Isolation and structure of two novel 6-kDa dimeric peptides from the corpora cardiaca of the insect Locusta migratoria. Molecular mass determination by mass spectrometry

We have isolated two major 6-kDa peptides from extracts of corpora cardiaca of adult females of Locusta migratoria. These peptides have been characterized by peptide sequencing and liquid secondary-ion mass spectrometry. They are structurally related dimers, one (6278.5 Da) being a homodimer (A-A chains), the other (6280.5 Da) being a heterodimer (A-B chains). A 60% similarity exists between the A and B chains. Both peptides have been chemically synthesized and the synthetic compounds appeared to be identical to the native ones. Polyclonal antibodies raised against each of these peptides demonstrated that they were contained within the secretory granules of the intrinsic cells of the glandular lobes of the corpora cardiaca. The physiological significance of these two peptides is unknown but, using the synthetic peptides, we are currently probing their biological role.     

Isolation and structure elucidation of a novel adipokinetic hormone (Lom-AKH-III) from the glandular lobes of the corpus cardiacum of the migratory locust, Locusta migratoria

A new adipokinetic hormone (named Lom-AKH-III) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N-terminus it is blocked by a 5-oxoproline (pyroglutamic acid) residue (less than Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be [less than Glu]-Leu-Asn-Phe-Thr-Pro-. Fast-atom-bombardment spectrometry (FAB-MS) revealed that the new hormone is an octapeptide, which is amidated at the C-terminus, and has a relative molecular mass of 1072. Based on the FAB-MS data the complete sequence is less than Glu-Leu-Asn-Phe-Thr-Pro-Trp-Trp-NH2, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB-MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom-AKH-I (less than Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High-[K+]-containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species. Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.     

cDNAs from neurosecretory cells of brains of Locusta migratoria (Insecta, Orthoptera) encoding a novel member of the superfamily of insulins.

From neurohaemal lobes of corpora cardiaca of Locusta migratoria a 5-kDa peptide has been isolated and its sequence established [see the accompanying paper, by Hietter et al. (1990) Eur. J. Biochem. 187, 241-247]. We have designed oligonucleotide probes from the peptide sequence of this molecule and screened a library prepared from mRNA of the neurosecretory cell region of the brain of this insect. Several positive cDNAs were isolated, the combined nucleotide sequences of which predict a large precursor of 145 residues (15770 Da) containing the newly isolated 5-kDa peptide. The peptide is flanked by regions homologous to the A and B chains of the superfamily of insulins. The overall organization of the precursor is as follows: signal peptide/domain homologous to the B chain of insulins/C (connecting)-peptide (corresponding to the newly isolated 5-kDa peptide)/domain homologous to the A chain of insulins. The numbers and relative positions of the cysteines of the Locusta peptide are equivalent to those of the other members of the insulin superfamily and most of the hydrophobic core residues are conserved.     

The role of the glandular lobes of the corpora cardiaca during flight in Locusta

ABSTRACT. Flight performance in Locusta is reduced following severance of the major afferent nerves to the corpora cardiaca or removal of the glandular lobes of the corpora cardiaca. These operations prevent the release of adipokinetic hormone and the consequent mobilization of stored lipid. However, locusts deprived of about 90% of their glandular lobe tissue, while flying poorly, did mobilize lipid. It is suggested that the remaining glandular parenchyma cells are capable of secreting enough hormone to stimulate lipid mobilization, but that the concentration may be inadequate to encourage lipid utilization. After removal of all the glandular lobe parenchyma, the blood carbohydrate concentration was temporarily depressed. Nevertheless flight performance was equally poor, both when haemolymph carbohydrate levels were low and when they had returned to normal. After the injection of trehalose into operated control locusts and locusts deprived of their glandular lobes, flight was still markedly poorer in the operated insects, even though the injection of trehalose prevented adipokinetic hormone release in the intact locust. It seems that the poor flight performance of locusts deprived of their glandular lobes cannot be fully explained by the simple absence of adipokinetic hormone.     

Locustakinin, a novel myotropic peptide from Locusta migratoria, isolation, primary structure and synthesis.

The isolated hindgut of the cockroach, Leucophaea maderae is a very efficient bioassay tool for the monitoring of certain structural types of insect myotropic peptides during HPLC purification. Using this detection system, a six residue peptide has been isolated from an extract of 9000 brain corpora cardiaca-corpora allata suboesophageal ganglion complexes of Locusta migratoria. Amino acid composition and sequence analysis combined with enzymatic digestion data established the structure of the novel peptide as Ala-Phe-Ser-Ser-Trp-Gly-amide. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. The carboxy-terminal pentamer sequence is the active core of leucokinins II, V and VII and of achetakinin III (myotropic neuropeptides isolated from Leucophaea m. and from Acheta domesticus; Holman et al., 1990). Furthermore, the octapeptide leucokinin VII contains the novel sequence as its carboxy-terminal hexamer and Achetakinin V (AFHSWGamide) differs from it by one residue. This new peptide designated as locustakinin I (locusts) may therefore represent an evolutionary molecular link between leucokinin VII (cockroaches) and achetakinin V (crickets). Using synthetic locustakinin, physiological studies will be performed in the locust. In view of the known effects of leucokinins, locustakinin may be important in the stimulation of ion transport and inhibition of diuretic activity in Malpighian tubules. This study indicates that the AFXSWGamide sequence appears to have been well conserved and that members of this peptide family may be widely distributed among insects and posses a number of functions.     

Isolation, identification and synthesis of locustamyoinhibiting peptide (LOM-MIP), a novel biologically active neuropeptide from Locusta migratoria.

A novel peptide termed locustamyoinhibiting peptide (LOM-MIP) was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. The primary structure of this nonapeptide has been determined Ala-Trp-Gln-Asp-Leu-Asn-Ala-Gly-Trp-NH2. LOM-MIP suppresses the spontaneous contractions of the hindgut and oviduct of Locusta migratoria and of the hindgut of Leucophaea maderae. This novel peptide is, however, structurally different from leucomyosuppressin, a hindgut suppressing peptide isolated from Leucophaea maderae heads. LOM-MIP has a Gly-TrpNH2 carboxy-terminal in common with APGWamide, a penis retractor muscle inhibiting peptide isolated from the snail, Lymnea stagnalis. In addition, it shows carboxy-terminal sequence similarities with locust AKH II which ends in AGWamide. No sequence similarities were found with other vertebrate or invertebrate peptides. Synthetic LOM-MIP showed biological as well as chemical characteristics indistinguishable from those of native LOM-MIP.     

Prepulse inhibition of the startle reaction in the locust Locusta migratoria (Insecta: Orthoptera: Acridoidea)

A fast startle reaction of unrestrained sitting locusts (Locusta migratoria) can be elicited by sound pulses of steep rise time above 80 dB. The reaction consists of a fast jerky movement of legs and body with a mean latency of 35 ms and graded amplitude. The fast startle reaction did not result in any positional change; this was in contrast to acoustically induced escape reactions of flying Orthoptera. The startle reaction could be inhibited by pure tone stimuli of much lower intensity (60 dB) presented 160 ms before the startle-eliciting noise. This type of reflex modification is a striking convergence to the well-known prepulse inhibition of the mammalian startle response where it has been used to assess sensory thresholds. In the locust, prepulses between 3 and 20 kHz suppressed the startle reaction completely, with thresholds in the locust's hearing range as known from tympanal nerve recordings. No inhibition could be observed at prepulse frequencies of 40 kHz, although this frequency lies within the locust's hearing range. The presence of prepulse inhibition in an invertebrate preparation shows that it is not restricted to vertebrates.     

Effects of an imidazole derivative (KK-42) on development and ecdysteroid production in Locusta migratoria (Insecta, Orthoptera)

The imidazole derivative KK-42 was applied in various experimental conditions to larvae and adult females of Locusta migratoria. The effect of this compound was monitored on the development of larvae, on oocyte growth in adult females and on the development of eggs laid by these females. KK-42 had only minor effects on postembryonic development; anticipation of imaginal moult was never observed. In contrast oocyte and egg development were markedly affected by KK-42: this effect is however not related to modifications of the synthesis of ecdysteroids in the ovaries.     

Chemosensory proteins of Locusta migratoria (Orthoptera: Acrididae)

We describe a family of proteins abundantly expressed in the chemoreception sensory organs, the antennae and legs, of the desert locust, Locusta migratoria (Orthoptera, Acrididae). Using polymerase chain reaction-based approaches and homology screenings, "OS-D"-like proteins were identified in L. migratoria. The different sub-types (LmigOS-Ds) are very similar to each other and share about 50-70% identity with OS-Ds from Drosophila melanogaster and Periplaneta americana. A similar degree of identity was also observed with moth OS-Ds. Northern blot analysis revealed a strong expression of the LmigOS-Ds in the antennae and legs, suggesting their involvement in chemosensory processes. Despite the lack of direct evidence for their role in chemosensation, LmigOS-Ds and their homologs seem to constitute a large protein family, characterized by a striking abundance and diversity among insect chemosensory organs.     

Isolation and identification of the AKH III precursor-related peptide from Locusta migratoria

We have isolated an 8770Da peptide from extracts of corpora cardiaca of adult male and female Locusta migratoria. The N-terminal amino-acid sequence as partially established by Edman degradation is Ala-Leu-Gly-Ala-Pro-Ala-Ala-Gly-Asp. These nine amino acids correspond to the first nine N-terminal amino acids of the adipokinetic hormone precursor-related peptide gamma-chain (APRP-gamma), a peptide that is predicted from the gene encoding the adipokinetic hormone III precursor. The APRP-gamma chain has a monoisotopic mass of 4387Da and contains two cysteine residues. It is known that both AKH I and AKH II precursors occur as dimers. After processing they give rise to the active hormones and three dimeric (two homodimers and one heterodimer) adipokinetic hormone precursor related peptides (APRPs). Based on the mass of 8770Da and the established N-terminal sequence tag, we conclude that the isolated peptide is a homodimer consisting of two APRP-gamma units, covalently linked to each other by two disulphide bounds. In analogy with the previous identified APRPs (APRP-1, APRP-2, and APRP-3), this APRP will be designated as APRP-4.     

Lom-AG-myotropin: a novel myotropic peptide from the male accessory glands of Locusta migratoria

A myotropic peptide, termed Lom-AG-myotropin, was isolated from extracts of 4400 accessory gland complexes of males of the locust, Locusta migratoria; the following sequence was derived: Gly-Phe-Lys-Asn-Val-Ala-Leu-Ser-Thr-Ala-Arg-Gly-Phe-NH2. This sequence is completely different from all presently known myotropic peptides from Locusta or other insects. The Lom-AG-myotropin is active on the oviduct and hindgut of Locusta migratoria and Leucophaea maderae. The stimulatory activity is, in both insects, 1000 times greater on the oviduct than on the hindgut, suggesting a specificity for the oviduct.     

On the pronymphal stage in the migratory locust (Locusta migratoria, Orthoptera, Acrididae)

The structure of the integument, somatic and visceral muscles, midgut, and Malpighian tubules were investigated at the late stages of the embryonic and early postembryonic development of the migratory locust, Locusta migratoria, to assess the organization of its pronymphal stage. In its morphogenetic features, the vermiform locust larva sometimes called the pronymph corresponds to the first nymphal instar covered with the second embryonic cuticle which has not been shed. Since the first-instar locust nymphs before and after the shedding of this embryonic cuticle differ significantly in many morphological characters, two consecutive phases of this nymphal instar can be distinguished: the first phase existing from the moment of development of the third embryonic cuticle to the shedding of the second one; the second phase existing from the shedding of the second embryonic cuticle to the formation of the cuticle of the second nymphal instar. Since the pronymphal stage should precede the nymph stage, it may be concluded that the pronymph of the locust is fully embryonized and covered with the second embryonic cuticle, which is also typical of other insects with hemimetabolous development (Konopová and Zrzavý, 2005). Therefore, it would be erroneous to refer to the vermiform first-instar nymph as the pronymph, because the two stages are separated by molting and formation of a new cuticle.     

Comparative study of neuropeptides from the corpora cardiaca of solitary and gregarious Locusta

Neuropeptide content of the corpora cardiaca (CC) was studied in crowded (gregarious phase) and isolated (solitary phase) Locusta migratoria migratorioides adults, using electrophoretic, chromatographic, and immunological techniques. Quantitative differences were found in the three neuropeptides investigated (neuroparsins, Lom-OMP, and APRP). The amount of neuroparsin A was higher in the CC of crowded locusts. Neuroparsin B content of the CC was quite similar in isolated and crowded locusts, or in some cases slightly higher in the latter. The comparative amounts of the ovary maturating parsin, Lom-OMP, in the CC were dependent on the sexual maturation of the locusts, being nearly similar in maturing isolated locusts and immature crowded locusts, but higher in crowded locusts when both phases were completely mature. The amount of AKH-precursor related peptides (APRP) was markedly and consistently higher in the CC of isolated locusts. These findings are discussed in relation to other physiological and ecological phase-dependent differences in locusts. © 1996 Wiley-Liss, Inc.     

Further experimental evidence for the involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta, Orthoptera)

Ecdysone has recently been shown to be able to trigger meiotic reinitiation in vitro in submature oocytes of Locusta. In the present study we have experimentally depressed (by 60-70%) ecdysone biosynthesis in the ovaries of adult females by rearing them on a diet with a modified sterol profile. Mature oocytes from such females fail to undergo normal reinitiation, but when incubated in vitro, can be induced to break their meiotic arrest by the addition of exogenous ecdysone. These results lend further support to the hypothesis that in Locusta, ovarian ecdysone is involved in the control of meiotic reinitiation.     

Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (Insecta, Orthoptera)

It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.     

Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera)

The morphology, ultrastrucure, effective elastic modulus, and adhesive properties of two different smooth-type attachment pads were studied in two orthopteran species. Tettigonia viridissima (Ensifera) and Locusta migratoria (Caelifera) have a similar structural organization of their attachment pads. They both possess a flexible exocuticle, where the cuticular fibrils are fused into relatively large rods oriented at an angle to the surface. The compliant material of the pad contributes to the contact formation with the substrate. However, the pad material structure was found to be different in these two species. L. migratoria pads bear a thick sub-superficial layer, as well as a higher density of rods. The indentation experiments showed a higher effective elastic modulus and a lower work of adhesion for L. migratoria pads. When the indentations were made at different depths, a higher effective elastic modulus was revealed at lower indentation depths in both species. This effect is explained by the higher stiffness of the superficial pad layer. The obtained results demonstrate a clear correlation between density of the fibres, thickness of the superficial layer, compliance of the pad, and its adhesive properties. Such material structures and properties may be dependent on the preferred environment of each species.