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1. Late in the fifth instar, Manduca sexta larvae cease feeding and become ‘wandering larvae’ which are morphologically characterized by an ‘exposed heart’ and the appearance of a pink pigment along the dorsal midline. Two days later ocellar retraction signals the beginning of the prepupal period and 3 days thereafter the pupal ecdysis occurs. 2. The timing of the endocrine events which are responsible for these changes was determined by ligaturing animals of the appropriate developmental stage at various times of day. The times of prothoracicotropic hormone (PTTH) release by the brain were determined by neck ligations. Estimates of the times of prothoracic gland activity were obtained through the isolation of abdomens. 3. It was found that the fifth stage larva releases PTTH on two occasions. The first release lasts approximately 3.5 h and triggers the transformation to the wandering stage. The second release occurs two days later, lasts at least 7 h, and provokes the onset of the pupal moult. 4. The prothoracic glands are involved in triggering the same two changes. In the first instance the glands apparently require the continuing influence of the brain and consequently secrete for about 3.5 h. During the stimulation of the pupal moult the prothoracic glands become ‘turned-on’ and continue to secrete for at least 10 h after the time when the brain is no longer required. In this latter instance the total time of prothoracic gland activity may be as long as 17 h.

When abdomens were isolated from larvae during a critical period of the molting cycle, only parts of the abdominal epidermis had seen sufficient ecdysone to render them capable of forming a new cuticle independent of the prothoracic glands. By isolating abdomens at various times, it was found that the epidermis of a segment gains independence according to a reproducible pattern. The pattern of independence seen for a larval molt is roughly the reverse of that seen for a pupal molt. The presence or absence of juvenile hormone commits the epidermis towards the synthesis of larval or pupal cuticle, respectively. Under the influence of juvenile hormone, the pattern by which the various areas of the epidermis become committed to make larval cuticle is very similar to the larval pattern of independence from the prothoracic glands. This commitment to larval differentiation occurs at approximately the time when the epidermis becomes independent. In the absence of JH (in preparation for the pupal molt), the pattern by which the epidermis is committed to synthesis of pupal cuticle is the reverse of that seen for the larval molt. Moreover, the epidermis becomes committed to pupal differentiation 2 days prior to the time that it actually becomes able to secrete a pupal cuticle in the absence of the prothoracic glands.

1. A preliminary analysis of the silkmoth eclosion clock demonstrated that the endogenous, free-running cycle of the clock could be divided into two parts: an initial synchronization period and a dark decay period. 2. A 1/2 hour light pulse applied during the synchronization period served to restart the free-running cycle. By proper timing of light pulses, a given cycle of the eclosion clock could be restarted ad infinitum. 3. When the length of the interrupting pulse was progressively increased up to 24 hours, the subsequent eclosion rhythms showed a constant phase relationship to the end of the interruption, irrespective of its duration. This phase relationship was the same as observed when the eclosion clock was started by transfer of moths from continuous light to continuous darkness. 4. It is concluded that the synchronization period involves a photoreversible process. During this period light rapidly stops the clock and the cycle can begin only with the onset of darkness.

1. In the giant silkmoths, adult eclosion is immediately preceded by a stereotyped series of abdominal movements - the pre-eclosion behaviour. The pattern of movements is species-specific and has a duration of about 1 ¼ h. 2. The pre-eclosion behaviour is followed sequentially by eclosion, the release of the labial gland secretion, the post-eclosion activity, and the spreading of the wings. These five elements of behaviour make up the emergence sequence. 3. The progression from one part of the behavioural sequence to the next is independent of stimuli provided by the pupal cuticle or cocoon, and therefore must be due to an internal programme. Thus, when the pupal cuticle was removed from pharate moths 12 h before their normal eclosion time, these ‘peeled’ animals continued to behave in a pupal fashion. But upon the arrival of the eclosion gate, the entire emergence sequence was displayed. 4. Through surgical manipulations the brain was shown to trigger the pre-eclosion behaviour. Moreover, this action of the brain was mediated by a neurosecretory hormone - the eclosion hormone. 5. Injections of extracts with eclosion-hormone activity triggered the precocious display of the emergence sequence by pharate moths. 6. When the eclosion hormone was injected into the isolated abdomens of pharate moths, these fragments performed the pre-eclosion behaviour and then shed the surrounding piece of pupal cuticle. The information for the pre-eclosion behaviour and for the abdominal movements associated with eclosion must therefore be programmed in the abdominal ganglia. Moreover, after its ‘activation’ the abdomen can switch from one behaviour pattern to the next without influence from the higher centres.

1. A semi-quantitative biological assay for the eclosion hormone is described. 2. In the pharate moth hormonal activity is confined to the brain and the corporacardiaca (CC). 3. During the life history of the moth, activity is practically absent from the brain and CC of larval and pupal stages and appears only in preparation for adult emergence. 4. The titer of eclosion hormone in the brain and CC of Pernyi moths was followed through adult development. In both structures activity first appeared on about day 7. Titers then increased for the next 3 to 5 days followed by a plateau. On the morning of the day of emergence, the titer in the brain fell and that in the CC correspondingly reached its highest level. At the time of eclosion in the evening, the CC titer then dropped and hormone appeared in the blood. 5. Tests on brains and CC from cockroaches and bugs failed to show activity. 6. It was concluded that the eclosion hormone is used only for the pupal-adult ecdysis and, thus, is probably restricted to the holometabolous orders of insects.

Juvenile hormone inhibits cuticular melanization in tobacco hornworm larvae. This action of juvenile hormone was used as the basis of a new bioassay which is sensitive to less than 0·01 ng (3 × 10−14 moles) of topically applied hormone and requires only 2 days.

1. Motor neurons in the fourth abdominal ganglion were identified by diffusing cobalt into the ganglion through the cut ends of the peripheral nerves. 2. The ganglion from the last instar larva had a total of 74 motor neurons. These were divided into 10 groups which were characterized by the position of the cell body in the ganglion and the root through which the axon travelled to the periphery. 3. In the transition to the pupal stage, 12 larval neurons were lost and 8 new motor neurons appeared. 4. There was no change in the number of motor neurons during adult development. Most of the larval neurons persist through metamorphosis and are used by the adult at emergence. At this stage there are 70 motor neurons. 5. After adult eclosion there was a loss of 40 motor neurons. Only “larval” neurons degenerated. Those which remained included both those which were larval in origin and those which appeared in the pupa. After emergence the interneuron number dropped from approximately 380 to 200. 6. At the time of emergence the eclosion hormone “turns-off” pupal behavior and “turns-on” adult behavior. It also triggers the breakdown of various muscle groups in the abdomen. It is concluded that the neural death was the end result of this aspect of eclosion hormone action.

1. In silkmoths the gating of adult eclosion by light-dark cycles persists after extirpation of the compound eyes, frontal ganglion, subesophageal ganglion, or corpora allata-corpora cardiaca complex. Removal of the brain abolishes gating and such moths emerge irrespective of time of day or night. 2. Implantation of a brain into the abdomen of a debrained animal restores the ability both to entrain to a light-dark cycle and to free-run under conditions of continuous darkness. 3. Implantation of pieces of brain which contain the cerebral lobes likewise restored to debrained moths the ability to entrain and to free-run. But the synchrony was not as sharp as that observed in moths which had received implants of the entire brain. 4. Transection of the brain lateral to each median neurosecretory cell cluster yields pieces which are apparently incapable of gating the eclosion of debrained moths. Both moths receiving the median piece and those receiving the lateral pieces showed a randomized distribution of eclosion. 5. Experiments involving brain transplantation and selective illumination of parts of the body showed that the brain directly receives the light information which is necessary for the entrainment of the clock. 6. In theCecropia moth, the lights-on signal serves to stimulate eclosion and thus partially masks the output of the clock. This effect is mediated solely by light which is perceived by the compound eyes.

1. The larval ecdyses of the tobacco hornworm, Manduca, and of the oak silkmoth, Antheraea, occur during specific times of the day. These times are instar-specific and species-specific and depend upon the photoperiod and temperature conditions. 2. The larval ocelli are not required for the synchronization of larval ecdysis with the photoperiod. 3. The synchrony observed in larval ecdysis does not arise from a gating of the ecdysial event per se by a biological clock. Rather, it is an outcome of the gating of the endocrine events which initiate the moulting process. 4. Application of neck ligatures to fourth-instar hornworm larvae at various times of day showed that prothoracicotropic hormone (PTTH) secretion occurred only during a gate in the early to middle portion of the night. The times of the gates for second- and third-instar larvae were similar. 5. When neck ligatures were applied to fourth-instar larvae within the first 1-5 h after PTTH release, the larvae underwent precocious metamorphosis rather than Moulting to the Fifth Instar. A Consideration of the Number of Larval-Pupal Intermetiates Produced Indicated that with in Span of 15 min Sufficiant JH is Liberatd from the Corpora Allata to Ensure Full Larval Differentiation. 6. Larval abdomens which were isolated during the period from 3 to 6 h after PTTH release underwent a mosaic larval moult-i.e. they became a patchwork of new and old cuticle. Thus, the epidermal structures of the abdomen vary as to the time that each becomes independent of the prothoracic glands. The first structures which continue fifth-instar differentiation in the absence of the prothoracic glands are the crochets. These are then followed in order by the prolegs, the ventral midline, the intersegmental areas, and, by 6 h after PTTH release, the remainder of the abdomen. During this 3 h period the prothoracic glands are presumably secreting ecdysone.

An adult moth sheds its pupal skin only during a specific period of the day. The brain is necessary for the synchronization of this behavior with the environmental photoperiod. This function is fully preserved when all the brain's nervous connections are severed or when a "loose" brain is transplanted into the tip of the abdomen. By appropriate experiments it was possible to show that the entire mechanism is brain-centered. The components include a photoreceptor mechanism, a clock, and a neuroendocrine output. The clock-controlled release of the hormone acts on the central nervous system to trigger a species-specific behavior pattern which culminates in ecdysis.

The emergence of the adult cecropia silk moth from the pupal skin involves a stereotyped series of abdominal movements-the pre-eclosion behavior. This behavior, triggered by a neurosecretory hormone, consists of three phases that are characterized by the relative frequency and pattern of movements. Electrical recordings from a nerve cord with severed peripheral nerves demonstrate that the pre-eclosion behavior is prepatterned in the abdominal ganglia. In response to the hormone, the entire 1.25-hour behavioral program can be activated and "read off" in the absence of sensory feedback.

When males of the silkmoth Antheraea pernyi were exposed to 12 degrees C diuring adult development and then tested for flight activity at 25 degrees C, the time of the onset of the activity was advanced to the early part of the night. This change in the time of activity was stable and persisted through the life of the moth. Females showed a corresponding shift in the time of sex pheromone release when they were treated in the same fashion.

1. In the tobacco hornworm, wing spreading involves a stereotyped series of movements which take 74 min to complete and which include two components, wing folding and wing inflation. 2. Moths decapitated at the moment of eclosion show neither wing inflation nor wing folding. If decapitation is delayed until 5 sec after emergence, then the full wing-spreading behaviour is displayed. 3. Newly emerged moths which are confined show intense digging-behaviour and delay the onset of wing spreading until after their release. Decapitated moths attempt to spread their wings immediately, regardless of whether or not they are confined. 4. Surgical experiments showed that the brain was not required in a neural capacity in order for wing spreading to occur; it was needed only as a source of the eclosion hormone. The neural influence of the suboesophageal ganglion was required until immediately after eclosion. 5. Newly emerged moths whose abdomens had been removed showed wing-folding behaviour. No inflation occurred and the duration of wing folding was much longer than normal. It was concluded that wing-folding behaviour was centrally programmed but that the abdomen could modify the length of the programme. 6. Injections of bursicon into abdomenless moths reduced the duration of wing-folding behaviour to almost normal levels. Therefore, in some manner, bursicon has an important role in determining when wing-folding behaviour will come to an end.

EXPOSURE of insect embryos to juvenile hormone or to any of its analogues can result in delayed effects which are realized later at metamorphosis1–5. These have been attributed to: (1) persistence of the applied hormone through larval life2; (2) interference with the overall programming of the embryo for postembryonic development1,3; and (3) the selective interference with the embryonic programming of the corpus allatum3,5,6. This study presents evidence that juvenile hormone disrupts the embryonic programming of the corpus allatum such that it does not cease secretion of the hormone as a prelude to metamorphosis.

1. In females of the Cecropia silkmoth, mating stimulates the rate of egg deposition.2. When virgin Cecropia are mated to castrated males, they receive an apparently normal spermatophore except that it lacks sperm. Such females show an oviposition pattern corresponding to that of unmated females. Thus, the normal increase in oviposition rate is triggered by the reception of sperm by the female.3. The corpora cardiaca are shown to be involved in this change in oviposition rate. After the corpora allata are removed from female pupae, the resulting moths respond normally to insemination. After removal of the corpora allata-corpora cardiaca complexes, the ovipositional response to mating is effectively abolished. Reimplantation of up to 5 pairs of corpora allata-corpora cardiaca complexes into pupae lacking their own complexes does not restore the normal oviposition pattern.4. It is concluded that the increase in oviposition rate is due to a hormone which is produced by the intrinsic cells of the corpora card...

Publisher Summary The behavioral responses of an animal to its environment can be drastically modified by changes in its hormonal milieu. Hormonal effects on behavior have been long established in vertebrates, and have been studied primarily with respect to the effects of hormones on sexual behavior. This chapter is concerned with actions of the endocrine system on the nervous system, which lead to overt behavioral acts. Hormones are well suited for modulating the general responsiveness of the nervous system because they have access to all of the neurons. For a widespread change to be mediated through purely neural means, requires an extensive series of excitatory and inhibitory networks. The extra neural circuitry will provide rapid and “fine-tuned” modulation of response thresholds, but it will also increase the complexity and size of the CNS. Hormonal modulation, by contrast, apparently either facilitates or suppresses neural pathways, according to the response characteristics of the particular neurons involved. The manner by which hormones exert their effects on the nervous system is unknown. They may alter the permeability characteristics of the neurons, affect the level of transmitter synthesis, facilitate, or block synaptic transmission, etc. Invertebrates, with their simplified nervous systems and their large, identifiable neurons, have proved to be excellent animals in which to answer many questions concerning the functioning of the nervous system.

The emergence of the Pernyi silkmoth from the pupal exuviae is dictated by a brain-centered, photosensitive clock. In continuous darkness the clock displays a persistent free-running rhythm. In photoperiod regimens the interaction of the clock with the daily lightdark cycle produces a characteristic time of eclosion. But, in the majority of regimens (from 23L:1D to 4L:20D), the eclosion clock undergoes a discontinuous “hourglass” behavior. Thus, during each daily cycle, the onset of darkness initiates a free-running cycle of the clock. The next “lights-on” interrupts this cycle and the clock comes to a stop late in the photophase. The moment when the Pernyi clock stops signals the release of an eclosion-stimulating hormone and is demonstrated to be a function of the time when the free-running cycle is interrupted by lights-on. Moreover, the width (duration) of the eclosion peak in a photoperiod is shown to be dependent upon the length of the dark phase, and, consequently, upon the amount of the free-running cycle that is completed. This relationship demonstrates that the free-running cycle may be divided into two parts. The attainment of maximal accuracy (and thus the narrowest eclosion peak) is dependent upon the completion of only the first 2 hr of the free-running cycle. The completion of succeeding portions of the cycle, while having an effect upon the time of eclosion, no longer affects the accuracy of the clock. A mechanistic model of the eclosion clock is presented.

In insects, hormones are involved in the regulation of behavior to a greater extent than in other invertebrate or vertebrate groups. This extensive adoption of hormonal involvement in the function of the nervous system may be partially due to size restrictions and to the rich behavioral repertoires which insects characteristically show. Hormones, with their ability to reach every cell in the nervous system, are capable of causing widespread changes in responsiveness and thus of radically changing behavior. The use of this kind of hormonal control instead of more complex neural mechanisms may be a major factor in packing a broad array of behaviors into a small, simplified nervous system.

Taxonomic work on the Culicidae is hindered by lack of a completely satisfactory method of preserving specimens. Mosquito larvae stored in fluid often lose hairs and gills, and also are difficult to manipulate and study. A more satisfactory method is to mount the larvae on slides, but the specimens often distort and can be viewed from only 2 sides. Preservation of adults raises more serious problems. Preservation in fluid is unsatisfactory, since this method destroys the scale patterns which are necessary for taxonomic work. Mounting on pins or points is not especially satisfactory because the specimens shrivel and become distorted as they dry. Freeze-drying of adults gives well-preserved specimens (Woodring and Blum 1963), but the apparatus required is expensive and not generally available, especially to field workers. This paper describes a technique for the treatment of adult and larval mosquitoes with acetone that results in dry specimens which are not distorted or shriveled.

Invertebrate Endocrinology and Hormonal Heterophylly. Edited by Walter J. Burdette. Pp. xviii + 438. (Springer: Berlin and New York, 1974.) $21.50.