet (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.

Nuptake Nt N0Dt

lnN0=

Nt

lnNt=N0(3)

where N0 and Nt are initial and final TBN inmg values, respectively

and*

N0 and*

Nt are initial and final atom %15

N excess values, t istime of the measure. For the sake of simplicity, average values of 3

replicate flies 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 flies

were destructively sampled and are thus single point measure-

ments. The percentage standard error of each replicate set of

measurements was generally far less than 10% and the com-

pounded uncertainty was generally less than 20%, even though the

data was not cleaned up to exclude outliers.

It was also possible to determinethe 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

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

22

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

2

22

22

2

2

22

2

22

2

2

2

2

2

2

2

2

2

2

2

2

2

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

G Model

IP 2546 19



http://www.mpbio.com/http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://www.mpbio.com/


4/9

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 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 hada significant effecton 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

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 significant

effect (F8,107 = 25.93, P< 0.001) and its interaction with metho-

prene alone, or methoprene plus diet including hydrolyzed yeast,

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).

3.2. Total body carbon and nitrogen

There were significant 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 significantly

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 flies at emergence

the C:N ratios started higher in females than in males. There was

an increase in C:N ratios in the first seven feeding days and thenC:N ratios of both sexes stabilised with a significant drop in C:N

ratios in the females at 35 days (Table 1).

Access to diet including hydrolyzed yeast (P+) had a significant

impact (F1,107 = 215.89, P< 0.001) on the total body carbon (TBC)

in males causing almost a doubling of TBC over the first seven days

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 decreasedto 1.2 mg C by the end

of the experiment (Fig. 2). There were no significant effects of

application of methoprene on TBCobserved 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 significantly different

from the M-P- treatment. Age had significant effects on male TBC

(F8,107 = 21.44, P< 0.001).

As expected there were significant differences (F1,107 = 358.81,

P< 0.001) between the total body nitrogen (TBN) of males withaccess 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 significantly over the 35

days (Fig. 3). However, there was no significant impact of the

3

3

3

3

3

3

3

3

3

3

3

3

3

33

3

3

3

3

Fig. 1. Meandry weight(SD, N= 27)ofBactrocera cucurbitae females with accessto 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. 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

andproteinsource (MP+),methoprene andonly sugar (M+P),or nomethopreneand

only sugar (MP). Females received nomethoprene, but had accessto protein source

in their diet (MP+).

Fig. 3. Mean total body nitrogen (TBN) (SD, N= 27) ofBactrocera 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

andproteinsource (MP+),methoprene andonly sugar (M+P),or nomethopreneand

only sugar (MP). Females received nomethoprene, but had accessto protein source

in their diet (M

P+).


G Model

IP 2546 19





5/9


6/9

region of 90mg N per day between days1 and 5 of age, which fell toaround 60mg N per day between days 5 and 8. There was nodifference 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 flies was also observed.

4. Discussion

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 first 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 significantly higher body weight than sugar-

fed males. In C. capitata, Yuval et al. (1998) reported that there was

no significant size difference between lekking and resting males.

However, lekking males were significantly heavier and contained

significantly 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.

TBCrelatesmainlytothelipidsinthebody(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

andYuval,1996; Pereira, 2005). Theinclusionof hydrolyzedyeast 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 similarto lipid accumulationinA. suspensa(Pereira, 2005).In

both cases therewas a rise in lipids andTBC 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 teneralstage.For sugar-fedA. 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, ourresults on the B. cucurbitae showed little decline in TBC after males

became sexuallymatureand TBCnever fell below thatof 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 ageof the

flies was the main factor responsible for significant differences in

TBC between days. However both sex and diet interacted with age,

and they influenced the behavioural activities of the flies. In males

having access to only sugar, TBC increased until males became

sexually mature and then decreased a little. Pereira (2005) arguedthe decline in lipids in sexually maturemales maybe dueto 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 etal.,2010b). Inthis study a slightdeclinein 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 dietsupplemented 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 fly 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 flies 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 fixation by bacteria in the gut (Behar et al., 2005). In our

excperiments, however, there was no significant increase in TBN in

the sugar-fed males suggesting that symbiotic nitrogen fixation by

gut flora was minimal. These findings contrast with those of

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

4

44

4

4

4

4

4

4

4

4

4

4

4

4

44

4

4

4

4

4

4

4

4

4

4

4

4

44

4

4

4

4

4

4

4

4

4

4

4

4

4

Fig. 5. Mean (SD) nitrogen uptake rate in Bactrocera cucurbitae males treated with or

withoutmethoprene and withaccess to hydrolyzed yeast(proteinsource)in theirdiet.

There was no nitrogen uptake in males with no access to hydrolyzed yeast in the diet.

Maleswere treated withmethoprene and proteinsource (M+P+) or nomethoprene 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+).


G Model

IP 2546 19





7/9

Pereira (2005), who reported a decrease in body protein in sugar-

fed males during the first 15 days of sexual maturation after

emergence and then an increase afterwards; however this increase

was much higherin males with access to diet includinghydrolyzed

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.

Forboth sexes there wasan increase in C:Nratioduringthe first

seven feeding days and then stabilization. However, the signifi-

cant 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 flies after the first 7 days were not

significantly influenced 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 specific

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 changesto reach

certain developmental thresholds. Once the flies attained thatthreshold, little stoichiometric variations were observed. Inter-

estingly there was a strong correlation between TBN and d 13C inthe male flies, probably reflecting the fractionation processes

associated with lipogenesis. Lipid synthesis discriminates against13C 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 inuptake patterns between the sexes and a higher content of

structural N was found in males as compared to females. This

likely reflects 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 uptakeallowing less than 50mg dailyuptakerates tobe estimated with acceptable uncertainty. HigherN uptakeduring

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 around6mg N per day at day 8, a third of that of the males fed onhydrolyzed yeast-diet, and continued to fall to less than 1 mg Nperday 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 reflects the well documented

large requirementof N foregg productionin females.It washigher

during the first 5 days after emergence (90 mg N per day) andstable thereafter (ca. 60 mg N per day), even though egg-layingonly started around 15 days of age and continued till day 35 with

daily N loss rates of 1015mg. This suggests a low N efficiency, asonly around 15% of the N taken up is converted to protein for egg

production. This could reflect 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 + egg-

laying), probably reflecting thedaily foodforagingand oviposition

activities of mature females in anautogenous tephritids (Hen-

drichs et al., 1991; Hendrichs and Prokopy, 1994) which donotgo

through thefeedingand egg-layingcyclesof periodicfeeders 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 yeastand sugar is used as a standard

diet to maintain adult colonies in the mass rearing of B. cucurbitaeand has been found to be a better adult food forincreasedfecundity

as compared to these components provided separately (Sugimoto,

1978; Nakamori and Kuba, 1990). On the other hand, at fly

emergence and release facility, B. cucurbitae flies are fed only sugar

and water for 34 days before release (Nakamori and Kuba, 1990),

but recent findings on significant 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 findings 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 fly emergence and

release facilities has a positive effect on adult weight, which can

directly influence malemale interactions (Sivinski, 1993). Theeffects 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 includinghydrolyzed yeast have higher TBC and TBN

levels, and application of methoprene accelerates sexual matura-

tion 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 switchingthemto 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 pollinatortype. In: Jones, C.E., Little, R.J. (Eds.), Handbook of Experimental PollinationBiology. Van Nostrand Reinhold, New York, USA, pp. 117141.

Bateman, M.A., 1972. The ecology of fruit flies. Annual Review of Entomology 17,493518.

Behar, A., Yuval,B., Jurkevitch, E.,2005. Enterobacteria mediated nitrogen fixation innatural populations of the fruit fly Ceratitis capitata. Molecular Ecology 14,26372643.

Biltz, R.M., Pellegrino, E.D., 1969. The chemical anatomy of bone. Journal of Boneand Joint Surgery (American) 51A, 456466.

Blay, S., Yuval, B., 1997. Nutritional correlates to reproductive success of maleMediterranean fruit flies. 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.

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

55

5

5

5

5

5

5

5

5

5

5

5

5

55

5

5

5

5

5

5

5

5

5

6

6

6

6

666666666666666


G Model

IP 2546 19





8/9

Cangussu, J.A., Zucoloto, F.S., 1995. Self-selection and perception threshold in adultfemales ofCeratitis capitata (Diptera, Tephritidae). Journal of Insect Physiology41, 223227.

Cayol, J.P., 2000. Changes in sexual behavior and in some life history traits oftephritid species caused by mass-rearing processes. In: Aluja, M., Norrbom, A.(Eds.), Fruit Flies(Tephritidae): Phylogeny and Evolution of Behavior. CRCPress,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 flies. Annual Review of Ento-mology 15, 171192.

Cordts, R., Partridge, L., 1996. Courtship reduces longevity of male Drosophilamelanogaster. 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 ofInsect Physiology 47, 11271132.

Deniro, M.J., Epstein, S., 1977. Mechanism of carbon fractionation associated withlipid synthesis. Science 197, 261263.

Drew, R.A.I., Yuval,B., 2000. Theevolution of fruit flyfeeding behavior.In: Aluja,M.,Norrbom, A.L. (Eds.), Fruit Flies (Tephritidae): Phylogeny and Evolution ofBehavior. CRC Press, Boca Raton, FL, USA, pp. 731749.

Drew, R.A.I., Courtice,A.C.,Teakle, D.S., 1983. Bacteria as a natural sourceof food foradult fruit flies (Diptera: Tephritidae). Oecologia 60, 279284.

FAO/IAEA/USDA, 2003.Manual for Product Quality Control andShippingProceduresfor Sterile Mass-reared Tephritid fruit flies, Version 5.0. International AtomicEnergy Agency, Vienna, Austria, p. 85.

Field, S.A., Yuval, B., 1999. Nutritional status affects copula duration in the Medi-terranean fruit fly, Ceratitis capitata (Insecta Tephritidae). Ethology Ecology &Evolution 11, 6170.

Garca, R., Municio, A.M., Viloria, D., 1980. Glycerol phosphate and monoacylgly-

cerol pathways of triacylglycerol biosynthesis in microsomes of Ceratitis capi-tata. Comparative Biochemistry and Physiology Part B: Biochemistry andMolecular 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. Influence of adult nutrition upon the reproduction of three fruitflyspecies. In:Third SpecialReport on theControlof theOrientalfruitfly (Dacusdorsalis) in the Hawaiian Islands, Senate of the State of California, USA, pp. 7276.

Hansen, E.,1970. Proteins.In: Hume, A.C. (Ed.),The Biochemistryof Fruitsand TheirProducts. 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 fly Bactrocera cucur-bitae (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 dietaryprotein on male melon fly 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 fly (Diptera: Tephritidae)

in nature: location and diel pattern of feeding and other activities on fruitingand nonfruiting hosts and nonhosts. Annals of the Entomological Society ofAmerica 83, 632641.

Hendrichs,J., Prokopy, R.J.,1994. Foodforaging behavior of frugivorousfruitflies. In:Calkins, C.O., Klassen, W., Liedo, P. (Eds.), Fruit Flies and the Sterile InsectTechnique. CRC Press, Boca Raton, FL, USA, pp. 3756.

Hendrichs, J., Katsoyannos, B.I., Papaj, D.R., Prokopy, R.J., 1991. Sex differences inmovement between natural feeding and mating sites and tradeoffs betweenfood consumption, mating success and predator evasion in Mediterranean fruitflies (Diptera: Tephritidae). Oecologia 86, 223231.

Hendrichs, J., Lauzon, C.R., Cooley, S.S., Prokopy, R.J., 1993. Contribution of naturalfood 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. Medfly areawide sterile

insect technique programmes for prevention, suppression or eradication: theimportance 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 fly, Dacus cucurbitae Coquillet(Diptera: Tephritidae). Applied Entomology and Zoology 24, 152154.

Hood-Nowotny, R., Knols, B.G.J., 2007. Stable isotope methods in biological andecological studies of arthropods. Entomologia Experimentalis et Applicata 124,316.

Hood-Nowotny, R., Mayr, L., Knols, B.G., 2006. Use of carbon-13 as a populationmarker for Anopheles arabiensis in a sterile insect technique (SIT) context.Malaria Journal 5, 6.

Hooper, G.H.S., 1987. Applicationof quality control proceduresto largescale rearingof the Mediterranean fruit fly. Entomologia Experimentalis et Applicata 44,161167.

IAEA, 2000. Use of Isotope and Radiation Methods in Soil and Water Managementand Crop Nutrition. Training course series No. 14, International Atomic EnergyAgency, IAEA, Vienna, Austria.

IAEA, 2009. Manual for the Use of Stable Isotopes in Entomology. InternationalAtomic Energy Agency, IAEA, Vienna, Austria.

Iwahashi, O., Ito, Y., Shiyomi, M., 1983. A field evaluation of sexual competitivenessof sterile melon flies, Dacus (Zeugodacus) cucurbitae. Ecological Entomology 8,4348.

Jacome, I.,Aluja, M.,Liedo, P.,Nestel, D.,1995. Theinfluenceof adult diet andage onlipid reserves in the tropical fruit fly Anastrepha serpentina (Diptera: Tephriti-dae). 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 parasitoidreleases in an areawide integrated melon fly (Diptera: Tephritidae) controlprogram 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 andControl, 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 melonfly from the Okinawa Islands, Japan. II. Actual control program. In: Aluja, M., Liedo, P. (Eds.), Proceedings ofthe 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 competi-tiveness but reduces longevity of mass-reared sterile male Mediterranean FruitFlies (Diptera: Tephritidae). Annals of the Entomological Society of America 93,949955.

Kehayias, J.J., Heymsfield, S.B., LoMonte, A.F.,Wang, J., Pierson Jr., R.N., 1991. In vivodetermination of body fat by measuring total body carbon. American Journal ofClinical Nutrition 53, 13391344.

Koyama, J., Kakinohana, H., Miyatake, T., 2004. Eradication of the melon fly,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. Thefeasibility of measuringtotalbody carbonby counting neutron in elasticscatter

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 fly (Ceratitis capitata): pupal metabolism inrelation to mass rearing techniques. Entomologia Experimentalis et Applicata15, 2334.

Maor, M., Kamensky, B., Shloush, S., Yuval, B., 2004. Effects of post-teneral diet onforaging success of sterile male Mediterranean fruit flies. Entomologia Experi-mentalis et Applicata 110, 225230.

Maynard Smith, J., Bozcuka, A.N., Tebbutta, S., 1970. Protein turnover in adultDrosophila. 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 fly,Bactrocera cucurbitae Coquillett (Diptera: Tephritidae). Annals of the Entomo-logical Society of America 97, 10261033.

Medeiros, L., Zucoloto, F.S., 2006. Nutritional balancing in fruit flies: Performanceof wild adult females of Anastrepha obliqua (Diptera: Tephritidae) fed onsingle-food or food-pair treatments. Journal of Insect Physiology 52, 11211127.

Municio, A.M., Odnozola, J.M., Pineiro, A., Ribera, A., 1973. In vitro and in vivo [I4C]

acetate incorporation during developmentof insects. Insect Biochemistry 3, 1929.

Nakamori, H., Kuba, H., 1990. Aerial distribution of sterile melon files, Dacuscucurbitae Coquillett, anesthetized by chilling. Japan Agricultural ResearchQuarterly 24, 3136.

Nestel, D., Galun, R., Friedman, S., 1985. Long-term regulation of sucrose intake bythe adult Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Journal ofInsect Physiology 31, 533536.

Nestel,D., Galun, R.,Friedman, S.,1986.Balance energetico on el adultoirradiado deCeratitis capitata (Wied.) (Diptera: Tephritidae). Folie Entomologica Mexicana70, 7585.

Nestel, D., Nemny-Lavy, E., Chang, C.L., 2004. Lipid and protein loads in pupatinglarvae and emerging adults as affected by the composition of Mediterraneanfruit fly (Ceratitis capitata) meridic larval diets. Archives of Insect Biochemistryand Physiology 56, 97109.

Pagani, R., Suarez, A., Municio, A.M., 1980. Fatty acid patterns of the majorlipid classes during development of Ceratitis capitata. ComparativeBiochemistry and Physiology Part B: Biochemistry and Molecular Biology

67, 511518.Parthasarathy, R., Tan, A., Sun, Z., Chen, Z., Rankin, M., Palli, S.R., 2009. Juvenilehormone regulation of male accessory gland activity in the red flour beetle.Tribolium castaneum. Mechanisms of Development 126, 563579.

Pereira, R., 2005. Influence of a juvenile hormone analog and dietary protein onmale Caribbean fruit fly, Anastrepha suspensa (Diptera: Tephritidae), sexualbehaviour. PhD Dissertation, University of Florida, Gainesville, FL, USA.

Pereira, R., Sivinski, J., Teal, P.E.A., 2009. Influence of methoprene and dietaryprotein on male Anastrepha suspensa (Diptera: Tephritidae) mating aggrega-tions. Journal of Insect Physiology 55, 328335.

Pereira, R., Sivinski, J., Teal, P.E.A., 2010. Influence of a juvenile hormone analog anddietary protein on male Anastrepha suspensa (Diptera: Tephritidae) sexualsuccess. Journal of Economic Entomology 103, 4046.

Perez-Staples, D., Prabhu, V., Taylor, P.W., 2007. Post-teneral protein feedingenhances sexual performance of Queensland fruit flies. Physiological Entomol-ogy 32, 225232.

Prokopy, R.J., Hsu, C.L., Vargas, R.I., 1993. Effect of source and condition of animalexcrement on attractiveness to adults of Ceratitis capitata (Diptera:Tephritidae). Environmental Entomology 22, 453458.

777777777777

7777777777777777777

7777777777777777777777777777777777777

7777777777777777777


G Model

IP 2546 19



http://dx.doi.org/10.1016/j.insphys.2010.04.018http://dx.doi.org/10.1016/j.insphys.2010.04.018http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://dx.doi.org/10.1016/j.jinsphys.2010.07.011http://dx.doi.org/10.1016/j.insphys.2010.04.018


9/9

Roessler, Y., 1989. Insecticidal bait and cover sprays. In: Robinson, A.S., Hooper, G.(Eds.), World Crop Pests: Fruit Flies, Their Biology, Natural Enemies andControl, 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. Per-gamon, Oxford, pp. 363389.

Sharp, J.L., Chambers, D.L., 1984. Consumption of carbohydrates, proteins, andamino acids by Anastrepha suspensa (Loew) (Diptera: Tephritidae) in thelaboratory. Environmental Entomology 13, 768773.

Shelly, T.E., Kennelly, S., 2002. Influence of male diet on male mating success andlongevity and female remating in the Mediterranean fruit fly (Diptera: Tephri-

tidae) under laboratory conditions. Florida Entomologist 85, 572579.Shelly, T.E., Edu, J., Pahio, E., 2007. Condition-dependent mating success in malefruit flies: ingestion of a pheromone precursor compensates for a low-qualitydiet. Journal of Insect Behavior 20, 347365.

Sivinski, J., 1993. Longevity and fecundity in the Caribbean fruit fly (Diptera: Tephri-tidae): effects of mating, strain and body size.Florida Entomologist76, 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 fly, Dacuscucurbitae Coquillett (Diptera: Tephritidae). Japanese Journal of Applied Ento-mology and Zoology 22, 6067.

Teal, P.E.A., Gomez-Simuta, Y., Proveaux, A.T.,2000. Mating experience and juvenilehormone enhance sexual signaling and mating in male Caribbean fruit flies.Proceedingsof theNationalAcademyof Sciencesof theUnitedStatesof America97, 37083712.

Teal, P.E.A., Gavilanez-Slone, J.M., Dueben, B.D., 2004. Effects of sucrose in adultdiet 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 ofPsyttalia fletcheri (Hymenoptera: Braconidae) andsterile flies to suppress melon fly (Diptera: Tephritidae) in Hawaii. Journal ofEconomic Entomology 97, 15311539.

Varttsky, D., Ellis, K.J., Cohn, S.H., 1979. In vivo quantification of body nitrogen byneutron capture prompt gamma-ray analysis. The Journal of Nuclear Medicine20, 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 optimalnutrient mix from defined 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 adultMediterranean fruit flies. Physiological Entomology 21, 151158.

White, I.M., Elson-Harris, M.M., 1992. Fruit Flies of Economic Significance: TheirIdentification 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 andControl, vol. 3A. Elsevier, The Netherlands, pp. 141151.

Yuval, B., Kaspi, R., Shloush, S., Warburg, M.S., 1998. Nutritional reserves regulatemale participation in Mediterranean fruit fly leks. Ecological Entomology 23,211215.

Yuval, B., Kaspi, R., Field, S.A., Blay, S., Taylor, P.W., 2002. Effects of post-teneralnutrition on reproductive success of male Mediterranean fruit flies (Diptera:Tephritidae). Florida Entomologist 85, 165170.

Zera, A.J., Zhao, Z., 2004. Effect of a juvenile hormone analogue on lipid metabo-lism in a wing-polymorphic cricket: implications for the endocrine-biochem-ical bases of life-history trade-offs. Physiological and Biochemical Zoology77, 255266.

888888888888

8888888888888888888


G Model

IP 2546 19


the melon fly Bactrocera cucurbitae: Effects of methoprene a juvenile hormone analogue and of diet supplementation with hydrolyzed

Documents

Ihsan.prot.Lipid.article