73
The influence of mature males on the neurosecretory
control of ovarian development in the desert locust
By K. C. HIGHNAM and O. LUSIS
(From the Department of Zoology, The University, Sheffield 10)
With i plate (fig. 4)
Summary
The ovaries of the desert locust are made up of about 100 ovarioles. When females are
reared with mature males, the terminal oocytes in the ovarioles develop very much
more rapidly than the terminal oocytes of females reared without males. In addition,
the percentage of terminal oocytes which are resorbed during development increases
more rapidly in females reared without males than in females reared with mature
males. This results in a significantly larger percentage of mature eggs being produced
by females reared with mature males.
The neurosecretory system contains little stainable material in females whose
terminal oocytes are developing rapidly in the presence of mature males, but contains a
large amount when the terminal oocytes are mature. In females reared without males,
whose terminal oocytes either develop slowly or remain undeveloped, the neurosecretory system shows a precocious accumulation of stainable material. It is concluded that the neurosecretory system is actively extruding secretion when the amount
of contained material is small, and that material is accumulating and not being released
when it is present in large amount. The presence of mature males somehow brings
about the release of material from the female neurosecretory system, with consequent
rapid development of terminal oocytes and the production of a larger number of eggs.
Introduction
S I N G H (1958) has noted that the number of degenerate terminal oocytes in the
ovaries of Schistocerca gregaria is greater in females reared without males than
in normal females. Norris (1954) has shown that the presence of mature males
and copulation are necessary for maturation and normal oviposition in female
desert locusts. Copulation results in the discharge of material from the neurosecretory system (Highnam, 1962). The neurosecretory system exercises a
positive control over ovarian development, and artificially induced discharge
of neurosecretory material is followed by rapid development of the terminal
oocytes (Highnam, 19616, 1962). Evidently some relationship exists between
the control of ovarian development by the neurosecretory system of the female
and the presence of males. The present paper is concerned with demonstrating
that histological differences in the neurosecretory systems of females reared
in the presence and absence of mature males can be correlated with the reproductive performance of the two groups of females.
Material and methods
Newly-emerged adult female desert locusts, S. gregaria, were reared under
conditions of constant temperature and humidity as previously described
[Quarterly Journal of Microscopical Science, Vol. 103, part 1, pp. 73-83, March 1962.]
74
Highnatn and Lusis—Influence of males on ovary of locust
(Highnam, 1961a). Mature males were added to the cages of half the females
to give a sex ratio of 1:1. When immature males and females are reared
together, the female maturation time may be very variable (Norris, 1954;
Highnam, 1961a). The use of mature males in the present experiments had
the advantage of eliminating a large part of the individual variation in female
maturation rates. Significant differences between females reared with and
without males could then be demonstrated even with the rather small sample
numbers used (usually 15 to 20 individuals per sample).
Samples were taken at regular intervals over a period of 28 days. Ovaries
were examined under a binocular microscope and the total number of ovarioles counted, as well as the numbers of developing and resorbed terminal
oocytes. The percentages of developing and resorbed terminal oocytes were
then calculated for each individual. The length of each developing terminal
oocyte was measured.
Heads were fixed in Bouin's fluid under reduced pressure, the cuticle dissected away, and serial sections cut in paraffin wax at 10 fj,. The sections were
stained in paraldehyde-fuchsin (PF) after permanganate oxidation to show
neurosecretory material. Five heads were sectioned from each sample, except
where the development of the terminal oocytes in a sample varied greatly: in
this case, 5 heads were sectioned of individuals with undeveloped terminal
oocytes and 5 of individuals with well-developed terminal oocytes.
Results
Ovarian development
The two panoistic ovaries of the desert locust each consists of about 50
ovarioles, arranged in line and opening into the paired oviducts. The mean
number of ovarioles (200 individuals) was found to be 100-1^0-7, ranging
from 72 to 121. The terminal oocyte (that nearest the oviduct) in each ovariole
developed more rapidly than the others. After it had been laid, the previously
penultimate oocyte developed to full size. Just after emergence of the adult,
the terminal oocyte was found to measure 07^0-02 mm in length; the length
of the fully developed oocyte was 7-9^0-08 mm. After the terminal oocyte
had been laid, its original position in the ovariole was represented by a white
corpus 'luteum', developed from the follicle cells which had surrounded the
oocyte (Singh, 1958). In S. gregaria the white corpus luteum later became
orange. With the exceptions to be described, the terminal oocytes in any
individual developed at the same rate.
Increase in length of terminal oocytes in the presence and absence of mature
males. The percentage of females with longer terminal oocytes was always
greater throughout the sampling period in those groups that were reared with
mature males (table 1, see p. 82). Further, the maximum length of the
terminal oocyte was attained by about 80% of the sample after only 14 days
(fig. 1). In the absence of males, no individual possessed terminal oocytes
longer than 6-o mm after 14 days, and only about45% of the sample possessed
oocytes between 6-i and 8-o mm at the end of 21 days (table 1; fig. 1). It
Highnam and Lusis—Influence of males on ovary of locust
75
follows from this that the rate of development of the terminal oocytes was
greater in females reared with mature males than it was in females reared
without males. In fig. 2, A, B, the mean length was calculated from the
terminal oocyte lengths of whole samples. Even when the females that possessed undeveloped terminal oocytes (fig. 1) were excluded from the samples
reared without males, the rate of development of the terminal oocytes in the
remaining females still proved to be less than that of females reared with
mature males (fig. 2, c). Some variation occurred in the lengths of terminal
2 weeks
3 weeks
V
\ I
© • > - " -
lengths of terminol oocyfes (ram)
FIG. 1. Histograms showing the percentages of terminal oocytes of different lengths in the
ovaries of female »S. gregaria sampled at weekly intervals. Upper row: females reared without
males. Lower row: females reared with mature males.
oocytes in all the samples measured, but the mean lengths of the terminal
oocytes in the groups reared with mature males were always significantly
greater than those of the groups reared without males (table 2, see p. 82).
It is concluded from these results that the presence of mature males both
increases the proportion of females with developing terminal oocytes, and
increases the rate of development of those oocytes that have begun developing.
Increase in number of terminal oocytes in females reared with mature males.
The maximum possible number of mature oocytes which can be produced by
an individual desert locust is about 100, i.e. the number of ovarioles in the
ovaries. This number was attained in only 8% of the individuals in the
present experiments. The numbers found in the majority of individuals fell
short of the maximum as a result of resorption of a varying percentage of
terminal oocytes by the follicle cells.
76
Highnam and Lusts—Influence of males on ovary of locust
The terminal oocytes which were to be resorbed by the follicle cells stopped
developing at various stages of growth. The yolk became granulated and was
largely resorbed by the follicle cells and these later invaded the degenerate
oocytes, which came to resemble rather large corpora lutea. Since different
terminal oocytes in an ovary stopped developing at different stages in their
o 2
0
7
14
21
28
time (doys)
FIG. 2. Increase in length of terminal oocytes of 5. gregaria with time. A, females reared with
mature males; B, females reared without males; c, females reared without males and with
individuals with undeveloped oocytes excluded.
growth, an ovary with otherwise fully developed oocytes possessed resorptionbodies of different sizes and degrees of degeneration. An account of the
histology and histochemistry of the process of resorption will be published in
a further paper.
The percentage of resorption bodies in the ovary increased as the normal
oocytes increased in length, both in the females reared with mature males and
in the females reared without males (fig. 3), but the increase was very much
more marked in females reared without males. Consequently, the percentage
of developing terminal oocytes was greater in females reared with mature males
at all stages of development (table 3, see p. 83; fig. 3), and the final percentage
of mature oocytes was significantly greater than in females reared without
males (table 3). When immature females were reared with immature males,
the proportion of mature oocytes was not significantly different from that in
females reared without males (table 3).
Highnam and Lusts—Influence of males on ovary of locust
77
These results show that rearing females in the absence of males has a threefold effect upon ovarian development, when compared with rearing females
with mature males:
1. The ovaries in a certain proportion of the females do not start developing.
2. Where the ovaries do start developing, the rate of development of the
terminal oocytes is low.
3. The proportion of resorbed oocytes increases more rapidly as development proceeds, so that the final number of fully developed oocytes is
significantly less.
100
§ 90
o
^80
•
oocytes from females
reare
ed with mature o*c?
oocytes from females
reared without rfo*
oocytes from females
reared with immature
° 70
o
c
'E
66
t
60
stoc,.
50
i
(10-30mm)
(3-1-4-SmrrO
C4-6-6- 5mm) ( 6 6 - 8-0mmJ
development of terminal oocvtes
FIG. 3. Percentages of terminal oocytes and resorption-bodies in females reared with
mature males, immature males, and without males.
Histology of the neurosecretory system
Females reared with mature males. The histology of the neurosecretory
system of the newly emerged female and of the female possessing almost fully
developed terminal oocytes has already been described (Highnam, 1961a). In
the immature female, the neurosecretory A-cells of the pars intercerebralis, the
nervi corporis cardiaci (NCC I), and the corpora cardiaca contain little PFstainable material. In females with well-developed terminal occytes, on the
other hand, the neurosecretory system contains large amounts of PF-stainable
material.
The neurosecretory systems of females reared with mature males contained
small amounts of PF-stainable material when examined 7, 10, and 14 days
after emergence (fig. 4, A-C). Twenty-one days after emergence, when the
eggs were fully developed and were in many cases in the oviducts, the neurosecretory system contained large amounts of PF-stainable material. Twentyeight days after emergence, many of the females had laid eggs; the neurosecretory systems of these females once more contained small amounts of
78
Highnam and Lusts—Influence of males on ovary of locust
material (Highnam, 1962). The neurosecretory systems of females 28 days
old which had not laid eggs remained full of PF-stainable material.
Females reared without males. Seven days after emergence, the neurosecretory systems of females reared without males contained small amounts
of material, being very similar to the neurosecretory systems of females reared
with mature males. Fourteen days after emergence, the sample could be
divided into two groups (table 1; fig. 1): that in which the terminal oocytes
were 4-0 mm or less in length (slow developers) and that in which the terminal
oocytes measured between 4 a and 6-o mm in length (fast developers). The
neurosecretory systems of the slowly developing females were found to contain large amounts of PF-stainable material (fig. 4, D-F); the more rapidly
developing females possessed neurosecretory systems containing little P F stainable material (fig. 4, G-J). The neurosecretory systems of females reared
without males examined 21 days after emergence were found to contain large
amounts of PF-stainable material, whether the terminal oocytes were well
developed or not. Similarly, all the females reared without males for 28 days
since emergence possessed neurosecretory systems containing large amounts,
of PF-stainable material. None of these females had laid eggs; some possessed well-developed terminal oocytes and others poorly developed oocytes
(table 1; fig. r).
These results show that PF-stainable material appeared in large amount in
the neurosecretory systems of females reared with mature males between 14
and 21 days after emergence, and remained in large amount while the females
retained their fully-developed terminal oocytes. The material disappeared
from the neurosecretory system when the eggs were laid and the next generation of terminal oocytes began development.
Large amounts of PF-stainable material appeared in the neurosecretory
systems of females reared without males at 14 days after emergence if the
terminal oocytes were undeveloped, or had developed very slowly. Females
FIG. 4 (plate). Photomicrogaphs of neurosecretory systems of female 5. gregaria reared
with and without mature males.
A, neurosecretory cells in the pars intercerebralis of a female 14 days old, reared with mature
males.
B, NCC I from the same female.
c, corpus cardiacum from the same female.
D, neurosecretory cells in the pars intercerebralis of a female 14 days old, reared without
males.
E, NCC I from the same female.
F, corpus cardiacum from the same female.
G, neurosecretory cells in the pars intercerebralis of a female 14 days old, reared without
males.
H, NCC I from the same female.
J, corpus cardiacum from the same female. Notice the small amounts of neurosecretory
material in A, B, c compared with D, E, F. In G, H, J, the amount of neurosecretory material is
intermediate, D, E, F are taken from a female with undeveloped terminal oocytes; G, H, j are
from a female whose terminal oocytes had developed to about 48 mm in length. Further
explanation in the text. All sections from heads fixed in Bouin's fluid. Sections were stained
with paraldehyde-fuchsin after permanganate oxidation.
neurosecretory
cells
neurosecretory
•naterial
200//
100//
FIG. 4
K. C. HIGHNAM and 0. LUSIS
Highnam and Lusts—Influence of males on ovary of locust
79
of this age which possessed terminal oocytes between 4-1 and 6-o mm in length
had neurosecretory systems containing small amounts of material. Twentyone and 28 days after emergence, all the females reared without males possessed neurosecretory systems containing large amounts of material; the
oocytes in some of these females were well developed and were similar to those
of females reared with mature males in this respect; the terminal oocytes in
many of the females were poorly developed. It is clear that the neurosecretory
system contained large amounts of material either when the terminal oocytes
had completed their development or when the oocytes were undeveloped.
Discussion
When immature females are reared with mature males, their neurosecretory
systems contain small amounts of PF-stainable material until some time
between the fourteenth and twenty-first days after emergence. During this
time, the terminal oocytes increase rapidly in length almost to their full size.
By the twenty-first day after emergence, PF-stainable material in the neurosecretory systems has increased greatly in amount, and the terminal oocytes
have completed their development and in many cases have passed out of the
ovarioles into the oviducts.
On the other hand, when females are reared without males, a certain proportion possess neurosecretory systems containing large amounts of material
by the fourteenth day after emergence; the terminal oocytes in these females
are undeveloped. By the twenty-first day after emergence, all females reared
without males have neurosecretory systems containing large amounts of
material, similar to the neurosecretory systems of females reared with mature
males at this time. But the terminal oocytes of the twenty-one-day-old
females reared without males differ from those of females of the same age
reared with mature males in being either undeveloped or less well developed.
Arvy and Gabe (1952; 1953 a, b, c, d) have concluded from their examination
of the neurosecretory systems of a number of insect species at different stages
in their life-histories that where there is a large amount of contained neurosecretory material, the system is very active. The system is thought to be
less active where the amount of neurosecretory material is small. A similar
interpretation of differences in the histological appearance of the neurosecretory cells in phasmids has been put forward by Dupont-Raabe (1951,
1954, 1956).
It is, however, reasonable to suppose that material would accumulate in the
neurosecretory system if it were not released. A small amount of material in
the system would then indicate that the system was 'active' in the sense that
extrusion from the system was occurring; a large amount of contained material
would imply that the system was 'inactive' in that secretion was not occurring
and that material was accumulating within it.
It is important to decide between these opposing ideas, since any attempt
to correlate the activity of the neurosecretory system with the progress of any
80
Highnam and Lusts—Influence of males on ovary of locust
developmental event will be profoundly influenced by the particular interpretation of the histological results.
Cautery of the neurosecretory cells in S. gregaria, or removal of the corpora
cardiaca, retards the development of the terminal oocytes, whereas the
implantation of whole brains into females whose neurosecretory cells have
been cauterized results in some oocyte development (Highnam, 1962). The
neurosecretory system therefore exercises a positive control over oocyte
development in S. gregaria. Since the present results show that the terminal
oocytes develop rapidly when the neurosecretory system contains small
amounts of material, it is very probable that the system is actively extruding
the secretion during this time. When females are reared without males, the
oocytes either do not develop, or develop slowly, and the neurosecretory
systems precociously come to contain large amounts of material, i.e. the
systems are not actively secreting. This result is supported by the precocious
accumulation of material in the neurosecretory system after ovariectomy
(Highnam, 1962). Further, the development of oocytes can be accelerated in
females reared without males by artificially inducing release of material from the
neurosecretory system (Highnam, 19616, 1962). In Calliphora erythrocephala
neurosecretory cells packed with inclusions have now been shown to be less
active than cells containing 'dispersed aggregates' of material (E. Thomsen,
personal communication).
As development of the terminal oocytes proceeds, the proportion of resorption-bodies increases in all S. gregaria females, although more rapidly in
females reared without males. This results in females reared with- mature
males, whose terminal oocytes develop rapidly, producing a significantly
larger percentage of eggs per individual. Similar resorption of developing
terminal oocytes occurs in Locusta migratoria (Singh, 1958). Further, it is
possible to calculate from the tables given by Singh the proportion of ovarioles
which produce mature eggs in females with rapid development (oviposition
within 10 days of emergence) and females with slower development (oviposition 11 to 15 days after emergence). In the former, 97-6±O'6% of the ovarioles produce mature eggs; in the latter only 84-5±3-3% of the ovarioles
produce mature eggs. These figures are statistically significantly different
(p < o-ooi). The presence of the male has no effect upon female maturation
in L. migratoria (Norris, 1954), so factors other than the presence of the male
must produce the effect. Haskell (personal communication) has shown that
non-specific stimulation of the sense organs in L. migratoria may affect the
reproductive performance of the females.
The increasing percentage of resorbed oocytes during ovarian development
of females reared without males suggests that control over ovarian development is a continuous process. It is possible that a minimum level of concentration of neurosecretory material has to be maintained while the oocytes develop.
It is difficult to test this hypothesis experimentally, since older females are not
able to survive for more than 48 h after cautery of the neurosecretory cells or
removal of the corpora cardiaca (Highnam, unpublished).
Highnam and Lusts—Influence of males on ovary of locust
81
Copulation by females previously reared without males results in gross discharge of material from the neurosecretory system, as does enforced activity
and excessive handling (Highnam, 1962). The greater activity of immature
females in the presence of mature males, and their earlier copulation, may
therefore both be factors in ensuring that the neurosecretory system remains
in a secretory condition for the time necessary to complete oocyte development.
The influence of the male pheromone in this context is a matter for speculation. Loher (i960) has suggested that during the maturation of the male, the
pheromone may stimulate chemoreceptors on the antennae and cause nervous
impulses which may affect a brain centre. Although a direct effect of this sort
upon the neurosecretory cells cannot be ruled out, there is the possibility that
the pheromone may lower the general sensory threshold of the female, and
the consequent increased reactivity of the insect to general non-specific stimuli
may be an important link in the chain of events leading to discharge of material
from the neurosecretory system.
I am grateful to the Anti-Locust Research Centre, London, for supplying
the desert locusts used in this study; to Dr. F. Segrove for his very helpful
criticism; and to Mr. W. Mosely for making the photomicrographs.
References
ARVY, L., and GABE, M., 1952. Ann. Sci. nat. (Zool.), 14, 345.
1953a. Z. Zellforsch., 38, 591.
19536. Arch. Zool. exp. ge'n., 90, 105.
1953c. Biol. Bull. Woods Hole, 106, 1.
1953^- C.R. Acad. Sci (Paris), 237, 844.
DUPONT-RAABE, M., 1951. Bull. Soc. Zool. (France), 76, 386.
— 1952. Arch. Zool. exp. gen., 89, 128.
— 1956. Ann. Sci. nat. (Zool.), 18, 293.
HIGHNAM, K. C, 1961a. Quart, J. micr. Sci., 102, 27.
— 19616. Nature, 191, 199.
— 1962. Quart. J. micr. Sci., 103, 57.
LOHER, W., i960. Proc. roy. Soc. B, 153, 380.
NORRIS, M. J., 1954. Anti-Locust Bulletin, No. 18.
SINGH, T., 1958. Trans. R. ent. Soc. Lond., n o , 1.
82
Highnam and Lusts—Influence of males on ovary of locust
Appendix
TABLE I
Percentage of terminal oocytes of different lengths in S. gregaria females reared
with and without mature males
Age of
sample
(days)
With or
without
males
% of sample with terminal oocyte length
2-1-4-0
1-0-2-0
4-1-6.0
6i-8-o
mm
mm
mm
mm
0
0
100
0
0
0
367
733
7
7
No. in
sample
20
15
without
with
14
14
23
15
without
with
47-8
0
217
67
304
13-3
0
800
21
21
3i
19
without
with
194
0
129
0
226
15-8
45i
84-2
TABLE 2
Mean lengths of terminal oocytes in S. gregaria females reared with and without
mature males
Age of
sample
(days)
Mean oocyte
length
(mm)
0-70
0-70
3
3
25
25
With or
without
males
without
with
7
7
20
15
without
with
1-09
1-69
±O-O2 J
II
10
with
442
±O-6 9
14
14
•14
23
15
12
without
with
without
297
703
441
±1-06 \
±0-40 j \
±0-17
/
p < 0001
p < o-ooi
21
21
•21
20
24
without
with
without
526
723
6-o6
±o-39 \
±0-46 / \
±0-32
/
p < o-ooi
p > o-oi <O-O2
28
10
without
6-41
±0-42
No. in
sample
Standard
error
±0-02 \
±O-O2 J
same sample
p < o-ooi
developed individuals excluded.
Comparison of
means
Highnam and Lusts—Influence of males on ovary of locust
83
TABLE 3
Percentage of developing terminal oocytes in the ovaries of S. gregaria females
reared with and without mature males
With or
Oocyte length
without
No. of
(mm)
males
specimens
with
i -0-3-0
3°
10-30
without
3°
3-1-4-5
3-1-4-5
15
16
with
without
*4-6-6-5
28
without
66-80
66-80
6-6-8-0
22
without
with
with
immature
28
15
Mean %
terminal
oocytes
per ovary
100
100
921
85-8
77-4
*#69-o
780
**7o-3
Standard
error
Comparison of
means
±o-o
±00
±4-3 \
±3-4/1
\
±i-7 "1 J
\
±2-5 1 "I
±i-8\/
±2'2/
p >
01
p > 0-02 < 005
p < o-oi
p <C O'ooi
p <C 0-001
* No value for females with mature males; this stage was passed through too quickly.
** Comparison of means: p > 0 1 .