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Running Head: Lipid and Protein Contents during Medfly Aging

Trends in Lipid and Protein Contents during Medfly Ageing: An Harmonic Path to Death

David Nestel, Nikos T. PapadopoulosZ, PabloLiedo3, Lilia Gonzales-Ceron4 and James. R. Carey5

El Colegio de la Frontera Sur (ECOSUR), Tapachula, Chiapas, Mexico

1 Corresponding Author, Institute of Plant Protection, The Volcani Center, P.O. Box 6, 50250 Beit-Dagan, Israel. nestel@agri.gov.il 2 University of Thessaly, Dept. of Agricultural Crop Production and Rural Environment, Phytokou st. N, 38446N Ionia Magnisias, Greece. 3 El Colegio de la Frontera Sur, Carretera Antigun Aereopuerto Km 2.5, 30700, Tapachula, Chiapas, Mexico. a Dept. of Parasitology, CIP-INSP, Tapachula, Chiapas, Mexico. 5 Department of Entomology, University of California, Davis, CA 95616, USA

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ABSTRACT Survival and egg-laying trends were investigated in Mediterranean fruit flies (Ceratitis

capitata) adults maintained on a sucrose only diet, or on a full diet which consisted of a 3:1

sucrose and yeast hydrolizate mixture. In addition, we followed the total individual lipid and

protein contents of aging flies. Survival trends, and life expectancy parameters at eclosion, for

males and females on full diet and for males on sucrose only were very similar. In contrast

mortality of females on sucrose only was high early in life, but then slowed down. Egg-laying

was ten times greater in female flies on full diet than in flies on sucrose only. Lipid contents in

males and females on both types of diets were very similar, and harmonically oscillated with

amplitude of approximately 10 days. Successive crests of lipids tended to be smaller with the

ageing of the cohort, and lipids contents significantly drop at very advanced ages and close to

the maximal age of the whole cohort. Protein contents of flies on full diet were maintained

high and at a constant level throughout the entire life of the cohort. Protein levels in males

and females on sucrose only dropped drastically during the first days of adult life, but then

stay stable at a low level until advanced ages. We propose that the synchronous rhythmic

oscillation in lipid contents of male and female flies seems to be independently set by an

internal clock. Protein reserves are allocated according to the access to protein food sources

and these levels of protein are closely associated to egg production and mortality. Our results

are discussed in view of resource allocation during reproduction and senescence. Keywords:

senescence, Ceratitis capitata, Tephritidae, rhythmic patterns, energy allocation, lipid, protein.

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INTRODUCTION

The allocation of energy between reproduction and somatic maintenance has been at

the center of theories related to the ageing process. Longevity has usually been associated

with reproductive tradeoffs (the "antagonistic pleiotropy theory") (Arking et al., 2002). That

is, the theory states that the organism will sacrifice energy aimed for reproduction and divert

this energy to increase longevity. Several studies have tried to demonstrate this theory by

comparing metabolic rates (measured by the production of COz and/or the consumption of 02)

in reproductive-young and aged organisms and by comparing reproductive parameters among

strains that have different longevity. In general, the empirical evidence has been unable to

proof this asseveration, and these studies have not been able to clearly show differences in

metabolic rate between aged and young organisms, or between strains selected for a longer

longevity (Arking et al., 2002; Promislow and Haselkom, 2002).

Recent studies showed that Mediterranean fruit fly (Medfly) (Ceratitis capitata)

females in the laboratory are able to shift from a waiting mode (low mortality and

reproduction) to a reproductive mode (where mortality accelerates at the onset of egglaying)

by changing the contents of their adult diets (from only sucrose to sucrose + protein) (Carey

el al., 1998) . Further studies showed that pulses of protein at different age intervals induce

cyclical egg production and strongly affects life expectancy of female Medflies (Carey et al.,

2002). That is, diet seems to play a key role in modulating age patterns of fertility and

mortality in fruit flies (Butov et al., 2003). We took advantage of this fact to study the effects

of constant diet conditions (sucrose only and sucrose + protein diets) on the pattern trends of

energetic metabolites throughout adult

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life of male and female medflies. We hypothesized that protein restrictions in the adult diet will

have an effect upon the allocation of energy in the flies, which we expected to be expressed in

the total lipid and protein contents of individual ageing flies. We also expected to indirectly

observe metabolic changes in ageing flies related to reproduction, senescence and death by

following the lipid and protein patterns throughout the entire life of the flies.

MATERIALS AND METHODS

Insects Flies used for this study were collected as pupae from the mass-rearing facility at

Metapa, Chiapas, Mexico, from the regular bisexual rearing strain (Schwarz et al., 1985).

Adult Maintenance and Food Experiments

Approximately 3000 pupae were placed in each 40 X 80 X 10 cm aluminum frame

mesh covered cage ("cohort cage"). Flies from these cohort cages were used to determine

mortality as affected by adult food and age. The number of dead flies per cage was recorded

daily. Flies at different ages were also sampled from these cages for lipid and protein

determination. For each type of treatment we established 10 simultaneous cohort cages.

Treatments consisted on allowing adult flies to feed on sucrose only or on a full adult diet

(sucrose and yeast protein hydrolyzate at a 3:1 ratio). Flies were allowed free access to water

during the whole experiment. Laboratory temperatures ranged from 25 to 27 °C. Relative

humidity was 65 to 85%. Light:Dark photoperiod was 15:9 h.

To determine egg-laying patterns, small plastic 5 cm in diameter by 10 cm long

cages ("couple cages") were used. These cages have a mesh cover where females laid their

eggs. A single pair of Medflies was placed in each cage and 100 couple cages were

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set up for each feeding treatment (sucrose only and full adult diet). The number of eggs laid

per each female was recorded daily

Mortality and fecundity recording was carried out until the last fly died. At 40 days,

most of the individuals, both in the large cohort cages and the small plastic couple cages,

were already dead. Therefore, sampling for lipid and protein analysis was stopped at age 35.

Lipid and Protein Determination Flies maintained on the cohort cages were sampled every two days, starting from the

2°d day after adult eclosion and until the age of 30 days. An additional sample was taken when

the flies were 35 days old. On every sampling date, a total of 20 living flies were removed

from each of the 10 cages (a total of 200 flies per sampling date and treatment) and

immediately freeze at -20 °C for later chemical analysis. Protein and lipid determinations were

performed on individual flies. For every date 9-10 flies were used for chemical determinations.

Protein contents were determined on individual flies with the Bradford method after extraction

on Phosphate Buffer Saline (PBS) (Yuval et al., 1998). Lipids were extracted from individual

flies with a chloroform-methanol separation method and quantified using the vanillin in

phosphoric acid colorimetric determination (Nestel et al., 2003).

Statistical Analysis Survival and reproductive parameters for flies maintained on the two types of diets

were calculated. Longevity per sex and diet for the whole cohort in the "cohort cages" was

obtained from the total group of flies in the 10 cages. Average and variance in life expectancy

at adult emergence (eo) for flies maintained in the large cohort cages were

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estimated from the 10 replicate cages established for each of the diet treatments. These

data were then used to compare effects of diet type and sex upon life expectancy using

a one-way ANOVA (Statgraphics Plus, 2000). Life expectancy at adult emergence was

also calculated for the 50 pairs of flies maintained on the small plastic cages ("couple

cages"). Mean number of eggs per female maintained on the two diet types was derived

from the 50 small couple cages. Differences in egg-laying between the two diets were

analyzed by Mann-Whitney analysis (Statgraphics Plus, 2000). General Linear Models

(Statgraphics Plus, 2000) were applied to the lipid and protein contents data to infer on

the effects of diet type, sex and adult age on these variables. To investigate harmonic

trends through time on the lipid and protein data, Fourier series up to 3 terms (e.g.,

sinus 3X and cosines 3X) were used (Israely et al., 1997). The ability of the predicted

models to describe the observed results was inferred from a multiple regression analysis

(Statgraphics Plus, 2000).

RESULTS

Demographic Parameters

Survival trends of males in cohort cages were similar for insects fed on sucrose

only and on a full adult diet mix (Fig. 1). In contrast, survival trends differed between

females fed on a full adult diet and those on sucrose only. Mortality in females on

sucrose only was stronger during the first days of adult life, but then those that survived

the first mortality wave, lived for a longer period of time than females fed on full adult

diet (Fig. 1). Females on a full adult diet maintained higher survival levels at the

beginning of adult life, but after two weeks their mortality rate sharply increased (Fig. 1).

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significant in the case of females, both in the cohort cages and couples cages. In the case of

males, this difference was considerable only in the case of the couples cages (Table 1). In

general, life expectancy values at adult eclosion in the couple cages hosting pairs of flies were

much higher than on large and densely inhabited cohort cages (table 1). However, survival

patterns as affected by sex and adult food were similar in both types of cages.

Egg production by females fed a full adult diet mix was 6-fold greater than the average

production of eggs by females fed sucrose only (Table 1). Females on a full adult diet mix also

started to lay eggs a day earlier than females fed on sucrose only (Table 1). Daily patterns of

egg-laying in females fed the two types of diets are shown in Fig. 2. Females on sucrose only

showed a small initial peak of egg-laying at the beginning of adult life, which rapidly declined.

Females on a full adult diet showed a greater fecundity throughout their life span, with a peak

between 8 and 20 days-old.

Lipid and Protein Patterns during Aging

Lipid trends throughout adult life in both sexes of flies fed the two types of diets are

shown in Fig. 3. Lipid contents, which oscillate with age (Fig. 3), were significantly

affected by adult age (F15,57] = 5 1.6, P < 0.01). Similarly, adult diet and fly sex significantly

affected lipid contents in the flies (for diet F1,571 = 8.9, P < 0.01; for sex F1,571 = 9.8, P <

0.01). In general, females had larger loads of lipids than males, and lipid contents were larger

in sucrose fed insects than in flies fed the full adult diet mix. Higher lipid contents in sucrose

fed insects was more obvious during the first days of adult life (Fig. 3). The effect of diet

type paralleled in both sexes (the interaction between these two variable was not significant:

F1,571 = 0.15, P = 0.7).

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The adjustment of Fourier series to the lipid data was in general good. The coefficient

of determination for lipid trends in males fed on sucrose only diet was RZ = 0.63, while that for

males fed on a full adult diet mix was Rz = 0.56. Lipid trends in females fed on a sucrose only

diet had a coefficient of determination for the adjusted model of RZ = 0.61, while lipids in

females fed on a full adult diet adjusted also well to the fitted model (RZ = 0.58). The expected

sinusoidal trends, which are based on the fitted models, for the two sexes and diets are shown

as inserts in Fig. 3. Besides the first wave in lipid contents in male and females fed on the two

diets, the model closely approaches the trends shown by the data. The divergence between the

model and data during the first days is due to a drop of lipids in the data set during the 2°d and

4`" day (Fig. 3). The model was not able to simulate this situation. Both, the data and fitted

models, show 3 distinguishable waves (e.g., crests and sills) during the lifespan period. In all

cases (e.g., males and females fed on the two diet types), waves have a more or less similar

longitude of approximately 10 days, and the amplitude of waves is quite close. Of interest is

the fact that the 4 models (and data) are very similar and males and females feeding on the

two types of diet showed a synchronous trend in lipid contents. In addition, and as shown by

the data, the models simulates a steady decline in lipids from crest to crest, and a sharp drop in

lipid contents at age 35.

Protein contents were significantly affected by adult age (F15,563 = 15.5, P< 0.01), by

sex (Fi,s63 = 90.3, P< 0.01) and diet (FI,s66 = 1141.2, P< 0.01). Protein contents in males

and females fed on a full adult diet were maintained at a high and constant level, while protein

contents in flies maintained on a sucrose only diet sharply drop during the first days of adult

life (Fig. 4). Afterwards, protein contents in flies fed on a sugar only

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diet continue to decline but at a lower pace (Fig. 4). In contrast to lipid content trends, there is a

significant interaction between sex and diet in protein contents (FI,563 = 8.49, P < 0.01),

suggesting that there is no synchrony in protein trends between sexes and diet type. The

adjustment of Fourier series for protein trends on male and female flies maintained on a

sucrose only diet was good (Rz = 78.9 for males and RZ = 52.5 for females). The adjusted

models are shown as an insert in Fig. 4. In contrast, model fitting to the protein content trends

in males and females fed on a full adult diet was low and non significant (RZ = 31.3 for males

and RZ = 21.7 for females). These results suggest that protein contents in flies fed a full adult

diet mix can not be satisfactorily explained by a rhythmic pattern which is based on modeling

with Fourier series.

DISCUSSION

As expected for Tephritidae fruit flies (see for example, Webster et al., 1979; Jacome

et al., 1995; Carey et al., 1998 and 2002), females feeding on a full adult diet mix, which

includes protein hydrolyzate, produced several folds more eggs than females fed on sucrose

only. Moreover and as previously shown (Carey et al., 2002), protein fed female medflies

where able to maintain egg production throughout most of their adult life, with an intensive

egg-laying stage early in adult life, which declined with age. Although life expectancy at

eclosion was different from what was reported by Carey et al. (1998), the trajectory of the

survival curves were similar in these two separated studies, with high mortality at early ages

for flies on sucrose only diet, a cross over at about middle age and lower mortality at older

ages. This discrepancy in the expectation of life at eclosion between these two studies could

be attributed to differences in the numbers of flies dying at each age interval, but the general

pattern was the same. Life

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expectancy differences between large crowded cohort cages and small couple cages are

consistent with previous studies (Carey et al., 1992).

Previous studies on adult medfly lipid patterns were taken up to the stage where

reproductive activity was at its maximum (Nestel et al., 1985; Warburg and Yuval, 1996).

Nestel et al. (1985) showed a decline of lipids during the first eight days of adult life, while

Warburg and Yuval (1996) showed a decline in lipids with a subsequent recovery of lipids to

teneral levels at day 5. Differences between the two studies were probably related to the

frequency of lipid determinations in time (e.g., once each 4 days in Nestel et al.) and to the

diet types (Nestel et al. were using suboptimal sucrose solutions). As far as we are concerned,

this seems to be the first study in Tephritidae that follows the lipid and protein patterns of

individual flies throughout most of their adult life with a very intense sampling frequency.

The results of the present study showed a very interesting, and unexpected, pattern of total

lipid contents throughout adult life. Regardless of the diet type, lipids were shown to

harmonically oscillate with a certain periodicity at more or less the same levels in males and

females (Fig. 3). Small statistically significant differences in lipid levels were only seen early

in life, and were related to diet: sucrose fed males and females showed a higher peak than

protein fed medflies during the first oscillation. This was probably related to a compensation

effect derived from the lack of protein in the sucrose only diet, and the intensive use of

endogenous proteins during these first days of adult life. Successive lipid crest-levels also

seem to decrease with age in the two sexes and diets, and average lipid levels close to the

maximal longevity age of most of the flies in the cohort drops to very low levels.

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Total lipids patterns in both sexes of Anastrepha serpentina, which were investigated

during the early stages of adult life, were shown to synchronously fluctuate when flies are fed

with a protein food source (Jacome et al., 1995). However, and in contrast to our result, lipids

in A. serpentina maintained on a food regime with no protein sources do not oscillate and

steadily decline (Jacome et al., 1995). While in the case of A. serpentina lipid trends on the

different diets were suggested to be linked to egg production waves, the results of the present

study does not allow such a straightforward conclusion. During vitellogeneis of insects

appreciable quantities of lipids are known to be deposited in the eggs (Downer and Matthews,

1976). Thus, it is logical to expect that the lipid waves seen in females fed protein hydrolyzate

are in fact related to the eggproduction waves observed in this study (Fig. 2). The fact that

lipid trends in males maintained in the two diet types and in females fed on sucrose (who

produced a single wave of eggs early in life, Fig. 2) are very similar and synchronous,

preclude us from accepting this simple link between egg-production and lipid trends.

Moreover, the results of this study suggest that the medfly seems to have a predetermined

mechanism for lipid regulation that functions independent of the capacity of the female fly

to lay eggs, and which is mutual to both sexes. Previous studies have shown the existence of

diel-rhythms of lipid and sugar levels in insects (see for example, Das et al., 1993). However,

as far as we are concern there are no reports on rhythmic processes of lipid regulation in

insects that oscillate in cycles longer than a day (i.e., circadian) throughout all their adult life.

Such an independent endogenous long-term regulation mechanism (i.e., "endogenous

biological clock") of lipids has been observed in trout fish (Wallaert and Babin, 1994). The

circannual variation in lipids and lipoproteins observed in this fish is independent of

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age, sex and sexual maturity, and seems to be synchronized by photoperiod (Wallaert

and Babin, 1994). The synchronization of medfly lipids in the two sexes and in the two

diet types may be related to their seclusion in the same environment: the whole

experiment was simultaneously run with cages laying side by side, resulting in an almost

equal exposure of the flies to the physical environment (i.e., photoperiod).

Synchronization of lipid levels could also result from the exposure of flies to a similar

chemical environment (e.g., pheromone concentration in the air). Recently, Weller

(1998) showed that humans can modify their ovulation clock when exposed to the

axillary odors of females passing through the follicular stage of the ovulatory cycle,

demonstrating that chemical communication in humans and animals can be an element in

the establishment of "oestrous synchrony". Chemical communication may have also play

a role in the synchronization of medfly lipid oscillations, and probably of other metabolic

and behavioral systems. The functionality of this synchronization is not yet known, but

may enhance the probability of both sexes to found themselves at the same sexual

maturation stage, thus increasing their chances of mating and reproducing.

The drop of lipids contents in the two sexes and diets close to the maximal

longevity of the cohort suggest that this is a change during senescence that seems to

precede death. During all the study we only sampled living organisms in order to avoid

decomposition effects upon the lipid and protein metabolites of the fly. Thus, we have no

information on the immediate metabolic changes related to lipids and protein occurring

before death of the flies. However, previous studies have shown that starved flies sharply

drop their total lipid contents, and that this drop seems to correlate with their survival

capacity (Nestel et al., 1985; Jacome et al., 1995). Moreover, Tower (1996) suggested

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that the capacity of metabolic reserves in Drosophila flies seems to limit the lifespan of the

flies. Based on these studies, it is reasonable to suggest that the sharp drop in lipids during

the last phase of the cohort lifespan is probably related to changes in senescence and may be

a symptom that precedes medfly death.

The fitting of the harmonic models to the protein data was low, and there does not

seem to be a clear rhythmic pattern of protein catabolism and anabolism. The effect of diet on

total protein loads was very significant and affected the two sexes similarly (Fig. 4). Although

autogeny exists in the tephritid fruit flies, and a few eggs are produced from protein sources

derived from the larval - pupal stage, continuous production of eggs and attainment of

maximal reproductive capacity requires the continuous ingestion of protein building-blocks

during adult life (Jacome et al., 1995; Wheeler, 1996, Carey et al., 2002). Our results support

this assumption. The inability of sucrose fed flies to replenish protein levels seem to affect their

capacity to produce eggs. In addition, the initial drop of protein levels in the two diets suggest

that there is a link between egg production and protein utilization, and that probably the first

batch of eggs is mainly produced from protein derived from the pupal stage. An additional

interesting result is the fact that male protein levels are affected by diet similarly to that seen

on females. This result suggests that males may also have energetic demands that parallel

those of females.

In contrast to lipid content patterns, protein trends do not provide any clue on changes

related to senescence and death. However, the maintenance of protein contents at a low, but

constant level in flies fed on sucrose only supports the view of Carey et al. (2002) regarding

the allocation of amino acid resources in protein deprived flies. Based on their study of food

pulses with protein, Carey et al. (2002) suggest that a small

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fraction of amino acids in protein deprived flies are always held as reserves in case that the

fly's nutritional environment improves later in life. These amino acids will become the basis

for late-age vitellogenesis, when the environmental conditions improve. This situation, thus,

will allow the flies encountering a shortage of protein early in their life to partially

accomplish their reproductive potential even at advanced ages.

Protein and lipid reserves are essential metabolites for life. Their patterns are expected

to express the energetic demands of the organism at a given time and situation. Lipid reserves

have been shown in previous studies to reflect the energetic status of the medfly: a decrease in

lipid levels have been observed when the developing fly is in high demands of energy and its

income of energy through food intake is deficient (Nestel et al., 1985; Nestel et al., 2004) or

absent, such as in the pupal stage (Nestel et al., 2003). If lipids are in fact a reflection of the

energetic balance of the medfly, thus, the results obtained in this study suggest that the flies

throughout all their adult life were not in a negative energetic balance, at least regarding the

caloric intake provided by sucrose and the energy required for egg-laying and maintenance.

If energetically the flies were balanced during most of their adult life, thus, the rhythmic

patterns in lipid contents observed in this study point at a unique endogenous regulation of

lipid reserves which has not been previously described in fruit flies. This unique patterns of

lipid regulation throughout medfly adult life adds to a recently described exceptional

regulation of lipids during the larval-adult transition of the fly: regardless of the original lipid

levels at the time of larval pupation, the pupae seems to regulate lipid loads towards a certain

optimum level for adult emergence (Nestel et al., 2004). The metabolic aim of this

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special regulation of lipid reserves during pupation and adult life is an intriguing question that

deserves to be further investigated.

ACKNOWLEDGEMENTS Our appreciation to A. Oropeza, S.E. Salgado, R.E. Bustamante, E. De Leon, R. Rincon, S.L.

Rodriguez, and G. Rodas for technical assistance. We acknowledge the support from the

Moscamed Program in Mexico (SAGARPA, DGSV) and the Centro de Investigacion Sobre

el Paludismo (INSP). Research supported, in part, by the U.S. National Institute on Aging

(POI AG022500-01; P01 AG08761-01).

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Butov AA, Carey JR, Volkov MA, Sehl ME, Yashin Al. 2003. Reproduction and survival in

Mediterranean fruit flies: a "protein and energy" free radical model of ageing.

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Carey JR, Liedo P, Orozco D, Vaupel JW. 1992. Slowing of mortality rates at older ages in

large Medfly cohorts. Science 258: 457-461.

Carey JR, Liedo P, Muller H-G, Wang J-L, Vaupel JW. 1998. Dual modes of ageing in Mediterranen fruit fly females. Science 281: 996-998.

Carey JR, Liedo P, Harshman L, Liu X, Muller H-G, Patridge L, Wang J-L. 2002. Food

pulses increase longevity and induce cyclical egg production in Mediterranean

fruit flies. Funct Ecol 16: 313-325. Das S, Meier OW, Woodring J. 1993. Dial rhythms of adipokinetic hormone, fat body

response, and hemolymph lipid and sugar levels in the house cricket. Physiol

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Israely N, Yuval B, Kitron U, Nestel D. 1997. Population fluctuations of adult Mediterranean

fruit flies (Diptera: Tephritidae) in a Mediterranean heterogeneous agricultural region.

Environ Entomo126: 1263-1269.

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Jacome I, Aluja M, Liedo P, Nestel D. 1995. The influence of adult diet and age on lipid

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Webster RP, Stoffolano JG, Prokopy RJ. 1979. Long-term intake of protein and sucrose in

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FIGURE LEGENDS Fig. 1. Survival trend of male (upper graph) and female (lower graph) Medflies

maintained on a sugar only adult diet (----) or on a complete mix diet which include

protein hydrolyzate from yeast sources ( ).

Fig. 2. Net Fecundity (lXmx) for Medfly in the "couples cages" (50 cages). Dark shade stands

for net fecundity by pair of flies feeding a complete diet mix (sugar + protein hydrolyzate),

while clear shade stands for flies fed only sugar. Fig. 3. Life trends in average lipid

contents in male (upper graph) and female (lower graph) Medflies fed a complete diet mix

( ) or a diet consisting of only sugar (----).

Inserts showed the calculated harmonic models (Fourier series) for each diet type and sex.

Fig. 4. Life trends in average protein contents in male (upper graph) and female (lower

graph) Medflies fed a complete diet mix ( ) or a diet consisting of only sugar (----).

Inserts showed the calculated harmonic models (Fourier series) for each diet type and

sex.

i

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Table 1. Demographic parameters for adult Medfly male and female maintained on sucrose only diet and a full adult diet mix which included sucrose

and brewers yeast hydrolyzate enzymatic (3:1). Parameters relate to two types of cages and population densities: Large "cohort" cages, with

approximately 3000 individuals, and small "couple" cages holding only a pair, one male and one female, of flies. Life expectancy (and its variance) in

"cohort" type cages was calculated from 10 replicate cages running simultaneously. Life expectancy for couple cages was derived from the mortality

patterns of organisms in 50 individual cages (no variability measures are included with this data). Reproductive parameters were derived from the 50

individual cages containing each one pair of flies.

Life Expectancy at Adult Reproductive Parameters

Eclosion eo (±SD)

Cohort Cage Couple Cage Mean No. of eggs (±SD) Age (days) for egg

laying onset (50% of

females) Sucrose Only Diet Male 16.90 (±1.14) a 28.36

Female 12.40 (±1.18) b 19.96 95.25(±75.3) b 4

Full Adult Diet Mix Male 18.05 (±2.66) a 34.94

Female 19.02 (±1.65) a 26.80 659.7(±316.8) a 3

Statistics* F= 48.1 W= 108 P < 0.01 < 0.01

*Life expectancy data was analyzed with a one-way ANOVA, while differences in number of eggs per female between treatments were analyzed by the Mann-Whitney non-parametric test. Within columns, means with different letters significantly differed at P < 0.01.