Post on 30-Apr-2023
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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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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