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.     

Cockroach allatostatin-immunoreactive neurons and effects of cockroach allatostatin in earwigs

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to demonstrate the presence of allatostatin-immunoreactive cells and fiber tracts in the neuroendocrine system of the earwig Euborellia annulipes. The corpora cardiaca cells were not immunoreactive, nor were the neurosecretory endings of fiber tracts from the brain to the corpora cardiaca. No immunoreactive material was detected in the corpus allatum, although the corpus allatum contained neurosecretory endings, and some cells of the brain, including medial and lateral protocerebral cells, showed immunoreactivity. In addition, the recurrent and esophageal nerves were allatostatin-positive. The last abdominal ganglion contained immunoreactive somata, and immunoreactive axons of the proctodeal nerve innervated the rectum, anterior intestine, and posterior midgut. We did not detect reactive endocrine cells in the midgut. Allatostatin I at concentrations of 10^–5 and 10^–7 M did not inhibit juvenile hormone biosynthesis by E. annulipes corpora allata in vitro. This was true for glands of low activity from 2-day females and brooding females, as well as for relatively high activity glands from 10-day females. In contrast, 10^–7 M allatostatin I significantly and reversibly decreased hindgut motility. Motility was decreased in hindguts of high endogenous motility from 2-day females and in those of relatively low activity from brooding females. These results support the notion that a primary function of allatostatin might be to reduce gut motility. Arch. Insect Biochem. Physiol. 38:155–165, 1998. © 1998 Wiley-Liss, Inc.     

Effects of Manduca allatotropin and localization of Manduca allatotropin-immunoreactive cells in earwigs

Manduca sexta allatotropin (Manse-AT) was first isolated on the basis of its ability to stimulate production of juvenile hormone in that insect. We examined whether this neuropeptide affects corpus allatum activity and visceral muscle contraction in adult females of the earwig, Euborellia annulipes. We also assessed the presence of allatotropin-like material in tissues using immunocytochemistry. Manse-AT at 1 nM to 10 muM stimulated juvenile hormone production in vitro by glands of low activity from 2-day virgin females. In glands of high activity from 12-day mated females, 1 and 100 nM allatotropin were effective, but 10 muM was not. Similarly, hindguts of 2-day and 12-day females significantly increased in motility in vitro in response to Manse-AT. A monoclonal antibody to Manse-AT was used to demonstrate allatotropin-like material throughout the nervous system of 2-day, virgin females. Immunoreactivity was most pronounced within varicosities of the corpora cardiaca and perisympathetic organs. No immunofluorescence was observed in gut tissue. Lastly, we showed that extract of retrocerebral complexes also enhanced in vitro hindgut motility from 2-day virgin females, in a dose-dependent manner. These results indicate material similar to M. sexta allatotropin in female earwigs and that such peptides may modulate juvenile hormone biosynthesis and visceral muscle contractions. Sensitivity to the peptides may change with physiological stage.     

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.     

Control of reproduction in female cockroaches with special reference to Nauphoeta cinerea—I. First pre-oviposition period

Prior to the first oviposition, a receptivity centre, perhaps neurosecretory cells in the brain, controls the female's acceptance of courting males. In L. maderae this centre is affected by starvation. A brief exposure to food can induce mating but is inadequate for oöcyte development. Before the first ovulation starvation has no effect on receptivity in N. cinerea.

In N. cinerea mechanical stimulation caused by the firm insertion of the spermatophore in the bursa copulatrix releases stimuli via the nerve cord to the brain which render the female unreceptive and, at the same time, increases the activity of the corpora allata resulting in rapid development of the oöcytes.

The mechanical presence of the oötheca in the uterus also has two principal effects. Like spermatophore insertion, it inhibits mating. But its effect on the corpora allata is inhibitory, rather than stimulatory, and, consequently, the oöcytes remain underveloped for almost the entire gestation period. The effectiveness of inhibitory stimulation from the stretched uterus depends upon the period in the reproductive cycle in which it occurs-i.e. on the physiological state of the female. In N. cinerea uterine stretching inhibits mating and oöcyte development after oviposition or during gestation but is not effective when exerted during the first pre-oviposition period. In P. surinamensis, uterine stretching does not inhibit the corpora allata prior to the first ovulation but does prevent oöcyte development during gestation.

In fed L. maderae and N. cinerea there appears to be a synergistic action of nutrition and mating in controlling the rate of oöcyte development. Mating (mechanical) and feeding (chemical) stimuli are both usually required for activating the corpora allata to their fullest extent so that the oöcytes mature at their maximum rate. There is some indication that mating stimuli in N. cinerea and L. maderae are effective in further stimulating the corpora allata only if the corpora allata have reached a certain level of activity, if activating stimuli have begun to occur in the brain, or if the mating stimulus occurs in combination with nutritional factors. Thus, the corpora allata in starved virgin females of N. cinerea become sufficiently active so that some yolk is deposited in the oöcytes but these oöcytes do not mature; mating is effective in further stimulating the endocrine glands in these starved females and oviposition occurs in about the normal period. In starved virgins of L. maderae the corpora allata are virtually inactive and yolk is not deposited in the oöcytes; mating has no effect on oöcyte development in starved females. D. punctata differs from both the above species in that the corpora allata in the virgin female usually remain inactive whether she feeds or starves. Mating stimuli alone can activate the corpora allata, in fed or starved females, and consequently the oöcytes mature.     

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 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.     

Controle neuroendocrine du fonctionnement du corpus allatum chez Rhodnius prolixus

Cauterization of the dorsal portion of the protocerebrum effected immediately after cessation of feeding in adult females blocks vitellogenesis. The growth of the oöcytes can be re-established by the implantation of corpora allata or by the application of juvenile hormone analogue, and it is suggested that the protocerebrum has a corpus allatum stimulating influence. The corpus allatum deprived of all its cerebral nervous connexions by cutting the aorta just behind the corpora cardiaca induces abundant vitellogenesis. Females with denervated corpora allata or pars intercerebralis cauterized 24 hr after a meal show a phase of vitellogenesis more active than that in normal female adults. This suggests the existence of a cerebral inhibitory centre: its activity through a nervous or endocrine path is discussed.     

The mode of regulation of the corpus allatum activity during starvation in adult females of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

When two-day-old female Leptinotarsa decemlineata were starved, their corpus allatum activity, as measured by the radiochemical in vitro assay, was significantly reduced after 24 hr. Such a reduction was not observed when the nerve connections between the central nervous system and the retrocerebral complex were severed and the beetles starved up to 5 days. In some experiments, the rate of juvenile hormone biosynthesis in vitro, was substantiated by measurement of the juvenile hormone titre in the haemolymph by physico-chemical methods. It is concluded that intact nervous connections between the central nervous system and the corpora allata are essential for restraining the juvenile hormone biosynthesis during the initial stages of starvation.Corpora allata from 1-day starved insects were considerably stimulated in vitro by farnesenic acid indicating that juvenile hormone synthesis is controlled enzymatically at a stage prior to the final two steps in the pathway. However, on day 5 of starvation, rate-limitation may occur after formation of this intermediate, since farnesenic acid stimulation was much less at this time.Corpora allata of adult females newly emerged from the soil were activated within 4 hr regardless of feeding.     

Humoral inhibition of the corpora allata in larvae of Diploptera punctata: Role of the brain and ecdysteroids

Juvenile hormone synthesis by adult female corpora allata was inhibited following implantation into final-larval-instar males; inhibition was prevented by decapitation of the larval hosts on day 11 (prior to the head critical period for moulting), but not by decapitation on day 13. Implantation of one larval protocerebrum restored inhibition of implanted corpora allata, demonstrating that the brain releases an inhibitory factor. Corpora allata implanted into larvae decapitated on day 11 were inhibited by injections of 20-hydroxyecdysone. Since treatment of corpora allata with 20-hydroxyecdysone in vitro did not inhibit juvenile hormone synthesis, ecdysteroids probably act indirectly on the corpora allata. Juvenile hormone synthesis and haemolymph ecdysteroid concentration were measured following implantation of corpora allata along with two larval brains into larval hosts. Brain implantation did not affect ecdysteroid concentration, but did inhibit juvenile hormone synthesis, even in animals with low haemolymph ecdysteroid concentration. Incubation with farnesoic acid stimulated juvenile hormone synthesis by corpora allata from males early in the final larval stadium, but not after day 8, showing that one of the final two reactions of juvenile hormone synthesis is rate-limiting in larval corpora allata at this stage. Adult female corpora allata which had been humorally inhibited by implantation into larvae were stimulated by farnesoic acid.     

Stimulatory activity of cysteamine on juvenile hormone release in adult females of the cockroach, Blattella germanica

1. A study of the activity of cysteamine in relation to juvenile hormone (JH) production in adult females of Blattella germanica was carried out. 2. In vivo assays showed that cysteamine stimulates protein synthesis in the left colleterial gland and, in some instances, enhances oocyte growth. 3. In vitro assays demonstrated that cysteamine enhances JH release by incubated corpora allata (CA), and that this effect is more pronounced when using CA from 10-day-old females (period of ootheca transport), either connected to the corpora cardiaca (CC) or to the CC and to the brain. 4. Possible antiallatostatic effects of cysteamine are discussed.     

Induction of egg diapause by implantation of corpora cardiaca and corpora allata in Bombyx mori

Diapause egg production was examined in non-diapause egg producers by implantation of various cephalic organs into pharate adults 4 days after larval-pupal ecdysis. The implantation of five pairs of corpora cardiaca or corpora allata induced a great amount of egg diapause. Implantation of these organs was effective in inducing egg diapause even when the suboesophageal ganglion of the recipients had been removed, although the effect of the corpora allata decreased moderately. The injection of juvenile hormone into 4-day-old pharate adults did not greatly increase production of diapause eggs.     

Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata

Summary A monoclonal antibody against allatostatin I was used to demonstrate the allatostatin-immunoreactive pathways between the brain and the corpus cardiacum-corpus allatum complex in the adult cockroach Diploptera punctata. The antibody was two to three orders of magnitude more sensitive to allatostatin I than to the other four known members of the allatostatin family. Whole and sectioned brains in which immunoreactivity was localized with horseradish peroxidase-H₂O₂-diaminobenzidine reaction showed strongly immunoreactive cells in the pars lateralis of the brain with axons leading to and arborizing in the corpus cardiacum and the corpus allatum. Although many neurosecretory cells of the pars intercerebralis project to the corpora allata only, four strongly immunoreactive cells were evident here (two pairs on either side), and these did not project to the corpus cardiacum and corpus allatum but rather terminated within the protocerebrum in areas in which lateral cells also formed arborizations. Immunoreactivity was found in many other cells in the brain, especially in the tritocerebrum.     

Effects of selected neuropeptides, mating status and castration on male reproductive tract movements and immunolocalization of neuropeptides in earwigs

In earwigs, the male reproductive system is complex, comprising accessory glands and long dual intromittent organs for transfer of materials to the female and for removal of rival sperm. We investigated potential factors altering contractions of the male reproductive tracts in vitro. Tracts from 0-day (newly emerged) males displayed relatively little motility in vitro; however, those from 5-day (intermediate stage of sexual maturity) and 8-day (fully mature) males pulsed vigorously. Both 1 and 100 nM proctolin (RYLPT-OH) stimulated the rate of contraction of reproductive tracts from both 5-day and 8-day males. In contrast, 1 nM and 100 nM FGLa AST (cockroach allatostatin) did not affect pulsations. However, 10 microM FGLa AST decreased activity of reproductive tracts. Mating decreased motility of tracts from 5-day old males, but did not alter motility of tracts from 8-day old males. Castration of larvae significantly suppressed reproductive tract motility in subsequent 8-day old adults compared with those of intact or sham-operated adults. Castration also suppressed seminal vesicle size. Lastly, we assessed the presence and distribution of proctolin-like and allatostatin-like immunoreactivity in tissues. Immunoreactivity to FGLa AST and proctolin was widespread, occurring in the brain and ventral ganglia. Surprisingly, we did not detect immunoreactivity to either FGLa AST or proctolin within the reproductive system; however, proctolin immunoreactivity was evident in nerves extending from the terminal ganglion of 8-day, but not 0-day, males. Collectively, these experiments demonstrate that the male earwig reproductive system is an appropriate model for use in addressing sexual maturation and activities in male insects.     

Structure-activity studies reveal two allatostatin receptor types in corpora allata of Diploptera punctata

Synthetic variants of the octadecapeptide amide ASB2 (AYSYVSEYKRLPVYNFGL-NH(2)), a cockroach allatostatin, were assayed in vitro on corpora allata (CA) from 2-day-old (vitellogenic) and 10-day-old (post-vitellogenic) female Diploptera punctata. The analogs [(17)psi(18),CH(2)-S]ASB2, [D-Trp(17)]ASB2 and [Ile(18)]ASB2 inhibited juvenile hormone (JH) synthesis with simple dose-response curves on sensitive CA from 10-day-old females. These analogs were fully effective but less potent than ASB2. When tested on CA from 2-day-old mated females, which are only partially (65-70%) sensitive to ASB2, the three analogs gave biphasic dose-response curves and elicited a maximal effect only at higher concentrations. The dose-response curve for ASB2 on CA from 2-day-old females had a Hill plot slope of only 0.78+/-0.03. These findings suggested that the observed CA sensitivity to ASB2 may be the result of two partial responses having an IC(50) of approximately 0.35 and 3nM respectively. One partial response, or receptor type, appeared more sensitive than the other to adverse modification of the "message" segment of the peptide. The activity of shorter allatostatins was also studied, indicating that pentapeptides of the YXFGL-amide structure are fully effective, albeit at low potency, as inhibitors of JH biosynthesis.     

Juvenile Hormone biosynthesis and utilization of farnesoic acid during the final larval stadium of the ring-legged earwig

Abstract. The regulation of Juvenile Hormone (JH) HI biosynthesis and release by the corpora allata (CA) was studied in final instar male and female larvae of the earwig, Euborellia annulipes , using a radiochemical assay in vitro. In males, maximal biosyntiiesis of JH IH occurred on day 1, then declined to virtually undetectable levels for the following 12 days of the stadium, and finally increased on days 14–16. In females, peaks of biosynthesis were detected on days 0–1 and on day 12. A further investigation of the 12-day-old larvae demonstrated mat in nonmoulting males and females, JH UJ biosynthesis was undetectable. However, for males and females undergoing ecdysis, the biosynthesis of JH III was detected and quantified.
The addition of 60 μM farnesoic acid to the incubation medium significantly increased the production of JH III by CA taken from females from day 8 until the end of the stadium. Glands from 12-day old females that had initiated ecdysis were stimulated by farnesoic acid. By contrast, we could detect no stimulation of production of JH III by farnesoic acid in CA taken from males, even very late in the stadium. CA from newly emerged adult males and females were more active than those of larvae, and were greatly stimulated by farnesoic acid. CA from females immediately after emergence were stimulated significantly more by farnesoic acid man were glands from newly emerged males. These results suggest fundamental differences in the synmetic activity of CA for males and females in this insect.     

In vivo and in vitro study of the action of dopamine on oocyte growth and juvenile hormone production in Blattella germanica (L.) (Dictyoptera; Blattellidae)

In vivo, within the first ovarian cycle of B. germanica, dopamine (1 microgram/cockroach) enhanced oocyte growth when injected on day 1 or 2, just before vitellogenesis, whereas it induced the inverse effect on day 6-7, at the end of vitellogenesis. In vitro, dopamine (10(-4) M) stimulates juvenile hormone production by incubated corpora allata from 2-day-old females, and inhibited it on 6-day-old glands. The physiological significance of these effects is discussed.