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Research Article
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Age and Sex Related Variations in Protein and Carbohydrate Levels
of Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae)
in Constant Lightness and Darkness |
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Yesim Koc
and
Adem GUlel
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ABSTRACT
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Variations in protein and carbohydrate levels correlated
with the age and sex of Galleria mellonella (Linnaeus, 1758) (Lepidoptera:
Pyralidae) in constant lightness and darkness were investigated. Tests
were conducted under laboratory conditions at 28 ±2 °C temperature
and 65 ± 5% relative humidity. Insects were fed on combs without
honey. Protein level in 100 mg of adults increased in the first days of
adult life of females in connection with their age and then decreased.
No difference was observed in males. Carbohydrate level in 100 mg of adults
increased in both sexes in connection with their age. In all tests carbohydrate
and protein levels of females were found higher than males. Protein and
carbohydrate levels of adult G. mellonella varied in connection
with the photoperiod regimes implemented. Decrease in the nutrient levels
was observed in constant darkness.
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INTRODUCTION
Protein, lipid and carbohydrate metabolism plays role in
many vital activities of insects. Many other factors such as sex (AktUmsek,
1996; Ito and Nakata, 1998), age (Jacome et al., 1995; Seker and
Yanikoglu, 1999; Akman, 2004), developmental stages (Bozkurt, 2003), diapause
(Pullin, 1992), nutrient quality and level (Yanikoglu, 1985; Jacome et
al., 1995; Ozalp and Emre, 1998; Socha et al., 1998; George
et al., 2002), seasonal conditions (Ito, 1989; Ito and Nakata,
1998), temperature (Varer, 2005), host type in some species (AktUmsek,
1996; Akman, 2004), sexual activity (Warburg and Yuval, 1996), use of
insecticide (Sak, 2004), photoperiod (Shuxia and Adams, 2000; Nakasuji
and Mizumoto, 2001; Barsagade and Tembhare, 2002; El-Aw, 2003) are effective
on the levels of these substances.
Adult insects need certain chemicals and energy in large
quantities to maintain vital activities such as mating, food searching,
oviposition and parasitism. Therefore, having basal nutrients such as
carbohydrates, lipids and proteins in certain quantities is a necessity
(England and Evans, 1997; Olson and Andow, 1998; Ozalp and Emre, 1998;
Meats and Leighton, 2004). Necessary nutrients can be stored in larva
or pupa stages or adults can synthesize them by taking related precursor
compounds. Morales-Ramos et al. (1996) found that Catolaccus
grandis (Hymenoptera: Petromolidae) females without a carbohydrate
source die in 2.5 days at 30°C but when they are given a mixture of glucose
and fructose in equal quantity each, they can survive 18.27 days under
the same conditions. Many studies on Trichogramma show that carbohydrate
source in adult nutrition prolong life (Olson and Andow, 1998).
Proteins are the last option among the substances to be
used as fuel. Proteins are effective especially on metamorphosis, growth,
cocoon and cuticula formation and flying. Protein level varies especially
before or during metamorphosis in the developmental stage (Socha and Sula,
1992; Shuxia and Adams, 2000; El-Aw, 2003; Meats and Leighton, 2004).
Many insects need sugars for metabolic processes or for
using them as the precursor compounds. These are sensitive to carbohydrate
levels (England and Evans, 1997; Olson and Andow, 1998; Olson et al.,
2000). Carbohydrate sources, which are used by insects in the nature,
such as nectar can be instantly used in metabolic activities or converted
to glycogen or trehalose and reserved for using later (Olson et al.,
2000).
Some insects use carbohydrates as the primary source of
energy and keep excess carbohydrate as lipid and protein. Generally Diptera
and Hymenopera use carbohydrates as a main energy source (Bailey, 1975)
Factors such as photoperiod (Pullin and Wolda, 1993), age (Seker and Yanikoglu,
1999), nutrition (Yanikoglu, 1985; Ozalp and Emre, 1998) and host (Akman,
2004) are effective on carbohydrate metabolism.
G. mellonella is a harmful species for combs and
its larvae are known as causing serious damages in beekeeping (Chang and
Hsieh, 1992; Haewoon et al., 1995; Charriere and Imdorf, 1999;
Hood et al., 2003). There are not many studies available regarding
the effects of photoperiod, age and sex on carbohydrate and protein levels
of this insect. Since significant variations in carbohydrate and protein
levels can be observed in connection with age and sex, the effects of
constant lightness and darkness as well as the effects of age and sex
on quantities of the substances in question will be focused in this study.
MATERIALS AND METHODS
Large wax moth Galleria mellonella (Linnaeus, 1758)
(Lepidoptera: Pyralidae) was employed for the tests. Tests were conducted
under laboratory conditions at 28±2°C; 65±5% relative humidity and in
constant lightness or darkness. Studies started by establishing successive
laboratory stock cultures of G. mellonella. Methods of Koc and
GUlel (2006) were used for establishing the stock culture, distinguishing
adults by sexes and grouping by age.
Insects were let to proliferate in jars at predefined constant
temperature and humidity in constant darkness (DK) or constant lightness
(DA). One day old adults were weighed and stocked on the first day of
their adult life. Ten adults matured on the same day from five and fifteen
days old groups put into a jar and were fed on comb without honey. Five
of them were weighed five days later and another five of them 15 days
later, respectively. Processes for the mentioned three age groups were
repeated three times with samples taken from the population at different
times. For each analysis, 15 adult insects from a certain age group were
weighed and stocked in 1.5 mL Eppendorf tubes at -50°C until they had
been analyzed.
Biochemical analysis: For carbohydrate and protein analysis, one,
five and fifteen days old adults stocked at -50°C were used.
Protein analysis: First of all a standard protein graph was created
for specifying protein values that will be obtained through protein analysis.
About 0.1% bovine serum albumin was used. The standard stock solution
in 1 mg mL-1 concentration was prepared. Then standard protein
solutions in 10, 20, 25, 30, 40, 50, 75 and 100 µg mL-1 concentrations
were prepared through serial dilutions of the standard stock solution.
Lowry method was applied to these solutions and the absorbance values
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Fig. 1: |
Standard protein graph |
monitored by a spectrophotometer at 695 nm wavelength against
blank. These processes were repeated three times for each concentration
of the standard solution. Standard protein graph was created by using
the absorbance values obtained (Fig.
1).
Total protein analysis in the stocked samples was based
on the method developed by Lowry et al. (1951). The samples weighed
and kept at -50°C were transferred into larger tubes for homogenization
after they had been kept at room temperature for a while. Each tube had
one insect in it and 5 mL work buffer was added to the tubes. Each insect
was homogenized at 8,000 rpm for 7 min. The homogenate was centrifuged
at room temperature at 3,500 rpm for 15 min. About 100 µL of the supernatant
generated in the tube at the end of the centrifuging was processed with
Lowry method and monitored with a spectrophotometer at 695 nm wavelength
against blank. The monitored absorbance values were evaluated by using
the standard protein graph.
Carbohydrate analysis: First of all a standard carbohydrate graph
was created for specifying carbohydrate values that will be obtained through
carbohydrate analysis. 0.1% glucose solution was used. Then glucose solutions
in 25, 50, 75 and 100 µg mL-1 concentrations were prepared
through serial dilutions of the stock solution. These standard solutions
were put into reaction with Anthron reactive agent and heated at 90°C
for 15 min. The color was changed. Then the tubes were cooled, stirred
and the absorbance values were monitored at 695 nm wavelength with a spectrophotometer.
These processes were repeated three times (Fig.
2).
Van Handel (1985) Anthrone test was employed for measuring
the carbohydrate level in the samples stocked for analysis. The samples
stored in a deep freezer at -50°C were kept at room temperature for a
while and each of them homogenized at 8000 rpm for 7 min in 2.5 mL 2%
Sodium Sulfate. One hundred and fifty microliter supernatant was taken
from the samples centrifuged at
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Fig. 2: |
Standard carbohydrate graph |
room temperature at 16000 rpm for 2 min after they had been stirred.
2 mL Anthrone was added to the samples and the tubes heated at 90°C for
15 min. The color was changed. Then the tubes were cooled on ice, stirred
and monitored at 625 nm against blank. Carbohydrate levels were measured
by using the standard carbohydrate graph.
Statistical assessment of the data collected: One Way Analysis
of Variance (ANOVA) was used for the comparison of more than two groups.
Averages were assessed by using Student-Newman-Kuel (SNK) Test, when the
test results were significant. Independent Two Samples t-test was employed
for the comparison of two groups and a = 0.05 confidence limit was taken
as basis.
RESULTS
Other results in connection with the protein analysis: Result
of age and sex related protein level analysis on adult G. mellonella
kept in constant darkness is given in Table 1.
SH-standard error: As it is shown in Table 1,
protein levels are different in male and female adults. In females, the
protein level was increased in the first five days of adult life but then
decreased afterwards. In males, the protein level kept increasing after
the fifth day of adult life. The average protein level was 4.31 mg in
100 mg of adult females one day older than adults from the same age group,
while the males from the same age group had 4.01 mg protein, in average.
The average protein levels of five days old females and
males were 5.51 and 5.11 mg, respectively, while the levels were 4.91
and 5.43 mg in fifteen days old ones. The difference of protein levels
between sexes for one and five days old adults are statistically insignificant
(p>0.05), whereas the difference between sexes is significant for fifteen
days old adults (p<0.05). The difference in protein levels between the
first day of adult life and the 15th day is significant (p<0.05) for both
sexes of G. mellonella adults.
Table 1: |
Protein level of adult G. mellonella
in constant darkness |
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*Average of 3
repeated processes each with 5 beings, **Significance level between
the protein levels of males and females from the same age group,
Average differences in the same column with the same letter(s)
are not significant (p<0.05) |
Table 2: |
Protein level of G. mellonella
in constant light |
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*Average of 3
repeated processes each with 5 beings, **Significance level between
the protein levels of males and females from the same age group,
Average differences in the same column with the same letter(s)
are not significant (p<0.05) |
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Fig. 3: |
Age and sex related
protein level of G. mellonella in constant darkness |
The protein level of males in constant darkness was increased in connection
with age, whereas it was decreased in females after the fifth day of adult
life ( Fig. 3).
Results of age and sex related protein level analysis on adult G.
mellonella kept in constant lightness are given in Table
2.
In females, the protein level was increased in the first five days of
adult life but then decreased afterwards (Table 2). In
males, the protein level kept increasing after the fifth day of adult
life. The average protein level was 4.91 mg in 100 mg of adult females
one day older than adults from the same age group, while the males from
the same age group had 4.61 mg protein, in average. The difference of
protein levels between sexes for five and fifteen days old adults are
statistically insignificant (p>0.05), whereas the difference between sexes
is significant for one days old adults (p<0.05). The difference in protein
levels the first day of adult life and the 15th day is significant (p<0.05)
for both sexes of G. mellonella adults.
Table 3: |
Carbohydrate level
of adult G. mellonella in constant darkness |
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*Average of 3
repeated processes each with 5 beings, **Significance level between
the protein levels of males and females from the same age group,
Average differences in the same column with the same letter(s)
are not significant (p<0.05) |
Table 4: |
Carbohydrate level of adult G. mellonella
in constant light |
 |
*Average of 3
repeated processes each with 5 beings, **Significance level between
the protein levels of males and females from the same age group,
Average differences in the same column with the same letters are
not significant (p<0.05) |
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Fig. 4: |
Age and sex related
protein level of G. mellonella in constant light |
Results in connection with the carbohydrate analysis: Results
of age and sex related carbohydrate level analysis on adults G. mellonella
are given in Table 3 and 4.
SH-standard error: As it is seen in Table 3 carbohydrate
levels in G. mellonella adults increased in both sexes connection
with their ages. The difference of carbohydrate levels between in both
sexes and in all age groups is statistically significant (p<0.05).
The carbohydrate levels in G. mellonella adults increased in both
sexes connection with age (Fig. 4).
The carbohydrate levels in G. mellonella adults increased in both
sexes connection with age (Table 4). The difference of
carbohydrate levels between in both sexes and in all age groups is statistically
significant (p<0.05).
DISCUSSION
Age and protein level relations, variations in protein level accompanied
by aging and different protein necessities of females and males were studied
by various researches (Shuxia and Adams, 2000; Akman, 2004; Meats and
Leighton, 2004). In this study, variations in protein levels of adults
in accordance with the adult age are observed (Table 1,
Fig. 3). For this variation it was observed that in females
increase in the beginning was followed by a decrease, whereas the increase
kept going in males. Protein levels of females first increased in the
beginning of adult life for high rate of egg production. However, as the
reproduction potential declined by aging in the following days of life,
the protein demand necessary for the egg production was also declined.
Protein levels of adults were decreased when this condition was united
with the decreased anabolic reactions. Since the protein used by males
for sperm production is not consumed in large quantities as by females,
the decrease observed in the protein level of males was less than the
decrease observed for females. The results obtained from this study of
the age related protein level variation are in agreement with the results
of other researches. In a research with Spodoptera littoralis (Lepidoptera:
Noctuidae) the effects of host-plant, photoperiod, day time, developmental
stage and sex on the protein band number and concentration were studied
and it was found that the protein band number of the last instar larvae
change (El-Aw, 2003).
Age-related changes in hemolymph free amino acids and proteins
were examined by Shuxia and Adams (2000), when Colorado Potato Beetle-Leptinotarsa
decemlineata was reared under both short-day (8:16) (L:D) and long-day
(17:7) (L;D) conditions. Under a short-day photoperiod, the total free
amino acid concentration in the hemolymph increased gradually up to 20
days of adult life, but the long-day beetles showed marked increases during
the first 10 days and then decreased afterwards. Proline, glutamine and
valine were the most abundant free amino acids in both sexes.
In a study with Bactrocera tryoni (Diptera: Tephritidae)
it was revealed that for egg production, protein consumption of sexually
matured adults are higher than non-sexually matured females (Meats and
Leighton, 2004). High protein levels of the females began to decrease
especially after the second week. The increase in the protein level until
becoming sexually matured and then the decrease in the protein level with
the start of egg production and age related protein issues, which were
observed in this study with G. mellonella, are in line with other
studies with different insects. Protein levels derived from adults were
found different in males and females as well as in lightness and darkness.
These differences might
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Fig. 5: |
Age and sex related
carbohydrate level in G. mellonella in constant darkness |
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Fig. 6: |
Age and sex related
carbohydrate level of G. mellonella in constant light |
be due to photoperiod induced endocrine imbalance changing
the protein level.
In this study with G. mellonella the relation between adult age
and carbohydrate levels and age related gradual increase in carbohydrate
level were observed (Table 2, Fig. 4).
This increase might be in connection with the food as well as the inhibition
of extreme carbohydrate consumption in the body due to Lepidoptera`s lipid
preference as the primary energy source. Increase in the carbohydrate
level is not related to the glucose synthesis from lipids because animals
do not have the glyoxylate converting enzyme and glucose can not be synthesized
from lipids. Besides, age related carbohydrate increase might be in connection
with the components of the wax, which is the food, as they are employed
in glucose synthesis. Carbohydrate level of females, which is higher than
males, can be linked to females` ability of accumulating and synthesizing
more carbohydrate from the food they were given and higher carbohydrate
consumption of males. It is proven by various researchers (Olson et
al., 2000; Giron and Casas, 2003) that in pre-adult and post-adult
phases, females generally keep more protein, carbohydrate and lipids than
males. Age related increase in glycogen levels were observed in the studies
on age related carbohydrate metabolism with other insects (Seker and Yanikoglu,
1999; Akman, 2004; Varer, 2005).
Variations in nutrient levels between G. mellonella
adults kept in constant lightness and constant darkness
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Fig. 7: |
Age and sex related
carbohydrate and protein levels of G. mellonella in constant
darkness |
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Fig. 8: |
Age and sex related
carbohydrate and protein levels of G. mellonella in constant
light |
depend on the variations between insects` activity and energy requirement
according to day length (Fig. 5-8).
In this study, age and sex related variations in the insect
metabolism in two different photoperiods and the effects of these variations
on protein and carbohydrate levels were observed. The results of our study
are in line with the results of other studies on other insects in this
scope (Seker and Yanikoglu, 1999; Shuxia and Adams, 2000; Olson et
al., 2000; El-Aw, 2003; Giron and Casas, 2003; Akman, 2004; Meats
and Leighton, 2004; Varer, 2005).
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