The Variation of the Amount of the Neurosecretory Materials in the Corpus Allatum of Pimpla turionellae L. (Hymenoptera: Ichneumonidae) During Oocyte Maturation
The purpose of this study is to examine the effects of neurosecretory material (NSM) in Corpus Allatum (CA) of endoparasitic Pimpla turionellae L. on reproduction. For this purpose, the egg maturation of the insect and the amount of the NSM in CA of the serial section of the brain were studied. Egg maturation was determined by measuring the terminal oocyte length. In order to determine the amount of the NSM in CA the neurosecretory granules of which were stained by paraldehyde fuchsin are considered. The egg maturation of P. turionellae have performed twice. The NSM intensity in CA continued increasingly from the first day of egg development until the first oviposition days; and reached the maximum level before the second egg maturation phase. These observations showed that the NSM in CA of this insect may be related to egg development.
Received: April 19, 2010;
Accepted: July 12, 2010;
Published: August 05, 2010
The median neurosecretory cells of the brain were shown as the control center
of reproduction (Toyoda et al., 1999) and other
metabolic activities (Shimokawa et al., 2008;
Broughton et al., 2010). These cells in different orders were studied
during the reproduction period of female insect (Juberthie
and Caussanel, 1980). Formerly, it was specified that the brain cells which
are stained purple by paraldehyde fuchsin techniques include neurosecretory
material (Peric-Mataruga et al., 2001). There
are many researches on these neurosecretory cells which are stained purple by
paraldehyde fuchsin (Toyoda et al., 1999). These
cells were searched concerning egg maturation on Labidura riparia (Juberthie
and Caussanel, 1980) and Lymantria dispar (Leob
and Hayes, 1980). The fact that, the NSM which is produced in the median
neurosecretory cells of the brain reaches to the corpora allata by passing through
the corpora cardiaca via the nervi corporis cardiaci is well known. In CA of
some insects, the NSM which is stainable with paraldehyde fuchsin positively,
was presented (Sakiko, 2003). In insects, corpora allata
are known to be involved in metabolic activities (Weaver
and Edwards, 1990; King et al., 2005; Tombes
and Smith, 2005). Although, some authors demonstrated the presence of the
NSM within the CA only a few of them provided some detailed account of the functional
significance of the NSM in the CA. In some studies it was mentioned that CA
also has correlation with egg maturation (Toyoda et al.,
1999; Huerta and Martinez, 2008).
As seen above, the relationship between the neuroendocrine system of the insects
belonging to different orders and the reproduction activities were studied and
various results were obtained. Previously the head endocrine system of P.turionellae
had been given morphologically, histologically (Ozluk, 1991)
and ultrastructurally but the physiological relationships between the endocrine
system and the reproduction activity had not been mentioned. On the insects
merged to Hymenopter order, it is rare to encounter studies that show the correlation
between the brain neurosecretory cells and the reproduction (Ozluk,
1993b). Especially, there is no study about the reproduction period which
is related to the correlation between the NSM in CA and the egg development
of endoparasitic P. turionellae.
In this study, the variation of the NSM intensity in CA during the reproduction period of female P. turionellae was observed. Thus, it was aimed to enlighten the relationship between the neurosecretory material in corpora allata and reproduction in P. turionellae.
MATERIALS AND METHODS
Experimental animals: Individuals of female P. turionellae was reared under laboratory conditions between years 2003-2005 in the Insect Culture Laboratuary at Ankara University, Faculty of Science, Department of Biology. The female experimental animals were obtained from these stock culture.
P. turionellae was reared at the Experimental Animals Laboratuary of
the Ankara University Biology Department between 2003-2005 years. The female
experimental animals were obtained from the stock culture. The continuity of
the stock culture was supplied from the greater wax moth, Galleria mellonellae,
reared in the semi-synthetic diet (Bronskill, 1961).
All P. turionellae females were kept at temperature of 25±2°C
with relative humidity of 75±5% in a 12: 12 (L: D) photoperiod. They
were fed with cotton pieces absorbed with 50% honey solution and to each insect
was given a pupa of G. mellonellae every two days in order to satisfy
their host haemolymph needs. All the experimental virgin females were reared
without males to eliminate possible effects of mating (Pratt
and Davey, 1972b; Acle et al., 1990). They
were selected on the 0, 3, 6, 9, 12, 15, 18, 21, 24 and 27th days, after adult
emergence. All experimental females were selected in the same body size to eliminate
the difference in terminal oocyte length (Briegel, 1990).
Microscopy: All females were decapitated at the same time (14.00-17.00)
to eliminate the possible diurnal changes in the neurosecretory system (Engelmann,
1968). The dissected brain of P. turionellae were fixed in Bouin's
solution for 12-18 h, sectioned serially at 7 milimicron after being embedded
in paraffin and were stained with paraldehyde fuchsin after potassium permanganate
oxidation (Gomori, 1950). The rest of the body of the
decapitated animals was dropped into isotonic insect saline and was left for
no longer than 3 h. The ovaries were dissected out, for microscopic calculation,
under a binocular microscope and were fixed in Bouin's fixative 4 h. Dissected
ovaries were sectioned serially at 10 milimicron. The sections were stained
with Ehrlich's haemotoxylin and eosin.
Measurements: In order to point out the changes in the intensity of
NSM on the experiment days, the paraldehyde fuchsin positive granules in CA
were counted. The NSM in the gland was numbered 0 to 5 increasingly according
to its staining intensity (Adams et al., 1975).
An average value for each day investigated was obtained by dividing the sum
of the staining intensity of each CA into the number of CA of each the day.
These calculations were done for each insect separately.
The lengths of the terminal oocyte were measured with an ocular micrometer.
The developmental stages of the oocyte growth were recorded as the length of
the biggest terminal oocyte in ovariole (Pratt and Davey,
1972a). All measurements were made in the largest area in the cross sections
of the terminal oocytes. Only one biggest terminal oocyte length from each of
at least 10 animals in each studied day was measured and their averages were
recorded (Table 1).
||The NSM intensity of CA and the terminal oocyte length change
of P. turionellae on different maturation days
|*Only one terminal oocyte were measured in each female; **There
is no terminal oocyte in ovariole on the day of emergence; ***Means compared
vertically. Means not followed by the same superscript are significantly
different from each other at p<0.05 by Fisher's t-test
The measurements in the terminal oocytes were done by Fisher's
(1948) method of significance control test (p<0.05) between two means.
Terminal oocyte growth: The larger oocytes in any ovariole represent
a longitudinally arranged series in which the terminal oocyte nearest the base
of the ovariole is the oldest and largest oocyte representing successively earlier
stages in the oocyte maturation (Pratt and Davey, 1972a).
In all P. turionellae females, there is no differentiation in the ovariole
on the day of emergence (Table 1). The terminal oocytes continue
to increase in length between the 3rd day and 15th day. Between the 12th day
and 15th day, the length of the terminal oocyte reaches to maximum level which
is called primary egg maturation stage. Between the days 15th and 21th, the
length of the terminal oocyte decreases to minimum. These days on which the
terminal oocyte length decreased to minimum are called the first oviposition
stage. The terminal oocyte again continues to increase after the 21th day on
the stage of secondary egg maturation.
Neurosecretory materials in corpus allatum: The NSM intensity in corpus allatum had different levels during the reproductive period of the females of P. turionellae (Table 1). The NSM intensity of newly emerged insects was on a limited scale and the level was low. This intensity level increased on the first days depending on the oocyte maturation, showed fluctuation on the following days. The NSM intensity which decreased the minimum level before the first egg laying phase days, reached a high intensity on the first egg laying days. After the first egg laying days, the NSM amount decreased considerably, but on the second egg maturation phase, it reached to the maximum level. This level of the NSM intensity in CA during egg maturation can be mentioned as there is a correlation between the CA and egg maturation on the reproduction period in this insect.
A brain cell has been considered as neurosecretory if stained purple with paraldehyde
fuchsin and only the purple condition has been considered indicative of the
presence of NSM and has been called paraldehyde fuchsin positive. The fact that
the CA of insects includes NSM was shown by the studies done by means of light
and electron microscope (King et al., 2005;
Tombes and Smith, 2005; Bonetti et al., 2006).
In the mean time the NSM exists also in the CA of female of P. turionellae
was marked in the serial sections which were obtained from the head of the insect.
In many hitherto studies on insects, the neurosecretory cells have appeared
to regulate oocyte growth (Greenplate et al., 1985)
by activating the CA (Bhaskaran and Jones, 1980; Khan
et al., 1984; Ulrich et al., 1985)
to produce a hormone essential for protein transfer from haemolymph into the
developing oocytes for yolk deposition (Bell and Barth,
1971) and perhaps by controlling protein synthesis and metabolism themselves
(Osorio et al., 1997). In many studies, the brain
hormones have already been chosen to show their cyclic activity (Leob
and Hayes, 1980) correlated with the oocyte maturation (Tabakoglu,
1988; Toyoda et al., 1999). Cyclical changes
in the appearance and volume of the CA have been observed in many insects. The
volume has often been used as a measure of the gland activity (Osorio
et al., 1997; Huerta and Martinez, 2008).
Just as, the CA volume has been used as a criterion to show the cyclic changes
in the terminal oocyte maturation (Stay and Tobe, 1981;
Ulrich et al., 1985). In one of his studies on
P. turionellae, Ozluk (1993a) indicated a parallelism
between the volume of the gland and its NSM amount.
In this study, in P. turionellae females, the day of emergence was characterized
by the lack of differentiation in the ovariole. On this day, the amount of the
NSM in CA was at a certain level. During the first days of the terminal oocyte
growth, a permanent increase or decrease was not observed at the amount of the
NSM in CA. It was suggest that after the first days of egg maturation, this
level showed fluctuation. Indeed, the level which has the minimum value just
before the first egg laying days (on 12th day), increased considerably on the
first egg laying days (between 18th day and 21st day). This reduction which
occurred just before terminal oocytes reached to the maximum length was assessed
as there has been no more need to hormones since the egg maturation was completed.
It has been known that the NSM in brain passes to CA throughout corpora cardiaca.
Thus, the changes in the intensity of NSM in CA can be explained with the changes
in the intensity of NSM in brain. It was specified that the NSM which is secreted
from median neurosecretory cells, provides oocyte maturing (Adams
et al., 1968). They specified with another researcher group in 1975
that there is a feedback mechanism between the brain cells of M. domestica
and ovariums. Adams (1976) also specified that the oocytes
which complete the maturation, produce a hormone which blocks the secretion
activity of brain that is called oostatic hormone; and this hormone causes NSM
accumulation in brain on egg laying phase. I also found that the striking increase
in NSM of P. turionellae on the first egg laying days can be explained
with the suggestion of Siew (1965) which shows that
egg laying activity requires high level of hormone. When injected the brain
extract which is obtained from the egg laying females of Gryllus bimaculatus
to young mature females, Sefiani (1987) observed that
they also show egg laying activity and abdominal contractions starts. Thus,
it was recommended that more neurohormones are required for egg laying and this
neurohormone causes abdominal contractions and egg laying movements. After the
first egg laying phase, the NSM in CA of P. turionellae decreases seriously.
The NSM amount which decreases after the first egg laying phase of P. turionellae,
reached to its maximum level on the second egg maturation days. In fact
that the NSM intensity which has a considerably high level during the egg laying
phase, decreased after the egg laying activity completed in L. riparia
(Juberthie and Caussanel, 1980), in 9-24 h after the
egg laying activity in Galeruca tanaceti (Siew, 1965)
and in 12-36 h in M. domestica (Adams et al.,
1975). The present study suggests that this decrease which occurred at the
beginning of the second egg maturation might have occurred for the same reasons
as the first decrease in P. turionellae. The maximum increase in the
corpus allatum activity at the height of their gonadotrophic activity has been
described by a considerable number of authors and my findings are consistent
with those of given by Stay and Tobe (1981), Pratt
and Pener (1983). In this study this maximum increase may be true for P.
turionellae only in the second oocyte maturation phase, because its maximum
increase occurred after the 21th day. Dia and Gilbert (1993)
proposed that the largest volume of the CA was reached at the beginning of yolk
storage in the terminal oocytes. Also, it was purposed that the CA is especially
responsible for the second oocyte growth in Nauphoeta cinerae (Buschor
et al., 1984).
The results obtained from present study shows that the NSM amount in corpus allatum of P. turionellae changes during the reproductive activity. It is also estimated that the egg laying and the egg maturation phases of P. turionellae are controlled by corpus allatum hormones could be denoted from these results.
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