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Physiology of diapause in the adult Colorado beetle—II
Y. Ins. Pkyriol., 1961, Vol. 6, pp. 152 to 151. Pergmmm Press Ltd., London. Printed in Great Britain
PHYSIOLOGY OF DIAPAUSE IN THE ADULT COLORADO BEETLE-II DIAPAUSE AS A CASE OF PSEUDO-ALLATECTOMY J. Laboratory
DE
of Entomology,
WILDE and J. A. Agriculturai
DE
BOER*
University,
(Received 9 January
Wageningen,
Netherl~ds
1961)
Abstract-Short-day treatment in Lept?notursa inhibits the activity of the corpora allata. This is correlated with a standstill of ovarian function, a low rate of oxygen consumption, and the characteristic ‘digging’ behaviour of the beetle entering diapause. The same syndrome may be produced by surgical removal of the corpora allata. However, whereas allatectomy-diapause is easily reversed by implantation of two to four active corpora allata, this leaves normal (short-day) diapause unbroken.
1.
INTRODUCTION
IN a former paper (DE WILDE et al., 1959) we dealt with the influence of external factors on the incidence of diapause in L~p~~~~aysa. It was shown that the state of ovarian activity (standstill of yolk deposition, oijsorption) strongly suggested that the corpora allata would be inactive during diapause. We also have shown briefly (DE WILDE and STEGWEE,1958) that the low level of oxygen consumption in diapausing beetles is related to the inactive state of the corpora allata. Moreover, we were able to produce diapause behaviour in female beetles reared under long-day conditions by means of allatectomy. In the present paper we will give a more complete experimental proof for the endocrine origin of diapause in the Colorado beetle. To this purpose, the effect of ‘long day’ and ‘short day’ treatment on behaviour, ovarian development, and size of the corpora allata will be compared. Secondly we will study the effect of extirpation of the corpora allata on oviposition and behaviour of the adult beetle. In conclusion, we will compare the effect of implantation of active corpora allata in beetles under allatectomy-diapause and normal (short-day) diapause.
2. METHODS
Beetles were reared ab onto under 10 and 18-20 hr photoperiods at temperatures around 25°C. In the first category, diapause occurs for 100 per cent, in the second category for only 20-30 per cent of the population (DE WILDE et al., 1959). Before dissection, the beetles were decapitated under ether narcosis and immersed in Levy solution in a wax basin. Drawings of the circumference of the * Part
of the experiments were made by Mrs. C. S. MARLSGE~~~NUS-DUI~TJ~. 152
PHYSIOLOGY
OF DIAPAUSE
153
IN THE ADULT COLORADO BEETLE-11
corpora allata and ovaries were made immediately after dissection under microscopic enlargement by means of a Zeiss drawing mirror while the organs were placed in a hollow object carrier and immersed in a drop of Levy. The surfaces, representing the largest cross-section, were measured by means of a pole planimeter.
FIG. 1.
Method of king the Colorado beetle for allatectomy. Specially constructed of pincers is shown that is used to bend the head at an angle of 90”.
pair
Allatectomy was performed in the following way: After being etherized for precisely 2 min, the beetles are immersed in Levy solution in a wax basin. The head is clamped in the instrument shown in Fig. 1, and bent to an angle of 90” with the body axis. The cervical membrane is now stretched to the utmost. It is split in the median line, the cervical muscles are transected and the fragments of fat body removed. The haemocoel of the head is now strongly illuminated and put into focus. When operating carefully, the bluish-white corpora cardiaca are rendered visible. When these are taken by forceps, usually the corpora allata are also removed. This technique had been followed in the experiments dealt with by DEWILDE and STEGWEE(1958). In the present series of experiments we were able to remove the corpora allata, leaving the corpora cardiaca intact. The result of all operations was controlled microscopically afterwards. Implantations of active corpora allata were made by means of the Ephrusi and Beadle technique (EPHRUSI and BEADLE,1936). Usually the glands were placed under the ventral membrane between pro- and mesothorax. 3. EFFECT OF PHOTOPE~IOD ON OVARIAN DEVELOPMENT AND SIZE OF CORPORA ALLATA Sixteen adult beetles, seven of which were females, had been reared ab ova under a 10 hr photoperiod and were kept under the same condition. Twenty-eight
154
J.
DE WILDE
and J. a.
DE BORR
beetles, of which sixteen were females, were obtained from an 18 hr culture. Sixteen days after emergence the beetles were dissected. At this time, all beetles of the 10 hr and three of the 18 hr culture had entered diapause. A sample of six ovarioles was taken from each of the two ovaries of each beetle. These ovarioles were drawn and measured, as well as the two corpora allata. Of the ovarioles only the germarium was measured. The differences between the vitellarium and the largest ovum in each of the two groups were so clear that the images suffice. The data obtained are given in Table 1.
FIG. 2. Random sample of ovarioles of beetle reared under an 18 hr photoperiod. Developing ova are visible in different stages. Numbers indicate surface of pfane projection of germarium in mmp/3600.
Figs. 2 and 3 give an impression of the shape of the ovarioles in random samples taken from both long- and short-day beetles. Fig. 4 gives a full account of the largest cross-sections of the corpora allata in both males and females of the experiment mentioned above.
155
PHYSIOLOGY OF DIAPAUSE IN THE ADULT COLORADO BEETLE-II
TABLE I-h’bAN.9 AND STANDARD ERRORS OF SURFACBS OF LARGEST CROSS-SECTION OF GERMARIUM, LARGEST OWM (SAMPLE OF TWBLVE OVARIOLES PER BEETLE) AND CORPUS ALLATUM (TWO PER BEETLE) OF BBBTLES REARBD UNDER TWO DIFFERENT PHOTOPERIODS Photoperiod (hr/day)
No. of PO
Germarium (mm*/3600)
18 10
14 7
94.7 + 4.9 53.1 * 5.5
Mean largest Cc. allata (mma/lO,OOO) ovum (mma/3600)
23.4 + I.5 14.6 + I.8
.
I
lmm
2.2 0.1
I.888 I.1
8.8 + 2.3 -
41.6 + 7.4
Difference
Mean number of developing ova per ovariole
,
\
/
FIG. 3. Random sample of ovarioles of beetle reared under a 10 hr photoperiod, drawn on the same scale as Fig. 2. Note small size of germarium
and absence of o8genesis.
The general picture of the ovarioles in active and diapausing beetles has been mentioned in a former paper (DE WILDE et al., 1959). After it appeared, BONHAG (1959, personal communication) directed our attention to the fact that the germarium of Leptinotarsu probably closely resembles that of Tenebvio, which
J. DE
156
WILDE
and J. A.
DE
BOER
according to SCHLOTTMANand BO~JHAG(1956) is filled with nutritive cells, the oijcytes being limited to the basal part. We have indeed obtained histological proof that this is true in Leptinotarsa. It now appears that the nutritive cells are much
ci’CORPORA
ALLATA
CORPORA ACLATA
18 HR
18HR
0 28 G 28
20 30
29 08
lmm
FIG. 4. Size of comora allata of beetles reared under different photoperiods. in&ate
surface of largest cross-section
Numbers
in rnm~~l~,~.
1
SURFACE 40 35 30 25 20 15
C.ALLATUM
IN mm’xf~ I I
_*[email protected]___-__r + 0
+
+
I --___ I+
-_---___-7_+_$_____ +
10 5 30
40
50
60
70 80 SURFACE
SO 100 GERMARKJH
110 ml_! IN mm* x360
0
FXG.5. Relation of corpus allatum size to development of germarium as a function of photoperiod.
o = 10 hr photoperiod;
+ = 18 hr photoperiod.
PHYSIOLOGY
OF DIAPAUSE
IN THE ADULT COLORADO BEETLE-II
157
enlarged in ‘long-day’ beetles. This accounts for the remarkable increase in size of the germarium of these beetles as a whole. Correlated with the increased ovarian activity we observed an increase in volume of the corpora allata. It will be noticed from Fig. 4 that also in the diapausing males the corpora allata are significantly smaller than in the active state. In Fig. 5 the resuits of the above-named measurements are represented graphically. This graph demonstrates again that the germaria and corpora allata of long- and short-day beetles belong to distinctly different statistical populations. 4. REMOVAL OF POSTCEREBRAL GLANDS AND OF CORPORA ALLATA ONLY In the experiments to be described here, both corpora allata and corpora cardiaca were removed following the technique described under ‘Methods’. Operations were always made on long-day (20 hr) beetles within 1 day after adult emergence. The results were compared with the effect of a 10 hr photoperiod. A total of twenty-two male and eighteen female beetles were successfully operated. Their behaviour is shown in Fig. 6. As a comparison, forty-six beetles Of these beetles twenty-six were not were given a short-day (10 hr) treatment. operated, whereas in twenty beetles only the neck membrane and cervical muscles were transected, These two control groups behaved very much the same. NUMBER
OF BEETLES
ALLATECTOMY
2
6
10
14 DAYS A:8
22
(8 6 1
26
311
36
FIG. 6. Duration of pre-diapause in beetles subjected to a 10 hr photoperiod as compared with allatectomiaed 18 hr beetles and control operations. The graphs show the number of beetles above soil. All treatments were started the first day after adult emergence.
Both short-day treatment and allatectomy resulted in diapause, but surprisingly enough, in the last case with a much greater delay of time. We will deal with this under ‘Discussion’. None of the allatectomized females ever oviposited.
1.58
J.
DE
WILDE and J. A. DE BOER
The question now presented itself as to the separate effects of corpora allata and corpora cardiaca. There were two reasons for considering the removal of the corpora allata as relevant. In the first place, it was shown that the transected neurosecretory tract (nervi corporis cardiaci laterales and mediales are fused in Le$notursa) in the course of 2-3 weeks forms a new bulb-like structure with a bluish-white appearance. Histological details of this corpus cardiacurn-regenerate will be published later; it may suffice that its lamellar inner structure and thin outward membrane seem to render it suitable for the release of neurosecretory substance. In the second place, only re-implantation of active corpora allata may reverse the effect of removal of the postcerebral glands. This will be dealt with in the following section. Definite proof was had by allatectomy. Operating very carefully we succeeded in removing only the corpora allata. Of twelve beetles which survived TABLE %--TIME RELATIONS BETWEEN OPERATION ANDEFFBCTIN TWELVECASESOF ALLATECTOMY AND SUBEEQUENT REACTIVATION BY IMPLANTINGFOUR ACTIVECORPORAALLATA The rate of egg production (during 2 weeks) immediately following reactivation is shown in the last column
-
.-
-
Allatectomysoil-positive (days) 27 14 19 18 20 20 14 :: 15 14 12
Soil-positivere-implantation (days)
-
Re-implantationoviposition (days)
_-
Number of
,Eggs per day .12 25 7
1
7 6 9 8
; 16 13 11 11 11 4 6
1: 6 3
-
: 2
-
-
the operation, all entered diapause within 12-27 days (Table 2). These insects survived the operation for up to 157 days at room temperature. None of them ever resumed activity. 5. IMPLANTATION
OF CORPORA ALLATA
(a) During allatectomy-diapause A series of thirteen females, of which the postcerebral complex had been removed successfully, were implanted with four active corpora allata and corpora cardiaca obtained from ovipositing long-day females. The results are given in Fig. 7. As is shown in this figure, all of the females thus treated left the soil and started egg-laying. Some of them did so for 6 weeks, which is a normal oviposition period
PHYSIOLOGY
OF DIAPAUSE
1N THE
ADULT
COLORADO
BEETLE-II
YUMEER OF EGGS
‘- 60
- 60
- 60
-‘60
-
60
R-
40 20
2 -
I t
- 60
-‘60 - 40 cl t-20 ‘)
FIG. 7. Egg production of beetles which had entered diapause after removaI of the postcerebral complex and subsequentIy reimplanted with two active corpora allata obtained from ovipositing females.
159
160
J. DEWILDEand J. A. DEBOER
for long-day females of 25°C. One beetle, No. D, produced 1243 eggs, a productivity equal to that in normal beetles under Dutch summer conditions. Digging behaviour, characteristic of diapausing beetles, ceased within l-2 days after re-implantation and oviposition started after 5-12 days. In Table 2 an experiment is shown in which a number of females were first subjected’ to allatectomy, the corpora cardiaca being left intact. From l-3 days after the operated beetles had entered the soil four active corpora allata were re-implanted. It appeared that within these limits the length of the period between the beginning of diapause and the moment of re-implantation had no effect on the speed of reactivation nor on the rate of oviposition. Implantation of isolated corpora cardiaca in no case resulted in reactivation. (b) f)ur&g natural (short-day) diapause During 1960, three groups of female beetles were brought into diapause by rearing them under a 10 hr photoperiod, and after becoming soilpositive, were implanted with a varying number of active corpora allata obtained from ovipositing females. Group I consisted of seven females which had become soil-positive 14 days before implantation. Each beetle was implanted with five to six corpora allata and corpora cardiaca. Fifteen days after implantation six beetles were still soil-positive, one negative. Group II consisted of eighteen females, each of which was implanted with four to five corpora allata only. The beetles had become soil-positive 3 weeks before implantation. Controls were made from 5 to 8 days after implantation, when seventeen of the beetles were still soil-positive, one negative. Group II contained eight females, which had become soil-positive i-7 days before imphtntation. One beetle was implanted with two corpora allata, four with four to five corpora allata and corpora cardiaca, and three with five to six corpora allata and corpora cardiaca. Controls were made up to 16-3 1 days after implantation when the six surviving beetles all were still soil-positive. 5. DISCUSSION
In the adult ~t~~ota7sa the corpora allata not only control egg formation but apparently also those special elements of behaviour that are essential to diapause. The control of yolk deposition by the corpora allata is in accordance with many facts in the literature (WIGGLESWORTH,1936; JOLY, 1945; THOMSEN, 1942; PFLUGFBLDER,1937). A new element is the control of ‘diapause behaviour’ by these glands. One may ask if this control is effected via the ovaries or in a more direct way on the central nerve system. We hope to deal with this matter in a following paper. Suffice it to say here that our preliminary castration experiments ‘long-day’ female beetles seem to exclude the ‘indirect’ control via the ovaries. It is of importance here to note that before pupation the fourth larval stage burrows into the soil. This is also correlated with a low activity of the.corpora allata.
PHYSIOLOGYOF DIAPAUSE IN TN3 ADULTCOLORAM)BEETLF-II
161
Another interesting point is the similarity between allatectomy and ‘short-day’ Both result in the same characteristic behaviour changes, treatment in ~ti~taysa. arrest of yolk deposition, low rate of oxygen consumption, and, in witro, low rate of succinate oxydation (DE WILDE and STEGWEE,1958). The effect of a 10 hr photoperiod may be described as ‘pseudo-allatectomy’ in the sense of JOHANSSON (1958). It does not seem justified, however, to conclude that diapause in the adult Leptinotarsa is merely an endocrine deficiency syndrome of the corpora allata in the same way as pupal diapause in the silkworm Hyalophora cecmpia is regarded as an endocrine deficiency syndrome of the prothoracic glands FILLIES, 1951). Firstly, there is the much delayed effect of allatectomy in long-day adult beetles as compared with the effect of a 10 hr photoperiod (Fig. 6). This, however, could still be due to differences in larva1 treatment. The allatectomy group had been reared previously under a 20 hr photope~od, whereas the 10 hr group had been reared ab ovo under short-day conditions. In fact, in a former paper (DE WILDE et al., 1959) we have shown that a similar prolongation of the pre-diapause period occurs when adult beetles obtained from ‘long-day’ larvae are exposed to a 10 hr photoperiod (Zoc. cit., Fig. 3). Secondly, the results cited in Section 5(b) clearly indicate that normal diapause in contrast to allatectomy-diapause cannot be reversed by implantation of active corpora allata. This may be explained by assuming: (a) that during diapause the corpora allata are actively inhibited by a ‘diapause-hormone’ or (b) that the corpus allatum hormone, to activate the beetle, needs a substratum which is lacking during normal diapause. This question will be dealt with in a subsequent paper. REFERENCES EPHRUSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Amer. Nut. 70, 218-225. JOHANSSON A. S. (1958) Relation of nutrition to endocrine reproductive functions in the Milkweed bug, OncopeEtusfusciatus (Dallas). iVyIt. Mug. Zool. 7, 1-132. JOLY P. (1945) La fonction ovarienne et son contrBle humoral chez tes Dytiscides. Arch. zool. exp. g&n. 84, 49-164. ~FLUG~LD~ 0. (1937) Bau, En~icklung und Funktion der Corpora allata und cardiaca von ~x$$us morons Br. Z. r&s. Zool. 149, 477-512. SCHLOTTMAN L. L. and BONHAGP. F. (1956) Histology of the ovary of the adult mealworm, Tenetio molitor L. Univ. Calif. Pubf. Ent. 11, 351-394. THOMSENE. (1942) An experimental and anatomical study of the corpus allatum in the blowfly, CalZiphora erythrocephala Meig. Vidensk. Medd. dansk naturh. Foren. Kbh. 106, 320-405. WIGGLESWORTH V. B. (1936) The functions of the corpus allatum in the growth and reproduction of Rhodniusp~olixus (Hemiptera). Quart.J. mim. Sci. 79, 91-121. DE WILDE J. and SlaGWEE D. (1958) T wo major effects of the corpus dlatum in the adult Colorado beetle (Leptinotarsa decemlineata Say). Arch. m&l. Zool. 13 (Suppl.), 227-289. DE WILDE J., DUINTJER C. S., and MOOK L. (1959) Physiology of diapause in the adult Colorado beetle---I. The photoperiod as a controIling factor. J. Ins. PhysioL. 3, 75-85. WILLIES C. M. (1951) Biochemical mechanisms in insect growth and metamo~hosis. Fed. Proc. 10, 546-552.
PHYSIOLOGY OF DIAPAUSE IN THE ADULT COLORADO BEETLE-II DIAPAUSE AS A CASE OF PSEUDO-ALLATECTOMY J. Laboratory
DE
of Entomology,
WILDE and J. A. Agriculturai
DE
BOER*
University,
(Received 9 January
Wageningen,
Netherl~ds
1961)
Abstract-Short-day treatment in Lept?notursa inhibits the activity of the corpora allata. This is correlated with a standstill of ovarian function, a low rate of oxygen consumption, and the characteristic ‘digging’ behaviour of the beetle entering diapause. The same syndrome may be produced by surgical removal of the corpora allata. However, whereas allatectomy-diapause is easily reversed by implantation of two to four active corpora allata, this leaves normal (short-day) diapause unbroken.
1.
INTRODUCTION
IN a former paper (DE WILDE et al., 1959) we dealt with the influence of external factors on the incidence of diapause in L~p~~~~aysa. It was shown that the state of ovarian activity (standstill of yolk deposition, oijsorption) strongly suggested that the corpora allata would be inactive during diapause. We also have shown briefly (DE WILDE and STEGWEE,1958) that the low level of oxygen consumption in diapausing beetles is related to the inactive state of the corpora allata. Moreover, we were able to produce diapause behaviour in female beetles reared under long-day conditions by means of allatectomy. In the present paper we will give a more complete experimental proof for the endocrine origin of diapause in the Colorado beetle. To this purpose, the effect of ‘long day’ and ‘short day’ treatment on behaviour, ovarian development, and size of the corpora allata will be compared. Secondly we will study the effect of extirpation of the corpora allata on oviposition and behaviour of the adult beetle. In conclusion, we will compare the effect of implantation of active corpora allata in beetles under allatectomy-diapause and normal (short-day) diapause.
2. METHODS
Beetles were reared ab onto under 10 and 18-20 hr photoperiods at temperatures around 25°C. In the first category, diapause occurs for 100 per cent, in the second category for only 20-30 per cent of the population (DE WILDE et al., 1959). Before dissection, the beetles were decapitated under ether narcosis and immersed in Levy solution in a wax basin. Drawings of the circumference of the * Part
of the experiments were made by Mrs. C. S. MARLSGE~~~NUS-DUI~TJ~. 152
PHYSIOLOGY
OF DIAPAUSE
153
IN THE ADULT COLORADO BEETLE-11
corpora allata and ovaries were made immediately after dissection under microscopic enlargement by means of a Zeiss drawing mirror while the organs were placed in a hollow object carrier and immersed in a drop of Levy. The surfaces, representing the largest cross-section, were measured by means of a pole planimeter.
FIG. 1.
Method of king the Colorado beetle for allatectomy. Specially constructed of pincers is shown that is used to bend the head at an angle of 90”.
pair
Allatectomy was performed in the following way: After being etherized for precisely 2 min, the beetles are immersed in Levy solution in a wax basin. The head is clamped in the instrument shown in Fig. 1, and bent to an angle of 90” with the body axis. The cervical membrane is now stretched to the utmost. It is split in the median line, the cervical muscles are transected and the fragments of fat body removed. The haemocoel of the head is now strongly illuminated and put into focus. When operating carefully, the bluish-white corpora cardiaca are rendered visible. When these are taken by forceps, usually the corpora allata are also removed. This technique had been followed in the experiments dealt with by DEWILDE and STEGWEE(1958). In the present series of experiments we were able to remove the corpora allata, leaving the corpora cardiaca intact. The result of all operations was controlled microscopically afterwards. Implantations of active corpora allata were made by means of the Ephrusi and Beadle technique (EPHRUSI and BEADLE,1936). Usually the glands were placed under the ventral membrane between pro- and mesothorax. 3. EFFECT OF PHOTOPE~IOD ON OVARIAN DEVELOPMENT AND SIZE OF CORPORA ALLATA Sixteen adult beetles, seven of which were females, had been reared ab ova under a 10 hr photoperiod and were kept under the same condition. Twenty-eight
154
J.
DE WILDE
and J. a.
DE BORR
beetles, of which sixteen were females, were obtained from an 18 hr culture. Sixteen days after emergence the beetles were dissected. At this time, all beetles of the 10 hr and three of the 18 hr culture had entered diapause. A sample of six ovarioles was taken from each of the two ovaries of each beetle. These ovarioles were drawn and measured, as well as the two corpora allata. Of the ovarioles only the germarium was measured. The differences between the vitellarium and the largest ovum in each of the two groups were so clear that the images suffice. The data obtained are given in Table 1.
FIG. 2. Random sample of ovarioles of beetle reared under an 18 hr photoperiod. Developing ova are visible in different stages. Numbers indicate surface of pfane projection of germarium in mmp/3600.
Figs. 2 and 3 give an impression of the shape of the ovarioles in random samples taken from both long- and short-day beetles. Fig. 4 gives a full account of the largest cross-sections of the corpora allata in both males and females of the experiment mentioned above.
155
PHYSIOLOGY OF DIAPAUSE IN THE ADULT COLORADO BEETLE-II
TABLE I-h’bAN.9 AND STANDARD ERRORS OF SURFACBS OF LARGEST CROSS-SECTION OF GERMARIUM, LARGEST OWM (SAMPLE OF TWBLVE OVARIOLES PER BEETLE) AND CORPUS ALLATUM (TWO PER BEETLE) OF BBBTLES REARBD UNDER TWO DIFFERENT PHOTOPERIODS Photoperiod (hr/day)
No. of PO
Germarium (mm*/3600)
18 10
14 7
94.7 + 4.9 53.1 * 5.5
Mean largest Cc. allata (mma/lO,OOO) ovum (mma/3600)
23.4 + I.5 14.6 + I.8
.
I
lmm
2.2 0.1
I.888 I.1
8.8 + 2.3 -
41.6 + 7.4
Difference
Mean number of developing ova per ovariole
,
\
/
FIG. 3. Random sample of ovarioles of beetle reared under a 10 hr photoperiod, drawn on the same scale as Fig. 2. Note small size of germarium
and absence of o8genesis.
The general picture of the ovarioles in active and diapausing beetles has been mentioned in a former paper (DE WILDE et al., 1959). After it appeared, BONHAG (1959, personal communication) directed our attention to the fact that the germarium of Leptinotarsu probably closely resembles that of Tenebvio, which
J. DE
156
WILDE
and J. A.
DE
BOER
according to SCHLOTTMANand BO~JHAG(1956) is filled with nutritive cells, the oijcytes being limited to the basal part. We have indeed obtained histological proof that this is true in Leptinotarsa. It now appears that the nutritive cells are much
ci’CORPORA
ALLATA
CORPORA ACLATA
18 HR
18HR
0 28 G 28
20 30
29 08
lmm
FIG. 4. Size of comora allata of beetles reared under different photoperiods. in&ate
surface of largest cross-section
Numbers
in rnm~~l~,~.
1
SURFACE 40 35 30 25 20 15
C.ALLATUM
IN mm’xf~ I I
_*[email protected]___-__r + 0
+
+
I --___ I+
-_---___-7_+_$_____ +
10 5 30
40
50
60
70 80 SURFACE
SO 100 GERMARKJH
110 ml_! IN mm* x360
0
FXG.5. Relation of corpus allatum size to development of germarium as a function of photoperiod.
o = 10 hr photoperiod;
+ = 18 hr photoperiod.
PHYSIOLOGY
OF DIAPAUSE
IN THE ADULT COLORADO BEETLE-II
157
enlarged in ‘long-day’ beetles. This accounts for the remarkable increase in size of the germarium of these beetles as a whole. Correlated with the increased ovarian activity we observed an increase in volume of the corpora allata. It will be noticed from Fig. 4 that also in the diapausing males the corpora allata are significantly smaller than in the active state. In Fig. 5 the resuits of the above-named measurements are represented graphically. This graph demonstrates again that the germaria and corpora allata of long- and short-day beetles belong to distinctly different statistical populations. 4. REMOVAL OF POSTCEREBRAL GLANDS AND OF CORPORA ALLATA ONLY In the experiments to be described here, both corpora allata and corpora cardiaca were removed following the technique described under ‘Methods’. Operations were always made on long-day (20 hr) beetles within 1 day after adult emergence. The results were compared with the effect of a 10 hr photoperiod. A total of twenty-two male and eighteen female beetles were successfully operated. Their behaviour is shown in Fig. 6. As a comparison, forty-six beetles Of these beetles twenty-six were not were given a short-day (10 hr) treatment. operated, whereas in twenty beetles only the neck membrane and cervical muscles were transected, These two control groups behaved very much the same. NUMBER
OF BEETLES
ALLATECTOMY
2
6
10
14 DAYS A:8
22
(8 6 1
26
311
36
FIG. 6. Duration of pre-diapause in beetles subjected to a 10 hr photoperiod as compared with allatectomiaed 18 hr beetles and control operations. The graphs show the number of beetles above soil. All treatments were started the first day after adult emergence.
Both short-day treatment and allatectomy resulted in diapause, but surprisingly enough, in the last case with a much greater delay of time. We will deal with this under ‘Discussion’. None of the allatectomized females ever oviposited.
1.58
J.
DE
WILDE and J. A. DE BOER
The question now presented itself as to the separate effects of corpora allata and corpora cardiaca. There were two reasons for considering the removal of the corpora allata as relevant. In the first place, it was shown that the transected neurosecretory tract (nervi corporis cardiaci laterales and mediales are fused in Le$notursa) in the course of 2-3 weeks forms a new bulb-like structure with a bluish-white appearance. Histological details of this corpus cardiacurn-regenerate will be published later; it may suffice that its lamellar inner structure and thin outward membrane seem to render it suitable for the release of neurosecretory substance. In the second place, only re-implantation of active corpora allata may reverse the effect of removal of the postcerebral glands. This will be dealt with in the following section. Definite proof was had by allatectomy. Operating very carefully we succeeded in removing only the corpora allata. Of twelve beetles which survived TABLE %--TIME RELATIONS BETWEEN OPERATION ANDEFFBCTIN TWELVECASESOF ALLATECTOMY AND SUBEEQUENT REACTIVATION BY IMPLANTINGFOUR ACTIVECORPORAALLATA The rate of egg production (during 2 weeks) immediately following reactivation is shown in the last column
-
.-
-
Allatectomysoil-positive (days) 27 14 19 18 20 20 14 :: 15 14 12
Soil-positivere-implantation (days)
-
Re-implantationoviposition (days)
_-
Number of
,Eggs per day .12 25 7
1
7 6 9 8
; 16 13 11 11 11 4 6
1: 6 3
-
: 2
-
-
the operation, all entered diapause within 12-27 days (Table 2). These insects survived the operation for up to 157 days at room temperature. None of them ever resumed activity. 5. IMPLANTATION
OF CORPORA ALLATA
(a) During allatectomy-diapause A series of thirteen females, of which the postcerebral complex had been removed successfully, were implanted with four active corpora allata and corpora cardiaca obtained from ovipositing long-day females. The results are given in Fig. 7. As is shown in this figure, all of the females thus treated left the soil and started egg-laying. Some of them did so for 6 weeks, which is a normal oviposition period
PHYSIOLOGY
OF DIAPAUSE
1N THE
ADULT
COLORADO
BEETLE-II
YUMEER OF EGGS
‘- 60
- 60
- 60
-‘60
-
60
R-
40 20
2 -
I t
- 60
-‘60 - 40 cl t-20 ‘)
FIG. 7. Egg production of beetles which had entered diapause after removaI of the postcerebral complex and subsequentIy reimplanted with two active corpora allata obtained from ovipositing females.
159
160
J. DEWILDEand J. A. DEBOER
for long-day females of 25°C. One beetle, No. D, produced 1243 eggs, a productivity equal to that in normal beetles under Dutch summer conditions. Digging behaviour, characteristic of diapausing beetles, ceased within l-2 days after re-implantation and oviposition started after 5-12 days. In Table 2 an experiment is shown in which a number of females were first subjected’ to allatectomy, the corpora cardiaca being left intact. From l-3 days after the operated beetles had entered the soil four active corpora allata were re-implanted. It appeared that within these limits the length of the period between the beginning of diapause and the moment of re-implantation had no effect on the speed of reactivation nor on the rate of oviposition. Implantation of isolated corpora cardiaca in no case resulted in reactivation. (b) f)ur&g natural (short-day) diapause During 1960, three groups of female beetles were brought into diapause by rearing them under a 10 hr photoperiod, and after becoming soilpositive, were implanted with a varying number of active corpora allata obtained from ovipositing females. Group I consisted of seven females which had become soil-positive 14 days before implantation. Each beetle was implanted with five to six corpora allata and corpora cardiaca. Fifteen days after implantation six beetles were still soil-positive, one negative. Group II consisted of eighteen females, each of which was implanted with four to five corpora allata only. The beetles had become soil-positive 3 weeks before implantation. Controls were made from 5 to 8 days after implantation, when seventeen of the beetles were still soil-positive, one negative. Group II contained eight females, which had become soil-positive i-7 days before imphtntation. One beetle was implanted with two corpora allata, four with four to five corpora allata and corpora cardiaca, and three with five to six corpora allata and corpora cardiaca. Controls were made up to 16-3 1 days after implantation when the six surviving beetles all were still soil-positive. 5. DISCUSSION
In the adult ~t~~ota7sa the corpora allata not only control egg formation but apparently also those special elements of behaviour that are essential to diapause. The control of yolk deposition by the corpora allata is in accordance with many facts in the literature (WIGGLESWORTH,1936; JOLY, 1945; THOMSEN, 1942; PFLUGFBLDER,1937). A new element is the control of ‘diapause behaviour’ by these glands. One may ask if this control is effected via the ovaries or in a more direct way on the central nerve system. We hope to deal with this matter in a following paper. Suffice it to say here that our preliminary castration experiments ‘long-day’ female beetles seem to exclude the ‘indirect’ control via the ovaries. It is of importance here to note that before pupation the fourth larval stage burrows into the soil. This is also correlated with a low activity of the.corpora allata.
PHYSIOLOGYOF DIAPAUSE IN TN3 ADULTCOLORAM)BEETLF-II
161
Another interesting point is the similarity between allatectomy and ‘short-day’ Both result in the same characteristic behaviour changes, treatment in ~ti~taysa. arrest of yolk deposition, low rate of oxygen consumption, and, in witro, low rate of succinate oxydation (DE WILDE and STEGWEE,1958). The effect of a 10 hr photoperiod may be described as ‘pseudo-allatectomy’ in the sense of JOHANSSON (1958). It does not seem justified, however, to conclude that diapause in the adult Leptinotarsa is merely an endocrine deficiency syndrome of the corpora allata in the same way as pupal diapause in the silkworm Hyalophora cecmpia is regarded as an endocrine deficiency syndrome of the prothoracic glands FILLIES, 1951). Firstly, there is the much delayed effect of allatectomy in long-day adult beetles as compared with the effect of a 10 hr photoperiod (Fig. 6). This, however, could still be due to differences in larva1 treatment. The allatectomy group had been reared previously under a 20 hr photope~od, whereas the 10 hr group had been reared ab ovo under short-day conditions. In fact, in a former paper (DE WILDE et al., 1959) we have shown that a similar prolongation of the pre-diapause period occurs when adult beetles obtained from ‘long-day’ larvae are exposed to a 10 hr photoperiod (Zoc. cit., Fig. 3). Secondly, the results cited in Section 5(b) clearly indicate that normal diapause in contrast to allatectomy-diapause cannot be reversed by implantation of active corpora allata. This may be explained by assuming: (a) that during diapause the corpora allata are actively inhibited by a ‘diapause-hormone’ or (b) that the corpus allatum hormone, to activate the beetle, needs a substratum which is lacking during normal diapause. This question will be dealt with in a subsequent paper. REFERENCES EPHRUSIB. and BEADLEG. W. (1936) A technique of transplantation for Drosophila. Amer. Nut. 70, 218-225. JOHANSSON A. S. (1958) Relation of nutrition to endocrine reproductive functions in the Milkweed bug, OncopeEtusfusciatus (Dallas). iVyIt. Mug. Zool. 7, 1-132. JOLY P. (1945) La fonction ovarienne et son contrBle humoral chez tes Dytiscides. Arch. zool. exp. g&n. 84, 49-164. ~FLUG~LD~ 0. (1937) Bau, En~icklung und Funktion der Corpora allata und cardiaca von ~x$$us morons Br. Z. r&s. Zool. 149, 477-512. SCHLOTTMAN L. L. and BONHAGP. F. (1956) Histology of the ovary of the adult mealworm, Tenetio molitor L. Univ. Calif. Pubf. Ent. 11, 351-394. THOMSENE. (1942) An experimental and anatomical study of the corpus allatum in the blowfly, CalZiphora erythrocephala Meig. Vidensk. Medd. dansk naturh. Foren. Kbh. 106, 320-405. WIGGLESWORTH V. B. (1936) The functions of the corpus allatum in the growth and reproduction of Rhodniusp~olixus (Hemiptera). Quart.J. mim. Sci. 79, 91-121. DE WILDE J. and SlaGWEE D. (1958) T wo major effects of the corpus dlatum in the adult Colorado beetle (Leptinotarsa decemlineata Say). Arch. m&l. Zool. 13 (Suppl.), 227-289. DE WILDE J., DUINTJER C. S., and MOOK L. (1959) Physiology of diapause in the adult Colorado beetle---I. The photoperiod as a controIling factor. J. Ins. PhysioL. 3, 75-85. WILLIES C. M. (1951) Biochemical mechanisms in insect growth and metamo~hosis. Fed. Proc. 10, 546-552.