The roles of Dippu-allatostatin in the modulation of hormone release in Locusta migratoria

Dippu-allatostatins (ASTs) have pleiotropic effects in Locusta migratoria. Dippu-ASTs act as releasing factors for adipokinetic hormone I (AKH I) from the corpus cardiacum (CC) and also alter juvenile hormone (JH) biosynthesis and release from the corpus allatum (CA). Dippu-AST-like immunoreactivity is found within lateral neurosecretory cells (LNCs) of the brain and axons within the paired nervi corporis cardiaci II (NCC II) to the CC and the CA, where there are extensive processes and nerve endings over both of these neuroendocrine organs. There was co-localization of Dippu-AST-like and proctolin-like immunoreactivity within these regions. Dippu-ASTs increase the release of AKH I in a dose-dependent manner, with thresholds below 10(-11)M (Dippu-AST 7) and between 10(-13) and 10(-12)M (Dippu-AST 2). Both proctolin and Dippu-AST 2 caused an increase in the cAMP content of the glandular lobe of the CC. Dippu-AST 2 also altered the release of JH from the locust CA, but this effect depended on the concentration of peptide and the basal release rates of the CA. These physiological effects for Dippu-ASTs in Locusta have not been shown previously.     

Apparent inhibition of in vitro juvenile hormone biosynthesis by the corpus cardiacum of adult crickets (Gryllus bimaculatus and Acheta domesticus) is due to juvenile hormone esterase

When measuring the in vitro JH III-biosynthesis by corpora allata (CA) from adult female crickets in the presence of corpora cardiaca (CC), the amount of JH III in the medium decreased in a dose dependent manner. The CC of a 4-day-old female Gryllus bimaculatus contain 42 pmol.pair CC⁻¹ Grb-AKH, 0.62 pmol.pair CC⁻¹ octopamine, and a JH-esterase activity of 9.8 pmol JH.h⁻¹.pair CC⁻¹. Comparable values for Acheta domesticus are 21 pmol.pair CC⁻¹ Grb-AKH, 0.53 pmol.pair CC⁻¹ octopamine, and 6.5 pmolJH.h⁻¹.pair CC⁻¹ of JH-esterase activity. Even if the entire octopamine content of the CC were released into the medium, the concentration would be below the 10⁻⁵ M threshold for octopamine inhibition of JH synthesis. An in vitro AKH inhibition of JH III synthesis was observed, but only at a relatively high concentration (10⁻⁵ M). If the entire AKH content (10⁻⁶ M) of the CC were released into the medium, the AKH concentration would approach JH synthesis inhibiting levels. However, the rate of release of AKH in vitro was very low, and, therefore, AKH from the CC could not affect JH synthesis. In contrast, a specific JH-esterase, released by isolated CC into the medium, was sufficiently high in both cricket species to account for the observed decrease in JH III present. OTFP-sulfone (10⁻⁵ M) restored apparent JH synthesis of the CA to the control level. There was no reduction in the amount of JH released when CA were incubated with heat treated CC. The CA themselves contained almost no JH-esterase activity. © 1997 Wiley-Liss, Inc.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

Release of neurosecretory granules within the corpus allatum in relation to the regulation of juvenile hormone synthesis in Diploptera punctata

The release of neurosecretory granules within the corpora allata (CA) of the viviparous cockroach Diploptera punctata has been compared in glands with intact nerves from the brain (Brain-CA) and those detached from the brain. Measurements of juvenile hormone (JH) synthesis in vitro, comparing these two conditions of the CA at several stages of vitellogenesis in adult females, showed lower production of hormone in Brain-CA complexes than in CA alone. Glands treated with tannic acid to trap exocytotic granules before fixation for electron microscopical examination showed, in sample sections, 10 times more exocytotic profiles in the glands with intact nerves to the brain than in the isolated glands. Sections treated with antibody against allatostatin I (Dip 7), a member of the neuropeptide family that inhibits JH synthesis by CA in vitro, showed neurosecretory granules in allatostatin immunoreactive nerves to be 75+/-4% of the granules in the sample of sections of CA. Because the total quantity of allatostatin in CA was found by ELISA not to vary significantly with changes in JH synthesis, it is concluded that the lower rates of JH synthesis by glands with intact nerves to the brain are most likely due to the release of small amounts of allatostatin within the CA.     

Inositol phospholipid hydrolysis may mediate the action of proctolin on insect visceral muscle

The formation of inositol phosphates in response to the neuropeptide proctolin was studied in locust oviducts. Glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate were identified in the locust oviducts using anion-exchange chromatography. Proctolin stimulated the release of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate during a 5-min incubation. In the presence of lithium ions the effects of proctolin were enhanced, with elevations of 98%, 42%, and 45% of inositol 1-phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, respectively. Physiologically the effects of proctolin upon muscular contraction of locust oviducts were mimicked by the active phorbol ester, phorbol 12-myristate 13-acetate, and by the diacylglycerol analogue, 1-oleoyl-2-acetylglycerol. The inactive phorbol ester, 12-myristate 13-acetate 4-O-methyl ether, was without effect. The effects of the active phorbol ester and the diacylglycerol analogue were calcium-dependent requiring micromolar concentrations of calcium. The results indicate that the locust oviducts possess proctolin receptors that are linked to phosphatidylinositol metabolism and that inositol phospholipid hydrolysis may mediate the physiological action of proctolin.     

An allatostatic factor and juvenile hormone synthesis by corpora allata in Locusta migratoria

The hemolymph juvenile hormone (JH) titer in sexually immature female adults of Locusta migratoria (Ibaraki strain, Japan) was lower than in sexually mature females; nevertheless, JH synthetic activity by the corpora allata (CA) in vitro was considerably higher in immature females than in sexually mature females ([Okuda et al., 1996]). We carried out experiments to explain this contradiction. The CA activity of sexually immature female adults was very low when the CA were incubated as a complex together with the corpora cardiaca (CC) and brain. When the same complex was assayed after cutting the nerve cord connecting the CC and CA (NCA1), JH synthesis by the CA was enhanced tenfold. When this pair of CA was incubated in fresh medium without the CC and brain, JH synthesis was further increased. Therefore, the higher in-vitro JH production by CA from immature female adults was the result of isolation of the CA from the brain and CC. A methanolic extract of brain-CC complexes contained a factor that inhibited JH synthetic activity by CA in vitro in both immature and mature insects, and this inhibition was reversible. The factor was heat-resistant but lost allatostatic activity after pronase digestion. These results indicate that the allatostatic factor is probably a heat-stable peptide.     

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.     

Structure function analysis of an arthropod peptide hormone: proctolin and synthetic analogues compared on the cockroach hindgut receptor

Proctolin is a pentapeptide (arg-tyr-leu-pro-thr) found in nervous tissues throughout the phylum Arthropoda. Initially described as a peptidergic neuromuscular transmitter, it now appears that proctolin is a major arthropod neurohormone modulating nervous activity, muscle tonus and contractile force. Structure-function studies with synthetic analogues demonstrate diverse peptides which retain agonistic activity, but few exhibit a high degree of affinity for the cockroach hindgut receptor compared with proctolin (Kdapp = 2 x 10(-8) M). High affinity agonists (Kdapp less than or equal to 10(-7) M) are limited to [phe2]-proctolin, [lys1]-proctolin and specific N-terminal additions. In this regard the hindgut receptor differs in its ligand specificity from that reported for the locust extensor tibia receptor. Using the analogue studies to predict sequences which may act as agonists, we have examined the known vertebrate peptide hormones for proctolin-like sequences. A possible relationship between vasoactive intestinal peptide, proctolin and erythrophore concentrating hormone is critically evaluated.     

Proctolin-like immunoreactivity in the central and peripheral nervous systems of the locust, Locusta migratoria

Proctolin-like immunoreactivity (PLI) was widely distributed in the locust, Locusta migratoria, within the central, peripheral and stomatogastric nervous systems, as well as the digestive system and retrocerebral complex. Proctolin-like immunoreactivity was observed in cells and processes of the brain and all ganglia of the ventral nerve cord. Of interest, PLI was found in the lateral neurosecretory cells, which send axons within the paired nervi corporis cardiaci II (NCC II) to the corpus cardiacum (CC). The CC contained extensive processes displaying PLI, which continued on within the paired nervi corporis allata (NCA) to the paired corpora allata (CA) where the axons entered and branched therein. The frontal and hypocerebral ganglia of the stomatogastric nervous system contained PLI within processes, resulting in a brightly staining neuropile. Each region of the gut contained PLI in axons and processes of varying patterns and densities. The paired ingluvial ganglia contained PLI, including an extensively stained neuropile and immunoreactive axons projecting through the nerves to the foregut. The hindgut contained PLI within longitudinal tracts, with lateral projections originating from the 8th abdominal ganglion via the proctodeal nerve. The midgut contained PLI in a regular latticework pattern with many varicosities and blebs. No difference in PLI in cells and processes of the central nervous system (CNS) was found between males and females.     

A review of the involvement of proctolin as a cotransmitter and local neurohormone in the oviduct of the locust,Locusta migratoria

The pentapeptide proctolin, originally identified in the cockroach, has been shown to be widely distributed in many insects and to have a broad range of physiological functions. In the oviduct of the locust, Locusta migratoria, proctolin's role as a neurotransmitter/neuromodulator has been well documented; however, a neurohormonal role in the locust is less certain. This review will examine the various roles of proctolin in locust oviduct contraction and will present evidence that a substance chromatographically, immunologically and physiologically indistinguishable from proctolin is present in the hemolymph of the locust, L. migratoria. This material is concentrated in the plasma, rather than the hemocytes, and is present at concentrations ranging from 0.1 to 0.2nM. This review extends the role of proctolin in insects, and suggests that proctolin may play a neurohormonal role in the locust.     

Cell size control by ovarian factors regulates juvenile hormone synthesis in corpora allata of the cockroach, Diploptera punctata

The corpora allata synthesize and release juvenile hormone (JH) that in turn regulates insect growth, metamorphosis and reproduction. In the corpus allatum (CA) of the female adult cockroach Diploptera punctata, cyclic rise and decline in JH synthesis rates occur concurrently with cyclic growth and atrophy during an ovarian cycle. Here, we report that protein content decreases, whereas Golgi population, lysosomal content and autophagic activities increase with decrease in CA cell size. Also, the concentration of cyclic GMP (cGMP) is low in large cells and high in small cells. Results of treating CA with ovarian tissue suggest that a putative peptidergic growth regulator released from mature ovaries acts directly on active CA cells and induces the elevation of intracellular cGMP content. Consequently, elevated cGMP may inhibit protein synthesis or trigger massive and synchronous autophagic activities, resulting in cell atrophy and reduction of protein content. As a result of the depletion of cellular machinery, CA glands exhibit long-term depression in JH synthesis.     

Ultrastructure of the corpus cardiacum and corpus allatum of the house cricket Acheta domesticus

Summary The ultrastructure of the corpus cardiacum (CC) and corpus allatum (CA) of the house cricket, Acheta domesticus, is described. Axon profiles within the CC contain neurosecretory granules 160–350 nm in diameter which are indistinguishable from those found in type I neurosecretory cells of the pars intercerebralis and in the nervus corporis cardiaci I. The CC itself contains two cell types: intrinsic neurosecretory cells and glial cells. Intrinsic NSC cytoplasm contains Golgi bodies and electron dense neurosecretory granules 160–350 nm in diameter. Synaptoid configurations with 20–50 nm diameter electron lucent vesicles were observed within axon profiles of the CC. The structure of the CA is relatively uniform with one cell type predominating. Typical CA cells possess large nucleoli, active Golgi complexes, numerous mitochondria, and occassional microtubules. Groups of dark staining cells scattered throughout the CA of some animals were interpreted as evidence of cellular death.This work was done while JTB was supported by USPHS Training Grant HD-0266 from NICHDI wish to express my thanks to Dr. Richard A. Cloney for sharing his expertise in electron microscopy     

Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects.

Proctolin, a myotropic substance with potent activity on the proctodeal (hindgut) muscles of the cockroach, $\text{Periplaneta americana}$ (L.), has been identified by the use of Edman degradation and dansylation techniques as Arg-Tyr-Leu-Pro-Thr. Synthesis of the pentapeptide having this sequence and exhibiting the properties of natural proctolin confirmed the structure. Threshold activity on proctodeal muscle occurs at about 10⁻⁹ M proctolin.     

Determination of locust AKH-I by radioimmunoassay and the identification of an AKH-like factor in the locust brain

The N-terminal glutamic acid analog of the decapeptide locust adipokinetic hormone-I, [Glu¹]AKH-I, was prepared by solid-phase synthesis and derivatized to [4-hydroxyphenyl propionyl-Glu¹]AKH-I. This derivative was radioiodinated on the 4-hydroxyphenyl function ([¹²⁵I]AKH analog). [Glu¹]AKH-I was coupled to bovine serum albumin to produce rabbit antiserum. This, together with the [¹²⁵I]AKH analog was utilized to develop a highly selective radioimmunoassay procedure (RIA) for detecting AKH-I in locusts. Methanol extracts of locust brain contain two factors (BI and BII) that inhibit protein synthesis in excised fat body tissue of vitellogenic female locusts. BI also elicits lipid mobilization when injected into adult male locusts. The HPLC retention time of BI on an RP-18 column is identical to that of locust adipokinetic hormone (AKH-I), both synthetic and derived from adult corpora cardiaca (CC). The second brain factor (BII) does not exhibit lipid mobilizing activity. One tissue-equivalent of BI contains 33 ng of AKH-I as determined by RIA, compared to 450 ng native HPLC-separated AKH-I in locust CC.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

Allatostatins and allatotropin in the corpus cardiacum/corpus allatum complex of larval and adult lepidopterans studied by confocal laser scanning microscopy: correlation to juvenile hormone biosynthesis

Peptidergic innervation of the corpus cardiacum/corpus allatum (CC/CA) retrocerebral complex, and neurosecretory areas of the brain of the lepidopterans Lacanobia oleracea, Heliothis virescens and Manduca sexta was studied by immunocytochemistry linked to confocal laser scanning microscopy. The patterns of immunostaining resulting from the simultaneous application of fluorochrome-conjugated antibodies against Manduca sexta allatostatin (Mas-AS), M. sexta allatotropin (Mas-AT), and a representative of the –Y/FXFGL-NH₂ superfamily of allatostatins was correlated with the physiological effects of these putative allatoregulatory peptides on juvenile hormone (JH) biosynthesis by the corpora allata. Whereas the two types of allatostatin immunoreactivity are present in both larval and adult CA of the three species, allatotropin immunoreactivity occurs only in the adult gland. The conclusion that withdrawal of the stimulatory effect of allatotropin is unlikely to be involved in the downregulation of CA activity prior to the onset of metamorphosis, but that an inhibitory influence of at least Mas-AS is important, is borne out in physiological experiments on JH biosynthesis in M. sexta larvae (Mas-AS inhibitory, Mas-AT without effect). Immunoreactivity to the Y/FXFGL-NH₂ allatostatins is present in both larval and adult CA and CC, frequently co-localised with Mas-AS. The function of this peptide family in the retrocerebral complex remains enigmatic since experiments on JH biosynthesis, either when the peptide is administered alone, or together with Mas-AS, show no effect on JH biosynthesis.Financial support was provided by The Wellcome Trust (063367/Z/00) (to A.T.) and by the Pesticide Safety Directorate of the Department for Environment, Food and Rural Affairs (to N.A. and R.J.W.)     

Cockroach oviducts: The presence and release of octopamine and proctolin

The oviducts of the cockroach, Periplaneta americana, contain octopamine (determined by radioenzymatic assay) and proctolin (determined from proctolin-like bioactivity following separation on reversed-phase high performance liquid chromatography). Octopamine content is 5–10-fold greater than proctolin content. Both substances are released in a calcium-dependent manner following depolarisation with high potassium saline. This method resulted in the release of 14% of the total store of octopamine, but only 0.3% of the total store of proctolin. The results are discussed in relation to the recent demonstration of octopamine-containing and proctolin-containing neurones associated with locust oviducts.     

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.     

The molecular and metabolic essentials for long-distance flight in insects

Summary The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.Abbreviations AKII adipokinetic hormone - apoLp-III apolipophorin III - HDLp high-density lipophorin - LDLp low-density lipophorin - LTP lipid transfer particle - MW molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

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.