Effect of precocene II on the endocrine regulation of development and reproduction in the bug, Oncopeltus fasciatus

GENERAL

Effect

AND

COMPARATIVE

37, 156-166

ENDOCRINOLOGY

(1979)

of Precocene II on the Endocrine Regulation and Reproduction in the Bug, Oncopeltus P. MASNER. Biological

W. S. BOWERS,’

Laboratory

qf Dr. Accepted

M. KALIN,

R. Maag

AC.

September

of Development fascia&s

AND T. M~~HLE

8157 Dielsdorf,

Switzerland

29. 1978

Large milkweed bugs, Oncopeltus fasciatus, were exposed to precocene II for different times as larvae or adults. Short-term contact during the stages in which the corpora allata are active (1 hr in the second instar. 12 hr in the young adults), inactivated the gland completely. Treatment during the last instar, in which the activity of corpus allatum is limited did not injure the gland which was subsequently activated in the young adults. The inactivation of the corpora allata resulted in precocious metamorphosis and degeneration of the prothoracic glands in younger larval instars and inhibition of vitellogenesis in adult females. However, the males with inactivated corpora allata were fertile. The corpora allata of treated insects remained inactive even when transplanted into an untreated insect. The effects of precocene II can be counteracted by subsequent application of juvenile hormone 1. The irreversibility of changes induced by precocene II holds promise for the practical application of compounds of this type as insect growth regulators if substances with a broad spectrum of activity and good persistency can be found

The discovery of anti-juvenile hormones by Bowers (1976) gave fresh impetus to the development of insect growth regulators (IGRs) just at the time when the limits of the practical application of IGRs with juvenile hormone (JH) activity had been recognized. These compounds, precocenes, were characterized by Bowers (1976) as antiallatotropins, which limit in some way the presence of JH in the insect body. This gives them nearly ideal pesticide activity as they should in principle stop insect development at any stage and prevent egg production. The activity of these compounds is limited mostly to Heteroptem and several species of grasshoppers and roaches (Pratt and Bowers, 1977). However, the existence of other compounds with this type of activity which would also affect other insects is probable. For instance dimethylsciadinonate. which was isolated from avocado leaves, causes pupa’ tion,

New York State Cornell University,

Agriculture Geneva,

Experimental 14456.

Sta-

N.Y.

156 0016-6480/79/020156-l Copyright All rights

1$01.00/O

@ 1979 by Academic Press. Inc. of reproduction in any form reserved.

tion in the silk worm (Chang et al., 1975). The present paper analyses the degree and type of activity of naturally occuring precocene II (Bowers. 1976) which was tested on the large milkweed bug. Special attention was paid to the effect on the endocrine system. MATERIALS

AND METHODS

A sunflower strain of the large milkweed bug, 0~ copeltus fasciatits, was reared using the method of Gordon (1974) at 30”. 60% r.h., and IS hr daily illumination. Precocene II (6,7-dimethoxy-2,2-dimethyl-2HI-benzopyran: Bowers, 1976) and JH 1 (methyl10.1 I-epoxy-7-ethyl-3,l I-dimethyl-2,6-tridecadienoate, mixture of isomers) were applied either to the bottom of a glass petri dish (dimaeter = 9 cm) or to a strip of filter paper ( 16.5 x 6 cm) which was confined in a plastic cup (200 cm3). Ten insects were placed in each cup, 10 larvae or 6 adults were confined in each petri dish. The biological activity of the compound applied to the glass is about 10 times higher than by the contact with the treated paper. cu-Ecdysone was diluted in 10% ethanol and injected with a glass capillary. Each experiment was performed in triplicate, unless otherwise stated. Transplantation experiments were performed using the method of Novik (1951) on insects anaesthetized by submersion in water. Prothoracic glands were dis-

PRECOCENE

II IN &ZCo~f?kS

sected after vital staining with methylene blue (Scharrer, 1948) and whole mounts prepared. The material for histological processing was fixed in aqueous Bouin’s solution, embedded in paraffin, sectioned, and stained with azan, paraldehydefuchsin. or thionin. The volume of corpus allatum was measured by the method of Joly (1967).

Morphology

qf

RESULTS Precocious Adults

Precocious adults are insects which attain adult characteristics at any instar of postembryonic development prior to the final adult molt. The extent of this precocious metamorphosis can be easily determined by observing the length, shape, and coloration of the wing disks and of the scutellum. Most of the second-instar larvae which have been exposed to precocene ecdyse into the third instar (Table 2). The presence of orange spots on the black larval wing buds and the black coloration of the scutellum are the adult characters of forms intermediate between the third larval stage and adult. These precocious adult characters are generally more evident in the precocious adults of the fourth and particularly the last fifth instar exposed to precocene in earlier instars. However, the degree of the precocious adult development attained varies considerably in individual specimens as exemplified by the last instar. A trace of precocious metamorphosis is seen in individuals with wing buds which are still completely black but definitely larger than in the normal last stage larvae (Figs. 1 and 2). They overlap the sides of the abdomen and resemble the wing buds of the sixth supernumerary instar, produced by contact application of JH (Fig. 3). The advanced adult differentiation of the wing buds of precocious adults of the last instar, on the other hand, is characterized by a typical adult orange coloration (Figs. 4 and 5). of Different Del~eloptrzentrrl Stages to Precocene

Set?siti\ity

The development of second-instar larvae permanently exposed to precocene (0.1

fUX’iatL4S

157

wg/cm”) in a glass petri dish is completely stopped (Table 1). More than one-half of these insects died as precocious adults; the others died as larvae without any conspicuous morphogenetic change. Exposure to a higher concentration for 1 hr followed by transfer into an untreated dish also stopped the development in all treated larvae. Exposure for 10 min caused 50% mortality. The sensitivity of third-instar larvae was similar. Fourth-instar larvae were less sensitive and dosages 100 times higher had to be employed to obtain the full inhibition of larval development. Larvae exposed to the lower dosages developed into fertile adults. Treatment of the last-instar larvae with the highest nontoxic dose did not prevent development of fertile adults. Young adult females exposed to precocene in a glass petri dish (10 pglcm2) for 12 hr and then transferred into untreated dishes were permanently sterilized. The same treatment of males for 48 hr did not affect their fertility (Table 1). Even permanent contact with a strip of filter paper impregnated with the same dose did not sterilize the males. Young females exposed to precocene for 48 hr in this system were permanently sterilized. This procedure was frequently used to sterilize females for further experiments. ffO,mOtle

The larvae which were permanently exposed to JH impregnated filter paper (100 &cm’) from the beginning of the second instar did not reach metamorphosis (Table 2). They died mostly during the same stage. Some of the larvae which were exposed at the beginning of the second instar to precocene in glass petri dishes also died in the same stage but nearly 60% ecdysed into the precocious adults of the third instar. When precocene pretreated larvae of the second instar were exposed to permanent contact with the mentioned dose of JH, the mortality in the same instar was reduced to

1.58

MASNER

ET

AL.

1-4. Oncopeltus fasciatus. I. Larva of the last fifth instar. 2. Precocious adult of the fifth instar with black broad wing buds overlapping the sides of abdomen (arrow). Precocene treatment. FIG. 3. Supernumerary sixth instar bearing mostly larval characters. JH 1 treatment. Note the similarity of the wing buds of this specimen and the precocious adult on Fig. 2. FIG. 4. Precocious adult of the fifth instar with black-orange wing buds-the adult pattern of coloration. Precocene treatment. FIGS. FIG. FIG.

23% and the larvae died later in development, mostly without any signs of precocious metamorphosis. With the lower dosage of JH (10 &cm’) 30% of the pretreated

larvae underwent precocious metamorphosis. Females which had been sterilized by 2-day contact with precocene-treated filter

PREcocENE

II IN Oncopeltus TABLE

LARVICIDE

fascia&s

159

1

ACTIVITY (TOTAL AND PROPORTION OF LARVAE, WHICH DIED AS PRECOCIOUS AND FEMALE STERILITY IN 30 LARVAE OR 18 ADULTS EXPOSED TO PRECOCENE APPLIED TO THE BOTTOM OF GLASS PETRI DISHES FROM THE BEGINNING OF INDICATED INSTAR

ADULTS),

Larvicide activity (%) Treatment started

Dose t&cm’)

Larva 2

0.1 0.01 1 I 0.1 0.01 10 30 IO 3 1 IO 10 IO

Larva 3 Larva 4 Larva 5 Female

Male

Contact period

Total

Precocious adults

Resulting females

permanent permanent 1 hr IO min permanent permanent permanent permanent permanent permanent permanent 12 hr 6 hr 48 hr

100 7 93 53 100 3 100 100 0 -

51 0 43 0 83 3 43 0 0 -

0 fertile 0 fertile 0 fertile 0 0 fertile sterile fertile sterile fertile fertile

paper start to lay eggs when placed in contact with filter paper impregnated with JH

(10 pg/crn’). Egg production at least 2 weeks.

continued

for

Degeneration of Prothoracic Glands and Inhibition of Ecdysis in Precocious Adults The precocious metamorphosis indicated by the changes in the wing buds is accompanied by extensive alterations to internal organs which impair further development. None of the insects of the last instar with distinct imaginal orange coloration on the wing buds molted again. They died without any sign of apolysis (Table 3). However, most of the precocious adults of the last fifth instar with very slight imaginal characters (large black wing buds) were able to initiate molting into a supernumerary instar. The metamorphosis of these insects was further advanced (orange coloration of the wing buds) but they died enclosed in the old cuticle. The pharate body represented a supernumerary instar intermediate between larva and adult (Fig. 6).

Molting may be initiated in the precocious adults with distinct imaginal coloration of wings by the injection of 10 pg of ecdysone. This indicates that the cessation of ecdysis is connected with the changes in function of the ecdysone-producing prothoracic glands. This suspicion was confirmed by the study of the anatomy and histology of the well-developed glands of the last instar larvae (Fig. 7) and the small remnants of the glands found in the freshly ecdysed precocious adults (Fig. 8). These remnants disappear within 24 hr in the precocious adults with distinct imaginal coloration of the wings. However, they remain preserved in the specimens with the large black larval-like wing buds, where the precocious metamorphosis is very limited. Morphology Complex

of the Corpus Allaturn-Brain

The volume of the corpus allatum of 7-day-old fertile females (3.03 & 0.53 mm3) is about one-third larger than the volume of the glands of sterile females, which have been exposed to the filter paper impreg-

I’ In the combined

experiment

100 IO 100 IO

48 48 48

-

I I I

-

-

-

Dose

Dose (&cm’)

JH

the larvae

(&cm”)

Contact period W

Precocene

were

pretreated

Permanent Permanent Permanent Permanent

-

Contact period

with

precocene

3 87 10 43 23 6

2 0 0 50 0 3 15

4

and then

0 13 33 0 67 43

3

Larvae of instar

placed

0 0 7 0 0 6

5

0 0 0 57 0 30

3

0 0 0 0 7 0

4

with

Precocious adults

in contact

Percentage of insects dead as

TABLE 2 OF THE POPULATION OF 30 YOUNG LARVAE OF THE SECOND INSTAR EXPOSED IMPREGNATED WITH JH OR TO GLASS COATED WITH PRECOCENE”

Treatment

DEVELOPMENT

JH.

0 0 0 0 0 0

5

EITHER

3 100 100 100 100 100

Activity

PAPER

Larvicide total

TO FILTER

0 0 0 57 7 30

Precocene

(5%)

TABLE FREQUENCY

161

II IN Oncopeltus fasciatus

PruxocENE

3

OF ECDYSIS IN THE LARVAE AND PRECOCIOUS ADULTS OF THE LAST FIFTH INJECTED WITH 3 pl OF RINGER SOLUTION OR ECDYSONE SOLUTIONS

INSTAR

No. of insects Tested insects

Ecdysone (&specimen)

Larvae Precocious adults with black wings Precocious adults with black-orange wings

Dead in 5th instar

Dead in ecdysis

Ecdysed adults

10

2 4

0 16

18 0

-

4

16

0

20 9

0 11

0 0

IO

u Twenty insects tested.

nated with 10 pg/cm2 precocene for the first 2 days (2.10 5 0.65 mm3). This is consistent with the observations of Bowers and Martinez (1977). The corpus allaturn-brain complexes from control last-instar larvae, young and fertile females (7 days old) were compared with those of precocious adults of the last instar and precocene-sterilized females of the same age. The median neurosecretory cells of the treated insects usually contained a few cells with accumulated paraldehyde-positive material, whereas the cells of the control insects were nearly empty in most of the specimens examined. Under the light microscope, the corpus allatum showed no conspicuous changes. On the other hand, Unnithan et al. (1977) observed degenerative changes under the electron microscope in the glands of Sday-old precocene-sterilized females. Morphogenetic Activity of the Corpus Allaturn The activity of the corpus allatum and of the corpus allatum-brain complex was studied by transplantation experiments. In the first series of experiments the organ(s) of the donor was transplanted into young larvae of the last instar which lasts 7 days under our conditions. The outcome of the ensuing ecdysis of the host provided evidence about the morphogenetically active

JH output of the gland: an active gland caused the host larva to molt into a supernumerary instar with more or less preserved larval characteristics, whereas an inactive gland permitted normal metamorphosis into the adult stage. The results are summarized in Table 4, the percentage of host larvae ecdysing into a supernumerary instars indicates the percentage active glands. Corpora allata from the young last-instar larvae were morphogenetically inactive, but one-fourth of the glands from 3-day-old larvae of this instar was active. The glands from 6-day-old larvae, young or fertile females or males were in nearly all cases active. The brains of both larvae or fertile females were devoid of any morphogenetic activity. In young female adults, contact for 1 or 2 days with the same dose of precocene had none or very little effect on the morphogenetic activity of the gland when it was directly transplanted into the host larva. However. the same treatment of both females or males followed by 5 days incubation in an untreated dish resulted in the complete inhibition of nearly all of the glands examined. Even implants of three glands or of the gland-brain complex showed no activity in a host larva. A similar experiment with fertile females (treatment

162

MASNER

ET

AL.

7

FIGS.

5-8.

Oncopeltus

fasciatus.

5. Adult. 6. Precocious adult of the fifth instar with black wing buds molting into the supernumerary instar. The old cuticle from the left wing bud was peeled off to reveal the new cuticle with adult orange coloration. Precocene treatment. FIG. 7. Prothoracic gland (arrow) along the salivary gland of the fifth-instar larva. Methylene blue. FIG. 8. Remains of degenerating prothoracic glands of the freshly ecdysed precocious adult of the fifth instar. FIG. FIG.

5

I-2

-

3-7 -

-

1-2 7-8

1-2

CA 3 CA

7 7 7 7 7

I 14

3 7I

2

1 7 7 I

3 6 6 6

1

On Day

3 10 -

-

--

-

-

-

Precocene treatment Dose (dcm2)

l-6 1-6 -

-

-

-

-

-

Day

6 15 22 8 8

7 12

8 289

8

II 25 14 5

7 14 18 6

8

II

0 7 18 2 1

1 I

2 32

0

0 6 3 0

3 0 3 0

I

Dead

larva

(CAB)

The

active

16 100 75 100 0

0 55

50 0

75

82 95 0 100

25 79 85 0

0

were

84 0 25 0 100

100 45

50 100

25

18 5 100 0

75 21 15 too

100

into (%) Adult

substances

Larvae ecdysed Supernumerary instar

5

COMPLEX LARVAE

of the transplant.

Host-young

ALLATUM-BRAIN INTO YOUNG

into a supernumerary instar indicates the morphogenetic effect of donors and to a glass petri dish for the treatment of hosts.

CA CA CA CA CA

CAB CA

CA

-

1-3 1-2 l-2

-

1-2

1

CA CA B CA

-

-

-

CA CA 3 CA B

--

-

-

CA

-

Transplantation Organ(s)

TABLE 4 OF CORPUS ALLATUM (CA) BRAIN (B) OR CORPUS FROM CONTROL AND TREATED INSECTS (DONOR) OF THE LAST FIFTH INSTAR (HOST)”

-

Treatment (d) (IO Kg/cm’) Precocene JH

n The percentage of insects ecdysing applied to filter paper for the treatment

Male

Female

Larva

stage

ACTIVITY TRANSPLANTED

Donor

MORPHOGENETIC

2 2 cr,

$ g

z

0 s 2 m

z

3 =I

F;r

164

MASNER

ET AL.

at the seventh-eighth day and incubation in untreated dishes until the 14th day) revealed, however, inhibition of activity of only 50% of the transplanted glands. Glands of females which had been sterilized with precocene and in which vitellogenesis was then induced by incubation with the paper it treated were mostly inactive. Morphogenetic activity resulting from the implanted corpora allata of fertile females was not basically changed in the experiment where the host larvae were incubated in the petri dishes the bottom of which had been coated with 3- 10 pglcm2 precocene.

contained few previtellogenic oocytes at the beginning of the experiment. The host females were sacrificed 1 week after the implantation and the ovaries dissected. The percentage of fertile host females, i.e., females with the yolk in the oocytes, indicated the percentage of gonadotropically active, i.e., JH-producing glands (Table 5). Corpora allata of young last-instar larvae are devoid of any gonadotropic activity, whereas the glands from old larvae, or from young or fertile adult females are active in nearly 70% of the cases studied. The implantation of three such glands or of the corpus allatum-brain complex of these insects were almost always active. The brain alone of fertile females was inactive. However. even three corpora allata or the corpus allatum-brain complex from the females sterilized with precocene, were devoid of any gonadotropic activity.

Gonadotropic Activity of Corpus Allatrtrn In the second series of transplantation experiments the corpus allatum or corpus allatum-brain complex of the donor was implanted into 7-day-old sterile females. These females had been sterilized by 2-day contact with filter paper impregnated with 10 pg/cm2 precocene and then incubated for 5 days in untreated dishes, before using them as hosts. The ovaries of these females

DISCUSSION

The results presented corroborate the conclusion of Bowers (1976) that the precocenes terminate allatal function in both

TABLE 5 GONADOTROPIC ACTIVITY OF CORPUS ALLATUM (CA) BRAIN (B) OR CORPUS ALLATUM-BRAIN COMPLEX (CAB) TRANSPLANTED FROM CONTROL OR PRECOCENE-TREATED INSECTS (10 pg/cm* ON FILTER PAPER) INTO T-DAY-OLD STERILE FEMALES (CONTACT WITH PRECOCENE DURING FIRST 2 DAYS AFTER EMERGENCE)~ Donor Stage

Larva

Precocene treatment (days) 5

Female

1-2 l-2

u The females

were

sacrificed

Host-sterile Transplantation Organ(s) On Day

n

3 CA CA 3 CA CAB CA 3 CA CAB CA 3 CA B 3 CA CAB

8 6 7 4 15 9 3 14 5 5 5 10

1 week

1 6 6 6 I 1 1 7 7 7 7 7 after

the operation

female

Dead

Females Fertile

and the presence

0 0 0 0 2 0 0 0 0 0 0 0

of yolk

0

67 100 75 54 89 100 71 100 0 0 0 in the ovaries

(%) Sterile

100 33 0 25 46 11 0 29 0 100 100 loo registered.

PRECOCENE

II

IN

OmX@tuS

fU.WiUtus

165

immature and adult stages. Short-term con- 1960; Wigglesworth, 1936). The results pretact with rather low doses causes perma- sented demonstrate that the male of 0. f~snent changes, which preclude further de- ciatus is fertile even when its corpus alvelopment. This makes the precocenes par- latum was inactivated by precocene treatticularly interesting for practical application ment. In precocene-treated larvae the proas insect growth regulators. thoracic glands irreversible The antiallatotropic activity of precocene undergo precludes any is specific as it can be counteracted with change. Its degeneration further development of the precocious JH. JH treatment of precocene-pretreated larvae of the second instar precludes the adults. This means that the regulation of the precocious metamorphosis: similarly JH prothoracic gland is basically changed in treatment counteracts the sterilization of the larvae in which the corpora allata have been inactivated with precocene. precocene-treated females. The results presented corroborate the folThe observed accumulation of paraldehyde-positive material in the brain of lowing hypothesis (Masner et ul. 1976): JH the precocene-treated insects may indi- produced by the corpora allata limits the activity of the prothoracic glands during cate that the effect of precocene is mediated larval development. The low level of ecthrough changes caused in the neurosecretory cells, which could be the source of dysone safeguards a limited morphogenesis allatotropins. This would mean that the and ecdysis from instar to instar. The inaccorpus allatum is not the primary target of tivation of the corpus allatum and the abprecocene as suggested by Bowers (1976). sence of JH at the end of postembryonic development permits an intensive activity The inactivation of the corpus allatum seems to be a two-step process: the short- of the prothoracic glands which produce a term impulse of precocene triggers a large amount of ecdysone required for The prothoracic change which takes place over several days complete metamorphosis. even when the insects are no longer in con- glands, which have been hyperactive during tact with the treated substrate. This applies metamorphosis, disintegrate after the folto larvae of the second-fourth stages or lowing ecdysis. young adults in which the corpus allatum is active. However, in the larvae of the last ACKNOWLEDGMENTS stage where the corpus allatum activity is We wish to express our thanks to Mr. M. Dickenlimited, precocene treatment does not in- mann and Miss M. Lehner for technical assistance jure the gland, which will be activated in the and to Mr. P. J. Miiller, Dr. A. Pryde, Professor Dr. young adult. The precocene treatment inac- H. A. Schneiderman. and to Dr. W. Vogel for helpful discussion and correction of the text. tivates corpora allata of young females more readily than the glands of 7-day-old REFERENCES fully active females. Bowers, W. S. (1976). Discovery of insect antiallatoOnce the corpus allatum has been inactitropins. In “The Juvenile Hormones” (L. I. Gilvated it cannot resume its activity even vert. ed.), pp. 394-408. Plenum, New York. when transferred into an untreated body. Bowers, W. S., Ohta, T.. Cleere, J. S., and Marsella P. The gland remains inactive also in sterilized A. (1976). Discovery of insect anti-juvenile horfemales which started to lay eggs after JH mones in plants. Science 193, 542-547. Bowers, W. S., and Martinez, R. (1977). Antiallatotreatment. tropins: Inhibition of corpus allatum development. The corpus allatum of the male is active. Science 197, 1369-1371. This has been considered to be necessary for Chang. F. C., lsogai, A.. Kamikado, T., Murakoshi, the normal function of the accessory glands S., Sakurai, A., and Tamura, S. (1975). Isolation and structure elucidation of growth inhibitors for and mating in several insect species (Loher,

166

MASNER silk-worm

larvae

from

avocado

Chem. 39, 1167- 1168. Gordon, H. T. (1974). Cohort cold-storage of the sunflower

leaves.

rearing strain

Agr. Biol.

and prolonged of Oncopelrus

fasciatus. Ann. Entomol. Sot. Amer. 67,976-980. Joly,

P. (1967). physiologique

gratoria Loher,

W. maturation

Comparison du des corpora

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manica Novak,

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et de I’activite de Locusta

miAnn. Sot. Entomol. Fr. 3, 601-608.

The process

chemical and its

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volume allata

deficiency the German L. In “The

Gilbert. ed.). pp. V. J. A. (1951).

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of the in

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ET

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Pratt,

Dal.

G. E.. and Bowers, W. S. (1977). Precocene II inhibits juvenile hormone biosynthesis by cockroach corpora allata in vitro. Nature (London)

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548-550. B. (1948).

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(1977). corpus

C.,

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K.

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S. the

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V. B. (1936). in the growth

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