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.     

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.     

Quantification of allatostatin receptor mRNA levels in the cockroach, Diploptera punctata, using real-time PCR

The cockroach allatostatin receptor (Dippu-AstR) is a 425 amino acid G-protein coupled receptor that is related to the mammalian galanin receptor. Using relative standard curve real-time PCR analysis, changes in Dippu-AstR mRNA expression levels were examined in tissues of adult mated and virgin female Diploptera punctata. Tissues were chosen that were either known targets of allatostatin (Dippu-AST) action or sites of Dippu-AST localization. Tissues examined included brain, corpora allata (CA), gut, ovaries, testes and abdominal ganglia. Dippu-AstR was expressed in all tissues examined for 7 days after adult emergence. Juvenile hormone (JH) biosynthesis is known to peak on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA was at the highest levels on day 6 post-emergence in brain and CA and day 2 post-emergence in midgut. Dippu-AstR expression was found to correlate with the decline in JH biosynthesis noted on day 5 post-emergence and early inhibition of feeding. Dippu-AstR mRNA expression in virgin female midgut and CA was dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR mRNA was found to be similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declined to low levels by day 4. Testes exhibited maximal Dippu-AstR mRNA expression on days 4 and 7 of adult life. A role for Dippu-AST in testes of Diploptera is unknown.     

Effects of starvation and mating on corpora allata activity and allatotropin (Manse-AT) gene expression in Manduca sexta

The levels of three alternatively spliced mRNAs from the Manduca sexta allatotropin (Manse-AT) gene were determined following physiological manipulations during the larval, pupal and adult stages; starvation of larvae, induction of pupal diapause and adult mating experience. The juvenile hormone biosynthetic activity of the corpora allata (CA) was also determined in starved larvae and in mated and unmated females. Starvation of early fifth instar larvae specifically increased the amount of one Manse-AT mRNA that is predicted to encode Manse-AT and two related peptides, Manse-ATL-I and -II. The normal rapid decrease in the activity of the CA in last instar larvae was not observed in starved insects which maintained a relatively high rate of JH biosynthesis for at least 3 days. Diapause induction resulted in a small increase in one Manse-AT mRNA, but levels were much lower compared to those observed in larvae or adults. During the first 4 days of adult life, Manse-AT mRNA levels were not changed as a result of mating. However, in mated females, the rate of JH biosynthesis gradually increased, in sharp contrast to the relatively low level of CA activity seen in virgin females. These observations suggest the elevated activity of the CA in mated females is not simply due to the increased level of Manse-AT mRNA.     

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.     

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.     

Activity of the corpora allata of adult female Leucophaea maderae: effects of mating and feeding

Juvenile hormone III biosynthesis by corpora allata of adult female Leucophaea maderae was measured by an in vitro radiochemical assay. In fed females, JH III synthesis increases more than 20-fold after mating to a peak of 55 pmol/pair/h on day 9 and then rapidly declines. This increase in JH III synthesis concomitant with rapid oocyte growth in mated females is not observed in virgin females. The corpora allata from starved, virgin females appear to be inactive. The addition of 150 microM 2E,6E-farnesol (a) JH III precursor) to the incubation medium stimulates the corpora allata from starved, virgin females less than the corpora allata from starved, mated females. Both feeding and mating are necessary for the expression of a normal cycle of JH III synthesis in this cockroach.     

Feeding and activation of corpora allata in the cockroach Blattella germanica (L.) (Dictyoptera,Blattellidae)

Adult females of the cockroach Blattella germanica have clearly-defined feeding cycles related to oogenesis. In the first cycle, food ingestion precedes volumetric increase in the corpora allata, which in turn precedes juvenile hormone production, whereas starved females do not develop the corpora allata and produce very low amounts of juvenile hormone. When the second gonadotropic cycle is provoked by removing the ootheca, the first event observed is an increase in food consumption, followed by an increase in corpora allata volume and activity. However, this increase in corpora allata volume (and activity) does not occur if females are starved, thus indicating that the ootheca in the genital chamber inhibits primarily feeding, and indirectly corpora allata development and activity. Corpora allata volume in isolated heads from starved and decapitated females was able to increase to levels similar to fed controls, but this increase was abolished by allatostatin treatment. We suggest that a factor produced in the thoracico-abdominal compartment, which reaches the head mainly through a nervous pathway, is released during starvation and inhibits corpora allata development. This factor may stimulate allatostatin production or release, or may well be allatostatin itself.     

Influence of the ovaries on JH titer in Teleogryllus commodus

Mating and availability of substrate were found to have a dramatic effect on the level of egg production and juvenile hormone III (JH III) titer in Teleogryllus commodus. Mated females with egg laying opportunity produced twice as many eggs, and exhibited an 8-fold increase in JH titer, as compared with virgin females or mated females forced to retain most of their eggs due to the absence of substrate for egg deposition. Denervation of the ovaries, or nerve cord transection, also caused accumulation of eggs and lowered the JH level, suggesting a feedback mechanism in which the state of egg depletion of the ovaries determines the level of JH secretion by the corpora allata (CA). Results from ovariectomy of mated or virgin animals suggest that the feedback mechanism includes inhibitory humoral factors secreted by the ovary.     

Structure-activity relationships in corpora allata of the cockroach Diploptera punctata: roles of mating and the ovary

Morphometric studies were made on corpora allata of the cockroach Diploptera punctata from animals in which increasing gland size is not coupled to hormone synthesis (ovariectomized mated females; last-instar larvae) and in which gland size is coupled to hormone synthesis (normal mated and virgin females; penultimate-instar larvae). Cell number, gland volume, and juvenile hormone synthesis were measured. From electron micrographs, nuclear, cytoplasmic, and extracellular volumes; and cell membrane area were calculated; and fine structure described. Low-activity glands of ovariectomized mated females resembled high-activity glands from mated females in high cell number, large overall and cytoplasmic volume, and low nuclear-cytoplasmic ratio; they differed in having organelles typical of low-activity glands, mitochondria with dense matrices and large whorls of smooth endoplasmic reticulum. Inactive lastinstar larval glands resembled mated ovariectomized, female glands in increased cell number and organelles characteristic of inactive glands; however, their nuclearcytoplasmic volume ratio was much higher. Penultimate cytoplasmic volume ratio was much higher. Penultimate larval glands with high activity per cell resembled active glands of normal mated females. Ovariectomy did not change morphometric parameters of virgin female glands; thus mating results in increase in size of adult female glands whereas the growing ovary is needed for changes in mitochondria and endoplasmic reticulum associated with high juvenile hormone synthesis.     

Expression of juvenile hormone acid O‐methyltransferase and juvenile hormone synthesis in Blattella germanica

Juvenile hormone (JH), a sesquiterpenoid synthetized by the insect corpora allata (CA), plays critical roles in metamorphosis and reproduction. Penultimate or last step of JH synthesis is catalyzed by juvenile hormone acid O‐methyltransferase (JHAMT). Here we report the cloning and expression analysis of the JHAMT orthologue in the cockroach, Blattella germanica (L.) (BgJHAMT). BgJHAMT is mainly expressed in CA, with only expression traces in ovary. Three different isoforms, differing in the 3′‐UTR sequence, were identified. Isoform A shows between 35 and 65 times higher expression than B and C in CA from penultimate nymphal instar and adult females. RNAi‐triggered knock down of BgJHAMT produces a dramatic reduction of JH synthesis, concomitant with a decrease of fat body vitellogenin expression and basal follicle length. BgJHAMT mRNA levels in CA of females along the gonadotrophic cycle parallel, with a slight advancement, JH synthesis profile. BgJHAMT mRNA levels were reduced in starved females and in females in which we reduced nutritional signaling by knocking down insulin receptor and target of rapamycin (TOR). Results show that conditions that modify JH synthesis in adult B. germanica females show parallel changes of BgJHAMT mRNA levels and that the JH‐specific branch of the JH synthesis pathway is regulated in the same way as the mevalonate branch. Furthermore, we demonstrate that nutrition and its signaling through the insulin receptor and TOR pathways are essential for activating BgJHAMT expression, which suggests that this enzyme can be a checkpoint for the regulation of JH production in relation to nutritional status.     

Biosynthesis and titre of juvenile hormone during the first gonotrophic cycle in isolated and crowded Locusta migratoria females

Basal oöcyte length, corpus allatum volume and “in vitro” juvenile hormone biosynthesis were measured in isolated and crowded Locusta migratoria females at selected times during the first gonotrophic cycle. Using gas chromatography-mass spectrometry with selected ion monitoring, the juvenile hormone titre in the haemolymph of isolated and crowded females was also determined 1 and 4 days after fledging. The rate of oöcyte growth was more rapid in isolated females and a significant (P < 0.01) difference in mean length was apparent as early as 3 days after fledging. This early manifestation of a difference in rate of oöcyte growth was correlated with a difference in haemolymph juvenile hormone titre between isolated and crowded females. Whilst there was no difference in titre 1 day after fledging, by day 4 the juvenile hormone titre in isolated females was found to be approximately twice that in crowded females. There was no significant difference in the rates of juvenile hormone biosynthesis by corpora allata from isolated and crowded females on days 0 through to 6 after fledging. On day 8, however, the rates of juvenile hormone biosynthesis of corpora allata from isolated females were very high (mean value = 136 pmol/h/pair) and were significantly (P < 0.002) greater than those of corpora allata from crowded females. Day 8 was also the point in the first gonotrophic cycle at which the difference in the mean basal oöcyte length in isolated and crowded females was at a maximum. The mean volume of corpora allata from isolated females was greater than that of corpora allata from crowded females at all points at which measurements were taken during the first gonotrophic cycle.     

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 and reproduction in crickets, Gryllus bimaculatus De Geer: corpus allatum activity (in vitro) in females during adult life cycle

ABSTRACT. Incubation conditions were established for a short-term radiochemical assay of spontaneous juvenile hormone (JH) biosynthesis in vitro by corpora allata from adult female Gryllus bimaculatus. The only JH synthesized was shown by HPLC to be JH III. A further incubation product, predominantly extracted from the corpora allata, was thought to be the JH III precursor, methyl farnesoate. In adult females reared at a constant temperature of 27°C the synthetic activities of the corpora allata-corpora cardiaca complexes in vitro increase from almost zero to a high peak value 4 days after the imaginal moult. Thereafter the activity decreases to varying intermediate levels, but always lower than the first maximum. Two days after the first peak in corpus allatum activity, ovarian fresh weight increases dramatically and the first oviposition occurs 2 days later.
Topical application of JH III to females reared at 20°C, which usually have a low fecundity, causes a dose-dependent stimulation of egg production and oviposition.