The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust

The presence of juvenile hormone in the haemolymph of larvae of Locusta has been detected by a modified Galleria bioassay and these results are compared with indirect methods of estimating corpus allatum activity. Juvenile hormone is present in the haemolymph during the fourth larval instar except on the last day of the instar, and is absent from the haemolymph of the fifth and final larval instar except on the last day of the instar. Changes in the volumes of the corpora allata simply reflect changes in the growth of the whole insect and are of no value in predicting endocrine activity. Changes in the size of the cells of the corpora allata can be correlated with the presence of juvenile hormone in the haemolymph in the fourth larval instar, but similar changes in cell size occur in the fifth larval instar when no juvenile hormone is present in the haemolymph. The effects of the implantation of corpora allata are unreliable as estimates of corpus allatum activity as isolated corpora allata from fifth instar larvae release juvenile hormone. Indirect methods of measuring corpus allatum activity are thus shown to be unreliable. The R_f value of Locusta juvenile hormone as determined by thin-layer chromatography differs from that of Roeller's juvenile hormone, suggesting that the two hormones might be chemically distinct.     

Compactin inhibits insect HMG-CoA reductase and juvenile hormone biosynthesis

The activity of 3-hydroxy-3-methylglutaryl CoA reductase in homogenates of the corpora allata of the tobacco hornworm, $\text{Manduca sexta}$, was competitively inhibited by compactin. The K_I for the sodium salt form of compactin was 0.9 nM for the reductase from both male and female corpora allata. In intact female corpora allata juvenile hormone biosynthesis was also inhibited by approximately 50 percent at 10 nM compactin. Following injection with compactin, darkening of the cuticle, an indication of juvenile hormone deficiency, was observed in larvae after ecdysis from third to fourth instar. Hence, compactin shows potential as an inhibitor of insect growth and development.     

Improved assay conditions for measurement of corpus allatum activity in vitro in the adult Colorado potato beetle, Leptinotarsa decemlineata

Assay conditions for the short-term, radiochemical, in vitro determination of the spontaneous rate of juvenile biosynthesis by isolated corpora allata from Leptinotarsa decemlineata have been further improved, permitting the measurement of juvenile hormone biosynthesis by individual pairs of corpora allata. The final incubation product has been identified as juvenile hormone III with the aid of High-performance liquid chromatography (HPLC) and juvenile hormone esterase degradation. Using the new assay conditions, the activities of adult corpora allata during maturation were found to be significantly higher in reproductive, long-day animals than in pre-diapause, short-day beetles. During diapause no activity was detectable, whereas corpora allata from post-diapause beetles were reactivated totally after 5 days. Simultaneous determination of the in vitro rates of juvenile hormone biosynthesis and corpus allatum volumes revealed no clear correlation although the results suggest that the volume may be indicative of the maximal capacity for juvenile hormone production. Corpora allata from a population of beetles did not display any synchronous diurnal rhythmicity.     

Regulation of the corpora allata in the black mutant of Manduca sexta

Regulation of corpus allatum activity in the black mutant strain of Manduca sexta was studied in vivo and in vitro. Allatectomy, denervation, and implantation studies demonstrated that black mutant corpus allatum activity remains low in both wild-type and black mutant host larvae. Attempts to distinguish humoral control mechanisms versus mechanisms dependent on intact allatal nerves indicated that intact allatal nerves were not required for the reduced black mutant corpus allatum activity in vivo. Incubation of corpora allata, using [1-¹⁴C]propionate as a juvenile hormone biosynthetic precursor and haemolymph as culture medium, confirmed that black mutant corpora allata are suppressed by a factor(s) in the haemolymph. Under identical conditions wild-type corpora allata were unaffected. Finally, the lowered black mutant corpus allatum activity in haemolymph in vitro correlates with the lowered juvenile hormone titre in black mutant larvae.     

Improved conditions for culture of biosynthetically active cockroach corpora allata

Summary Currently, short-term culture of insect corpora allata is most often performed in TC199. We now show that L-15B, a medium widely used in arthropod tissue culture, is superior to TC199 for both short- and long-term culture of cockroach corpora allata. In 3-h and 48-h incubations, juvenile hormone biosynthesis by corpora allata from Diploptera punctata was significantly higher in L-15B than in TC199. In addition, in both media, corpora allata activity was significantly improved by flotation of glands at the medium surface. Characteristics of L-15B responsible for its superiority were examined by comparison of gland activities in several TC199 formulations that had been modified in different ways to be more similar to L-15B. Adjusting the osmotic pressure of TC199 (288 mOsm/l) to near that of L-15B (362 mOsm/l) and D. punctata hemolymph (360 mOsm/l) significantly improved gland activity during the second 12 h of a 36-h incubation. Increasing the concentrations of amino acids, sugars, and organic acids in TC199 to the same levels as in L-15B significantly improved gland activity during both the second and third 12-h intervals of a 36-h incubation. These results suggest that L-15B is superior to TC199 because L-15B is isoosmotic with D. punctata hemolymph and because L-15B, like cockroach hemolymph, contains a high level of organic constituents. It is therefore more appropriate to use L-15B than TC199 for short-term in vitro assays of juvenile hormone biosynthesis and for extended corpora allata culture.     

Activite de l'hormone juvenile en C17 (JH-II) chez Locusta migratoria

The effects of C₁₇ juvenile hormone (JH-II) have been investigated in Locusta on morphogenesis, ovarial development, and pigmentation, by means of injections in oil. These effects have been compared with those of injecting C₁₈ juvenile hormone (JH-I) and of implanting corpora allata into Locusta. JH-I and JH-II are similar in their effects upon morphogenesis and pigmentation, and also on ovarial development in which JH-III has been found to be more effective in other insects. Injections of JH-I and JH-II have similar effects to those seen after implanting corpora allata. However in experiments on heart beat (in which the corpora allata have been shown to be involved) JH-I is the only substance to increase the rate of heart beat in the same way as active corpora allata. These observations are discussed, and it is concluded that JH-I is the hormone with effects nearest to those of the corpus allatum hormone itself.     

NADPH dependent epoxidation of methyl farnesoate to juvenile hormone in the cockroach Blaberus giganteus L.

Corpora allata and corpora allata homogenates from the cockroach $\text{Blaberus giganteus}$ are able to convert tritium labeled $\text{trans, trans}$-methyl farnesoate into insect juvenile hormone III. The epoxidation appears to be enzymatic occurring largely in the 100,000 g precipitate or microsomal fraction of corpora allata homogenates. The epoxidation is NADPH dependent, requires molecular oxygen and is inhibited by carbon monoxide, methylene blue, SKF 525A, and piperonyl butoxide.     

Primer effect of queen pheromone on juvenile hormone biosynthesis in adult worker honey bees

Juvenile hormone synthesis in adult worker honey bees was measured by an in vitro corpora allata bioassay. Adult queenless workers exhibit higher rates of juvenile hormone biosynthesis than queenright workers. Hormone synthesis is not correlated with the volume of the glands. Extract of queen mandibular glands, applied to a dummy, reduces juvenile hormone biosynthesis in caged queenless workers to the level of queenright workers. The same result was obtained with synthetic (E)-9-oxo-2-decenoic acid, the principal component of the queen mandibular gland secretion. This pheromonal primer effect may function as a key regulating element in maintaining eusocial colony homeostasis. The presence of brood does not affect the hormone production of the corpora allata.Abbreviations BSA bovine serum albumin - CA Corpora allata - JH juvenile hormone - 9-ODA (E)-9-oxo-2-decnoic acid     

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.     

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.     

Juvenile hormone III biosynthesis by the larval corpora allata of Manduca sexta

A radioimmunoassay (RIA) for juvenile hormone III has been established which quantifies the biosynthesis of this hormone in vitro by the corpora allata of larvae and pupae of the tobacco hornworm, Manduca sexta. The specificity of the RIA for homologues and metabolites of juvenile hormone III was determined and it was found that the antibody was specific for juvenile hormone III and its acid. The juvenile hormone III RIA activity synthesized in vitro by corpora allata from day-5 last-instar larvae was identified as juvenile hormone III by high pressure liquid chromatography. The kinetics of hormone synthesis by corpora allata from selected stages during larval-pupal development revealed differential rates of synthesis, suggesting that juvenile hormone III may have a hormonal function in the larva and that regulation of its synthesis may occur. The significance of these developmental fluctuations in rates of juvenile hormone III synthesis by the corpora allata is discussed in relation to the haemolymph titres of the hormone.     

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.     

The effects of juvenile hormone, 20-hydroxyecdysone and precocene II on activity of corpora allata and the mode of negative-feedback regulation of these glands in the adult Colorado potato beetle

When the titre of juvenile hormone III in female Leptinotarsa decemlineata was elevated by the implantation of supernumerary corpora allata or by the injection of the hormone, the rate of endogenous hormone production by the host glands was significantly restrained, as determined by the short-term in vitro radiochemical assay. From denervation studies, it is suggested that during phases of elevated juvenile hormone titre, the corpus allatum activity is regulated via humoral as well as neural factors requiring intact nerve connections. Restrainment of gland activity appears to be mainly via the neural pathway. Isolated corpora allata were not influenced by 10^?5 M juvenile hormone III added to the incubation medium in vitro.Studies with farnesenic acid revealed that the final two enzymatic steps in the biosynthetic pathway of juvenile hormone are also diminished during prolonged neural inhibition of the corpora allata.20-Hydroxyecdysone and precocene II had no apparent effect on the corpus allatum activity of Leptinotarsa decemlineata.     

Adaptation of a radiochemical assay for juvenile hormone biosynthesis to study caste differentiation in a primitive termite

A radiochemical assay measuring juvenile hormone synthesis by corpora allata incubated in vitro was adapted for use with the termite Zootermopsis angusticollis. Corpora allata from 3–4-day old virgin female neotenic reproductives were used in these studies because this caste showed the highest rates of juvenile hormone synthesis (0.6 pmol h^?1 per pair corpora allata). Juvenile hormone-III synthesis was linear for up to 6 h over the range of concentrations of labelled l-methionine from 27–280 μM. Rates of juvenile hormone synthesis were stimulated up to 10-fold in a dose-dependent manner by the addition of farnesoic acid to the incubation medium. However, the relatively high concentration of 120 μM farnesoic acid reduced the rates of juvenile hormone synthesis. The radiochemical assay was used to determine rates of juvenile hormone synthesis in vitro by corpora allata from larvae with a queen and king vs orphaned larvae. The presence of reproductives resulted in a suppression of larval corpus allatum activity relative to orphaned controls.     

Quantitative studies on the activity of the corpora allata in adult male Locusta and Schistocerca

Juvenile hormone has been detected in the haemolymph and corpora allata of adult male Locusta and the haemolymph of adult male Schistocera by a modified Galleria bioassay. The hormone was readily detected in the haemolymph of insects immediately after the final ecdysis, but then became difficult to detect until 2 days prior to the onset of sexual maturation. In sexually mature insects the titre of juvenile hormone was maintained at a constant level. The corpora allata of adult male Locusta increased in size throughout adult life. The juvenile hormone content of the corpora allata was low during the period of somatic growth, but increased at the onset of sexual maturation. Sectioning of the nervi corporis allati I in insects immediately after the final ecdysis prevented the normal increase in size of the corpora allata, but did not render them inactive since juvenile hormone was detected in the haemolymph after the operation. The half life of juvenile hormone in the haemolymph of allatectomized adult male Locusta was 1 to 2 hr.     

Responsiveness of the adult cricket (Gryllus bimaculatus andAcheta domesticus) retrocerebral complex to allatostatin-1 from a cockroach,Diploptera punctata

Brain-retrocerebral complexes of female crickets,Gryllus bimaculatus andAcheta domesticus, treated with antibody to allatostatin-1 from a cockroach,Diploptera punctata, show extensive immunoreactivity. The results suggest that allatostatins or allatostatin-like molecules are produced in neurosecretory cells of the brain and are delivered to the corpora allata through nervous connections and/or via haemolymph. Radiochemical measurements of juvenile hormone III biosynthesis by isolated corpora cardiaca-corpora allata complexes from adultG. bimaculatus have been used to demonstrate an in vitro sensitivity of these glands to allatostatin-1 fromD. punctata. Allatostatin-1 is a relatively potent inhibitor of juvenile hormone III biosynthesis in corpora allata of both young adult females and males. In glands taken from 3-day virgin females, 50% inhibition of hormone biosynthesis is reached at ca. 3 nmol·l^-1 allatostatin-1. The inhibitory action of allatostatin-1 is rapid, dose-dependent and reversible. Addition of 200 mol·l^-1 farnesol to the incubation medium prevents inhibition of juvenile hormone III biosynthesis by allatostatin-1. Juvenile hormone III biosynthesis by isolated corpora allata of 3-day female house crickets,A. domesticus, is also susceptible to inhibition by 1 mol·l^-1 allatostatin-1.Abbreviations ASB2 Diploptera punctata allatostatin-5 - CA corpora allata - CC corpora cardiaca - Dip A-1 Diploptera punctata allatostatin-1 - HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid - JH juvenile hormone(s) - Mas-AS Manduca sexta allatostatin - MF methyl farnesoate - NCA nervus corporis allati - NCC nervus corporis cardiaci - SEM standard error of mean - TRIS Tris(hydroxymethyl)aminomethane     

Sex-specific developmental profiles of juvenile hormone synthesis in honey bee larvae

Summary Juvenile hormone synthesis in drone larvae of the honey bee was measured by an in vitro radiochemical assay. The developmental profile of corpora allata activity in male larvae showed considerable differences from queen larvae, the presumptive reproductive females, and was comparable to workers, the sterile female morph. Drone and worker larvae, however, differed drastically in the regulation of juvenile hormone biosynthesis, as revealed by the addition of farnesoic acid to the culture medium. This precursor stimulated juvenile hormone synthesis of drone glands nearly eightfold, whereas in worker larvae it is known to lead to an accumulation of methyl farnesoate. The sex-specific differences in endocrine activity indicate a role for juvenile hormone in the expression of genetically determined sexually dimorphic characters during metamorphosis, a role not currently accounted for in models describing endocrine regulation of insect development. Correspondence to: K. Hartfelder     

Partial characterization and isolation of earwig `allatostatins': biological activities in earwigs and cockroaches

Allatostatins are a family of neuropeptides first isolated from the cockroach, Diploptera punctata, that inhibit juvenile hormone production in that species (but do not do so in earwigs), and inhibit hindgut muscle contractions in some insects, including the earwig, Euborellia annulipes. We examined whether material from earwig brains is similar to cockroach allatostatins biochemically, immunologically and physiologically. Brain extracts from adult female earwigs were separated by high performance liquid chromatography (HPLC), followed by radioimmunoassay using antibodies to cockroach allatostatin (Dip-AST). Fractions that co-eluted with cockroach allatostatins were immunoreactive, and at least two peaks of immunoreactivity were detected. Material from each peak at 10 nM Dip-AST equivalents inhibited juvenile hormone biosynthesis in vitro by corpora allata of 2-day virgin D. punctata cockroaches; 1 nM was less effective, and non-immunoreactive fractions failed to inhibit juvenile hormone biosynthesis. Both crude and Sep-Pak (Waters) purified extracts of brains of earwigs containing 1 nM Dip-AST equivalents failed to suppress hindgut contractions in vitro of 2-day earwigs and of brooding female earwigs. In contrast, 1 nM cockroach allostatin 1 (Dip-AST 7) reversibly inhibited hindgut contractions in vitro. These results suggested the presence of another brain factor, such as proctolin, that counteracts the inhibitory effects of Dip-AST. In support of this hypothesis, proctolin stimulated hindgut contractions in vitro at 1 nM; the effects of equal concentrations of allatostatin and proctolin varied with the stage of the female. Furthermore, HPLC-separated fractions that co-eluted with cockroach allatostatin and were immunoreactive with antibodies to Dip-AST suppressed hindgut contractions in vitro of 2-day female earwigs. Finally, crude brain extracts of earwigs suppressed earwig juvenile hormone biosynthesis in vitro in glands of low, but not in glands of high, activity. Thus, earwig brain extract after HPLC separation has Dip-AST-like material that inhibits cockroach corpora allata and suppresses earwig hindgut contractions. Sep-Pak-extracted earwig brain material, however, does not inhibit earwig gut contraction. Although synthetic Dip-AST 7 does not inhibit juvenile hormone synthesis by earwig corpora allata, there is heat-stable material in earwig brain extract that does have this action.     

Activity of the corpora allata in the adult female migratory locust

Juvenile hormone was detected in the haemolymph of adult female Locusta by a modified Galleria bioassay. The hormone was present in the haemolymph immediately after the final ecdysis, but could not be detected after this time until the end of the period of somatic growth just before the start of ovarian development. During the first gonotrophic cycle the levels of juvenile hormone in the haemolymph could be related to the growth of the proximal oöcytes. The volumes of the corpora allata could be related to haemolymph juvenile hormone levels during the first gonotrophic cycle. Ovariectomy had no effect on haemolymph juvenile hormone levels or on the volumes of the corpora allata.     

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.