The allatostatic effect of 20-hydroxyecdysone on the adult viviparous cockroach, Diploptera punctata

The effect of 20-hydroxyecdysone upon the activity of corpora allata (CA) from female Diploptera punctata has been investigated. This ecdysteroid inhibits juvenile hormone (JH) biosynthesis by the CA, whether they have been implanted into a male, or remained in situ within the female. In the female, this inhibition is reflected in reduced oöcyte growth and vitellin content. The allatostatic effect of 20-hydroxyecdysone becomes apparent in vivo within 24 hr. However, no inhibition was observed when the CA were maintained in vitro for 42 hr in medium containing up to 1·10^?5 M 20-hydroxyecdysone. This suggests that the effect of the hormone upon the CA is indirect. These experiments raise the possibility that ecdysteroids play an allatostatic role during the normal gonotrophic cycle in Diploptera.     

Larval development of Diploptera punctata reared alone and in groups

Larvae of the cockroach Diploptera punctata were reared in isolation, in pairs, or in groups of 8–10. Duration of larval development, age at each ecdysis, weights at birth and ecdyses, and adult head-capsule width were measured. Duration of larval development was longer and adult size was larger in isolated animals than in animals reared in pairs and groups. The effect of isolation on development was more pronounced in males. All females had 4 larval instars, whereas males had 3 or 4 instars. The proportion of males with 4 larval instars was higher among animals reared in isolation. There was no difference in the duration of larval development or adult size between pair- and group-reared animals. The sex of animals in the group did not affect adult size or the duration of larval development. Males which underwent 3 or 4 larval instars had different schedules of moulting. Rates of growth of males of both instar types reared in isolation and pairs were similar. Greater adult weight of isolated animals and 4-instar-type males was a result of their longer duration of larval development. Both a higher rate of growth and longer duration of larval development contribute to the larger adult size of females than males.     

The inhibition of milk synthesis by juvenile hormone in the viviparous cockroach, Diploptera punctata

The brood sac of the viviparous cockroach, Diploptera punctata, synthesizes a protein rich milk which nourishes developing embryos. Milk is first detected in the brood sac (by immunoelectrophoresis) when the embryos begin drinking and continues to increase in parallel with total protein of the brood sac. When embryos cease drinking, both total protein and milk decline in the brood sac. Premature decline in protein and milk content of the brood sac has been observed after treatment with juvenile hormone (from implanted active corpora allata) or a juvenile hormone analogue (ZR 512 applied topically). The fine structure of the brood sac 7 days after corpora allata implant is consistent with that of gland cells which are not actively synthesizing milk. The effect of ZR 512 is detected in decreased milk content of the brood sac after 24 hr of treatment.     

Regulation of juvenile hormone synthesis during pregnancy in the cockroach, Diploptera punctata

Regulation of juvenile hormone synthesis during pregnancy was investigated after determining the normal rates of synthesis in pregnancy and the second gonadotrophic cycle in Diploptera punctata by direct in vitro radiochemical assay.The low rate of juvenile hormone synthesis during early pregnancy is maintained by three factors: (1) the small ovary which is incapable of eliciting increased rates of juvenile hormone synthesis (2) an inhibitory centre in the brain acting via intact nerves to the corpora allata (similar to that in virgin females) and (3) an inhibitory centre in the brain acting via the haemolymph (elicited by embryos in the brood sac).The existence of two inhibitory centres in the brain is supported by the additive effect of denervating the corpora allata and removing embryos. Whereas these operations alone activated the corpora allata in 54 and 31% of the females, respectively, together they activated 87%, similar to the 91% activated by denervation alone in late pregnancy.The inhibition which remains after denervation of the corpora allata can be removed by decapitation and restored by implantation of the protocerebrum from a pregnant female but not from one developing oöcytes.The inhibition elicited by embryos in the brood sac can be overcome by introduction of a stimulatory ovary and/or substitution of active corpora allata.     

Cellular and volumetric changes in relation to the activity cycle in the corpora allata of Diploptera punctata

In the corpora allata (CA) of the viviparous cockroach, Diploptera punctata, a cycle of juvenile hormone (JH) synthesis during ovarian maturation can be correlated with cyclical changes in CA volumes and cell numbers. Uptake of [³H]-thymidine occurs in nuclei of CA cells during periods of increase in cell number. Both members of a pair of CA maintain symmetry of volume, cell number and rate of JH synthesis. After a cycle of CA activity, the CA can be transplanted to a young, allatectomized female, where they support a second wave of oöcyte development.     

Bacteria-induced haemolymph proteins of Manduca sexta pupae and larvae

The haemolymph of Manduca sexta larvae and pupae has been analyzed for proteins potentially associated with the bacterial defence response of the insect. Five proteins, M13, M18, M20, M23, and M24 in pupae, and M4, M11, M13, M18 and M23 in larvae, are induced by the injection of bacteria into the haemolymph. Proteins M4 and M11 are always present at high levels in uninjected pupae. Proteins M20 and M24 could not be induced in larvae. These proteins, as well as those not showing a response to bacterial challenge or injury, were also analyzed for presence of disulphide bonds and carbohydrate moieties, and their apparent molecular weights determined.     

Eclosion hormone activity in haemolymph of eclosing silkmoth, Bombyx mori

The presence of eclosion hormone in the haemolymph of eclosing adults of the silkworm, Bombyx mori, was demonstrated to provide evidence that the hormone is involved in the induction of eclosion of this insect.The eclosion of the adult occurred within 55–75 min after light-on, when the pupae had been kept under conditions of 16 h light and 8 h dark during adult development. The hormone became detectable in the haemolymph 10–20 min after light-on, followed by eclosion 40 min later. The hormonal activity increased sharply to a maximal level (30 units/ml haemolymph) at 30 min prior to the eclosion and then fell rapidly: detectable eclosion hormone was present in the haemolymph for only 15–20 min. The hormone was partially purified from the haemolymph of eclosing animals and a 400-fold purification was achieved. The preparation was rather stable against heat treatment, and the molecular weight was estimated to be 5,000–10,000 by gel-filtration on a Sephadex G-50 (superfine). The origin of eclosion hormone in the haemolymph is discussed.     

The effects of nutrition and methoprene treatment on ovarian ecdysteroid synthesis in Drosophila melanogaster

Radioimmunoassay of in vitro culture medium from ovaries of Drosophila melanogaster indicates that detectable ovarian ecdysteroid synthesis begins between 6 and 12 h after eclosion and reaches a peak between 24 and 30 h, when animals are reared at 25°C, 12 h photophase. Analysis of 24 and 72 h medium by a combination of high-performance liquid chromatography and radioimmunoassay demonstrates three ecdysteroid regions, two comigrating with known standards of ecdysone and 20-hydroxyecdysone and a third highly polar region containing one or more unidentified radioimmunoassay-active ecdysteroids. In 72 h medium the polar region comprises the majority of radioimmunoassay-active material while in 24 h medium the majority is in the ecdysone region. Provision of a nutritionally deficient diet to females at adult eclosion prevents the normal increase in vitellogenic-stage follicles and ovarian ecdysteroid synthesis. Methoprene treatment of such females stimulates a transient burst of ovarian ecdysteroid synthesis and the production of near normal numbers of vitellogenic oöcytes by 24 h, although by 48 h the number of vitellogenic oöcytes is less than normal.     

Occurrence of immunoreactive estradiol-positive material in the haemolymph and ovary of the tasar silkworm,Antheraea mylitta

Summary

Estradiol from the haemolymph and ovaries of tasar silkworm, Antheraea mylitta, was assayed using radio immunoassay. The estimated concentration of estradiol was 143 pg/mg in the ovaries and 102 pg/ml in the haemolymph.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

Precocenes as pro-allatocidins in adult female Diploptera punctata: A functional and ultrastructural study

Adult mated females of the viviparous cockroach Diploptera punctata are moderately sensitive to precocenes. Oöcyte growth is inhibited and oviposition is delayed in insects topically treated with precocene II or precocene III. C₁₆ juvenile hormone release by corpora allata of precocene-treated insects is markedly inhibited when compared to corpora allata of acetone-treated controls. Electron microscopy of the corpora allata reveals that precocene treatment results in a disorganisation of the intracellular organelles. Topically applied precocene II reaches a high concentration in the haemolymph (0.5 mM 2 hr after topical application of 250 μg). C₁₆ juvenile hormone release by isolated corpora allata is inhibited by precocenes in vitro; half-maximal inhibition over a 3 hr period is obtained at 0.4 mM precocene II. In vitro inhibition of corpora allata by precocene II concentrations higher than 1 mM rapidly destroys the glands as evidenced by electron microscopy (total disintegration of cellular organelles) and by the virtual cessation of C₁₆ juvenile hormone synthesis by the corpora allata. Inhibition of C₁₆ juvenile hormone release by precocene is time-dependent and is not reversible over the short-term incubation in vitro. This inhibition does not appear to be related to the spontaneous activity of the glands in vitro, and it can be reduced by two epoxidase inhibitors. Precocenes are pro-allatocidins in this species: they are bioactivated within the corpora allata to cytotoxic epoxides.     

The mechanism of compensation in juvenile hormone synthesis following unilateral allatectomy in Diploptera punctata

Following unilateral allatectomy in the viviparous cockroach, Diploptera punctata, the remaining corpus allatum (CA) can synthesize juvenile hormone (JH) at rates equal to that of a pair of control CA. A single CA undergoes changes in volume and cell number similar in magnitude to those occurring during the normal cycle of JH synthesis. There is no hyptertrophy of the CA associated with compensation in JH synthesis. Therefore, the rate of JH synthesis per cell or per unit volume of single CA following unilateral allatectomy is twice that of a single control CA.     

Inhibition of the corpora allata of last-instar male larvae of the cockroach, Diploptera punctata

Normal rates of juvenile hormone synthesis, cell number and volume of corpora allata were measured in penultimate and final-instar male larvae of Diploptera punctata. The rate of juvenile hormone synthesis per corpus allatum cell was highest on the 4th day of the penultimate stadium, declined slowly for the remainder of that stadium, and rapidly after the first day of the final stadium.Regulation of the corpora allata in final-instar males was studied by experimental manipulation of the corpora allata followed by in vitro radiochemical assay of juvenile hormone synthesis. Nervous inhibition of the corpora allata during the final stadium is suggested by the observation that rates of juvenile hormone synthesis increased following denervation of the corpora allata at the start of the stadium; this operation induced a supernumerary larval instar. Juvenile hormone synthesis by corpora allata denervated at progressively later ages in the final stadium and assayed after 4 days decreased with age at operation. This suggests an increasingly unfavourable humoral environment in the final stadium, which was confirmed by the low rate of juvenile hormone synthesis of adult female corpora allata implanted into final-instar larvae. Thus, inhibitory factors or lack of stimulatory factors in the haemolymph may act with neural inhibition to suppress juvenile hormone synthesis in final-instar males.     

Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella

Approximately two-thirds of the total amount of ecdysteroids in late—pharate adults of the wax moth, Galleria mellonella, were found in the ovaries and one-third in the ovariectomized body. Chemical analysis of these ecdysteroids by thin-layer and high-pressure liquid chromatography, coupled with an ecdysteroid radioimmunoassay, revealed the presence of 2-deoxyecdysone, ecdysone and 20-hydroxyecdysone, as well as high and low polarity unknowns. The predominant identifiable ecdysteroid in both the ovaries and ovariectomized body was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone, respectively. Incubation of late-pharate adult ovaries in culture medium revealed that they synthesize and secrete ecdysteroids in vitro. The in vitro distribution of ecdysteroids between ovaries and incubation medium was similar to that observed between ovaries and ovariectomized bodies in situ and the predominant identifiable moiety both retained and released by the ovaries in vitro was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone. Collectively, these results support the idea that the ecdysteroids synthesized by the ovaries of late-pharate adult Galleria are both stored and secreted and that the quantity of a specifically secreted ecdysteroid is precisely controlled. This apparent regulation of the distribution of ovarian ecdysteroids raises the possibility that the stored and secreted forms have distinct functions in the reproductive physiology of this insect.     

Cellular events in the prothoracic glands and ecdysteroid titres during the last-larval instar of Spodoptera littoralis

Changes in prothoracic gland morphology were correlated to developmental events and ecdysteroid titres (20-hydroxyecdysone equivalents) during the last-larval instar in Spodoptera littoralis. After ecdysis to the last-larval instar the haemolymph ecdysteroid titre remained at about 45 ng/ml, when the prothoracic glands appeared quiescent. The first signs of distinct gland activity, indicated by increased cell size and radial channel formation, were observed at about 12 h prior to the cessation of feeding (36 h after the last-larval moult), accompanied by a gradual increase in ecdysteroid titre to 110 ng/ml haemolymph, at the onset of metamorphosis. During this phase ecdysteroid titres remained at a constant level (140–210 ng/ml haemolymph) and prothoracic gland cellular activity was absent for a short period. The construction of pupation cells occurred when haemolymph ecdysteroids titres increased to 700 ng/ml. A rapid increase in ecdysteroids began on the fourth night (1600 ng/ml haemolymph) reaching a maximal level (4000 ng/ml haemolymph) at the beginning of the fourth day. In freshly moulted pupae a relatively high ecdysteroid titre (1100 ng/ml haemolymph) was still observed, although during a decrease to almost negligible levels. The increase in ecdysteroid level during the third and the fourth nights of the last-larval instar was correlated with the period when almost all the prothoracic gland cells showed signs of high activity. Neck-ligation experiments indicated the necessity of head factors for normal metamorphosis up to the second to third day of the instar. The possibility that the prothoracic glands are under prothoracicotropic hormone regulation at these times is discussed.     

Influence of leg regeneration on ecdysteroid titres in Acheta larvae

The effects of regeneration on ecdysteroid levels and duration of the 10th larval instar of female house crickets were studied. Three experimental groups were used. The first consisted of larvae regenerating two legs cut off on the first day of this stage. The second group was made up of insects that underwent either an epidermal incision (sham-operated group I) or bleeding (sham-operated group II) at the same time as amputation in the first group. The last group was composed of normal insects.Larval stage length: in the first two groups, the duration of the 10th larval stage was significantly modified. It increased for regenerating animals as well as for sham-operated group I, but decreased for sham-operated group II.Ecdysteroid production: controls showed two periods of intense ecdysteroid activity. The first, which took place between days 4 and 6, was assumed to induce apolysis of the tegument and to render regeneration impossible (it coincided with the so-called critical period for regeneration). The second, which occurred between days 7 and 8 initiated cuticle synthesis. For sham-operated insects neither epidermal injury nor a loss of blood appear to change this hormonal profile significantly. Regenerating crickets however exhibited drastically reduced ecdysteroid peaks: the total apparent production of ecdysteriods was 50% below normal. Moreover, relative concentration of ecdysone to 20-hydroxy-ecdysone was greatly increased. Thus, regeneration obviously has a great and specific influence on ecdysteroid release in insect larvae. This effect may be related to changes observed in the prothoracic gland cycle which suggest that it plays a complex role in the regulation of the regenerative process.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

Variations in ecdysteroid levels in 5th instar larvae of Schistocerca gregaria in gregarious and solitary phases

Moulting hormone levels in whole bodies of 5th instar S. gregaria were determined throughout the 5th larval instar using gas chromatography with electron capture detection of the derivatized hormone. No differences were found between hormone levels in insects in the gregarious and solitary phase. No evidence for the existence of polar ecdysteroid conjugates used for storage or transport of these hormones was found. Approximately one tenth of the 20-hydroxyecdysone observed in the gregarious insects was detected in the faeces, almost equally divided between free hormone and polar conjugates of it.     

Histological and cytological studies on the ovary of Nauphoeta cinerea (Blattaria,Oxyhaloidea) during the first reproductive cycle

Histological studies were performed on the ovary of the ovoviviparous cockroach Nauphoeta cinerea during the first reproductive cycle by means of optical microscopy and histoautoradiography, and electron microscopy. The oöcyte chamber is composed of follicle cells, the oöcyte and a layer of symbiotic bacteria at the level of the microvillous border of the oöcyte. The first reproductive cycle begins with a short inactive period preceding the appearance of vitellogenin. During this period, the follicular epithelium achieves its development by a mitotic flare. From the 3rd day on, vitellogenin is synthesized by the fat body and large intercellular spaces appear between the follicular cells, in conjunction with a rapid growth of the oöcyte, which takes up selectively the vitellogenin by means of pinocytotic vesicles. These coalesce to give the yolk globules. Along with these phenomena, the proteosynthetic apparatus and its activity in the follicular cells increase slowly. After about the 12th day, the intercellular spaces disappear and the follicular epithelium which has now a very well developed proteosynthetic apparatus, begins to synthesize and lay down the egg membranes. After ovulation, the empty oöcyte chamber collapses and the follicular epithelium shows rapid degeneration processes (large cytolysosomes) that destroy the chamber completely during the gestation period. At the beginning of the 2nd cycle, there only remain a cell or two of the previous follicular epithelium and a very large annular piece of basement membrane.