Haemolymph levels of Schistocerca gregaria ion transport peptide and ion transport-like peptide

Abstract.  Titres of ion transport peptide (ITP) and ion transport-like peptide (ITP-L) in the haemolymph of the desert locust Schistocerca gregaria Forskål are investigated using a combination of enzyme-linked immunosorbent assay and reversed-phase high-performance liquid chromatography (HPLC). The estimated circulating levels of these two peptides are used to investigate their putative physiological roles. Haemolymph levels of ITP-L are significantly higher in fed and exercise-stressed locusts than in starved insects, suggesting a role for ITP-L in postfeeding osmoregulation and metabolism associated with stress. The higher titres of ITP-L in the final nymphal stadium could be related to increased levels of ecdysteroids over this same period. In addition detection of ITP-L in the brain and corpus cardiacum of the locust demonstrates for the first time that the ITP-L is expressed in these tissues. Immunoreactivity to antibodies raised against ITP is significantly higher in fed locusts compared with starved insects. However, >95% of this immunoreactivity elutes later than synthetic ITP when separated by HPLC. The results suggest that this more hydrophobic immunoreactivity is neither native ITP nor a metabolic breakdown product of ITP. Haemolymph levels of immunoreactivity to ITP during the last nymphal stadium are similar to ITP-L but, unlike ITP-L, there is no measured increase in immunoreactivity to ITP due to exercise stress.     

Signal transduction for Schistocerca gregaria ion transport peptide is mediated via both cyclic AMP and cyclic GMP

The second messengers involved in the signal transduction for Schistocerca gregaria, ion transport peptide (Schgr-ITP) that regulates ion and fluid transport across the ileum of the desert locust S. gregaria, were measured using competitive enzyme-linked immunosorbent assays (ELISAs). Synthetic Schgr-ITP elevates intracellular levels of both cyclic AMP and cyclic GMP, measured over a 15 min period in the presence of 3-isobutyl-1-methylxanthine, in a dose-dependent manner. Furthermore, crude corpora cardiaca (CC) extracts elevate intracellular cyclic AMP levels 2-fold greater than Schgr-ITP, suggesting that factors present in the CC, other than Schgr-ITP, also act via this second messenger. These results suggest that the interaction of Schgr-ITP with two separate receptors, most likely a G-protein coupled receptor and a membrane bound guanylate cyclase, elevates intracellular levels of cyclic AMP and cyclic GMP to regulate ion and fluid transport across the locust ileum. Cyclic AMP stimulates Cl⁻, K⁺ and Na⁺ reabsorption, whereas secretion of H⁺ into the lumen of the ileum is most likely mediated via cyclic GMP. Cyclic GMP also stimulates Cl⁻ uptake in a similar manner to cyclic AMP. The measurement of tissue (central nervous system) levels of Schgr-ITP using an indirect ELISA confirms that the peptide is only present in the locust brain and the CC. The amounts present are greatest in the CC, where the peptide is presumably stored for release into the hemolymph when locusts feed.     

Mutational analysis of the C-terminus in ion transport peptide (ITP) expressed in Drosophila Kc1 cells

Ion transport peptide (ITP) stimulates Cl(-) transport (measured as short-circuit current, I(sc)) and fluid reabsorption in Schistocerca gregaria ilea. We report that Drosophila Kc1 cells transfected with preproITP cDNA secrete a peptide (KcITP(75)) that, while cleaved correctly at the N-terminus, had reduced (10-fold) stimulatory activity on ileal I(sc) compared to both native ITP (ScgITP) and synthetic ITP (synITP). We provide evidence that the reduced activity of KcITP(75) is due to incomplete processing of the C-terminal sequence LGKK (KcITP(75)) to L-amide. In support of this, in vitro amidation of glycine extended ITP (i.e., KcITP(73) ending in LG) but not KcITP(75) (ending in LGKK) significantly increased specific activity in the bioassay. Further evidence for C-terminus involvement includes complete loss of stimulation by truncated mutants (e.g., KcITP(71) which lacks LGKK) and a mutant in which alanine is substituted for the terminal glycine in KcITP(73). Moreover a natural homologue (KcITP-L, which differs only in the C-terminal sequence) expressed by Kc1 cells does not stimulate ileal I(sc). Rather KcITP-L acts as a weak ITP antagonist, as does the truncated mutant KcITP(71). KcITP(70) has no antagonistic effect. A short synthetic peptide fragment of the C-terminus (VEIL-amide) does not stimulate ileal I(sc), indicating that other regions of ITP are also essential to biological activity. Arch.     

Biological actions of synthetic locust ion transport peptide (ITP)

Locust Ion Transport Peptide (ITP) a member of the arthropod neuropeptide family which includes hyperglycemic, vitellogenesis-inhibiting, and moult-inhibiting hormones (CHH, VIH, MIH, respectively) was synthesized as proposed by Meredith et al. (1996) with terminal amidation of amino acid residue 72 and with 3 disulphide bridges. This is the first member of this family to be synthesized. Biological activities of synthetic ITP (synITP) were very similar to those previously reported for ITP purified from Schistocerca corpora cardiaca (ScgITP) and partially sequenced by Audsley et al. (1992a, b). Dose-response curves for both synITP and ScgITP on ileal transport of Cl- (measured as increased short-circuit current, delta Isc), were similar with a EC50 of 1-2 nM. The Isc time course and maximum delta Isc across ileal epithelia at different dosages of synITP and ScgITP had similar patterns as did changes in transepithelial open-circuit potential (Vt) and resistance (Rt), reflecting changes in salt transport which drives fluid absorption. Disulphide bridges were shown to be required for biological activity of synITP, which caused the same 4-fold increase in ileal fluid transport rate (Jv) as previously reported for ScgITP. Both synITP and ScgITP caused only partial stimulation of rectal Isc and had no significant effect on rectal Jv. These results indicate that the structure of ITP predicted earlier from cDNA is correct.     

Mutational analysis of the N-terminus in Schistocerca gregaria ion-transport peptide expressed in Drosophila Kc1 cells

The functions of the 6-7 amino acid N-terminal domain conserved in insect and crustacean members of the hyperglycemic hormone (CHH) family were assayed by site-directed mutagenesis of Schistocerca gregaria ion-transport peptide (SchgrITP). Mutant peptides were expressed in Drosophila Kc1 cells and tested in a biological assay measuring stimulation of active Cl(-) transport across the locust ileum. We exchanged the N-terminal domain of SchgrITP with that of the shrimp Penaeus japonicus hyperglycemic hormone leaving the remainder of SchgrITP intact. The chimeric peptide was completely inactive in the ileal bioassay, showing that the N-terminus of SchgrITP is essential and that the 2 amino acids (phenylalanine-3 and aspartate-4) conserved in the shrimp and locust peptides are not sufficient for function. We made all possible alanine substitutions in the SchgrITP N-terminal domain. Only phenylalanines 2 and 3 were essential for function in the locust ileal bioassay. All N-terminal mutations were cleaved correctly from the prepropeptide, and expressed in similar concentrations as wild-type ITP suggesting the specific amino acids are not essential for these functions. Post-translational modification may explain a minor ITP isomorph observed in Drosophila Kc1 cell expression. Alanine substitution at position 2 produced a weak ITP antagonist. These structure-function studies, the first for any member of the CHH family, show that both conserved and unconserved amino acids contribute to SchgrITP ion-transport function and that the conserved aspartate in position 4 is required for a yet uncharacterized function.     

Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)

The monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K⁺] through both a furosemide-inhibitable and a vanadate-inhibitable transport system. Intracellular exchangeable [Na⁺] was slightly lower than the extracellular [Na⁺] and was maintained at this level through a vanadate-sensitive transport system. Na⁺ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide-sensitive Na⁺ uptake when compared with furosemide-sensitive K⁺ uptake indicated that these cations are not cotransported. 4,4′-Diisothiocyano-2,2′-disulfonic acid stilbene (DIDS) inhibited Na⁺, K⁺, and Cl^? uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na⁺, K⁺, Cl^?, H⁺ and HCO₃ ^? fluxes are regulated in these cells.     

Pattern formation in fragmented eggs of the short germ insect Schistocerca gregaria

Summary The effect of transverse fragmentation on the segment pattern of the short germ embryo of the locust Schistocerca gregaria has been investigated at two stages subsequent to the formation of the germ anlage. Following fragmentation both anterior and posterior partial embryos were observed, although rarely in a single egg. Anterior partial patterns usually terminated with a segment visible at the time of fragmentation or with the next segment due to appear. Posterior partial patterns began with a wide range of segments depending on the level of fragmentation.Anterior and posterior partial patterns developing in a single egg were usually not complementary and the segments missing sometimes included some segments visible when the embryo was fragmented. Non-complementary patterns resulted following fragmentation in all regions, while complementary patterns only occurred after fragmentation in the visibly-segmented region.The results suggest that following fragmentation isolated posterior portions of the embryo continue to form segments, while isolated anterior regions usually do not. This effect could result from variable damage to an existing pattern of unequally-sized segment primordia, or from the disruption of a process of sequential segmentation in the elongating posterior region of the embryo. The results are broadly compatible with the progress zone model proposed by Summerbell et al. (1973).     

Analysis of the mitochondrial genome of Schistocerca gregaria gregaria (Orthoptera: Acrididae)

In the present study, we present the full sequence of the mitochondrial genome of the African desert locust Schistocerca gregaria gregaria. The size of 15625 bp reported matches very well with mitochondrial genomes of other Orthopteriodea. The mitochondrial genome comprises 13 protein‐coding genes, two ribosomal RNAs and 22 t‐RNAs with two t‐RNA (trnD and trnK) rearrangements that are typical for the taxon Caelifera. We compared the sequence with 12 mitochondrial genes of Schistocerca gregaria flaviventris and Schistocerca americana and used some of these data to construct phylogenetic trees, which confirm the close relationship between the two subspecies S. g. flaviventris and S. g. gregaria. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 296–305.     

Immunological evidence for an allatostatin-like neuropeptide in the central nervous system of Schistocerca gregaria,Locusta migratoria and Neobellieria bullata

Methanolic brain extracts of Locusta migratoria inhibit in vitro juvenile hormone biosynthesis in both the locust L. migratoria and the cockroach Diploptera punctata. A polyclonal antibody against allatostatin-5 (AST-5) (dipstatin-2) of this cockroach was used to immunolocalize allatostatin-5-like peptides in the central nervous system of the locusts Schistocerca gregaria and L. migratoria and of the fleshfly Neobellieria bullata. In both locust species, immunoreactivity was found in many cells and axons of the brain-retrocerebral complex, the thoracic and the abdominal ganglia. Strongly immunoreactive cells were stained in the pars lateralis of the brain with axons (NCC II and NCA I) extending to and arborizing in the corpus cardiacum and the corpora allata. Although many neurosecretory cells of the pars intercerebralis project into the corpus cardiacum, only 12 of them were immunoreactive and the nervi corporis cardiaci I (NCC I) and fibers in the nervi corporis allati II (NCA II) connecting the corpora allata to the suboesophageal ganglion remained unstained. S. gregaria and L. migratoria seem to have an allatostatin-like neuropeptide present in axons of the NCC II and the NCA I leading to the corpus cardiacum and the corpora allata. All these data suggest that in locusts allatostatin-like neuropeptides might be involved in controlling the production of juvenile hormone by the corpora allata and, perhaps, some aspects of the functioning of the corpus cardiacum as well. However, when tested in a L. migratoria in-vitro juvenile hormone-biosynthesis assay, allatostatin-5 did not yield an inhibitory or stimulatory effect. There is abundant AST-5 immunoreactivity in cell bodies of the fleshfly N. bullata, but none in the CA-CC complexes. Apparently, factors that are immunologically related to AST-5 do occur in locusts and fleshflies but, the active protion of the peptide required to inhibit JH biosynthesis in locusts is probably different from that of AST-5.     

Pharmacological study of the second messengers that control rectal ion and fluid transport in the desert locust (Schistocerca gregaria)

The involvement of second messengers in modulating Schistocerca gregaria rectal ion and fluid transport was investigated using two in vitro bioassay systems: everted rectal sacs and rectal flat sheets. Various agents known to block or activate specific signal transduction pathways were employed in these bioassays. Cyclic AMP stimulated rectal fluid reabsorption (J_v) and Cl⁻ transport (I_sc) to the same extent as aqueous extracts of corpus cardiacum storage lobes. Cyclic GMP also partially (50%) stimulated both rectal J_v and I_sc. Exogenous Ca²⁺ was not required for the maintenance of rectal transport, indeed Ca²⁺ free conditions increased the amount of stimulatable J_v. There was some evidence indicating intracellular Ca²⁺ may play a minor role in controlling rectal transport. The phospholipase C mediated signal transduction pathway was not involved with the stimulation of rectal transport, and appeared to have an inhibitory role. Neuroparsins, antidiuretic neuropeptides from Locusta migratoria, showed no activity upon either S. gregaria rectal ion or fluid transport. The findings of this study show that the two closely related insects possess discrete antidiuretic factors which stimulate rectal transport via different signal transduction pathways. © 1996 Wiley-Liss, Inc.     

Growth-inhibition effects of pacifastin-like peptides on a pest insect: the desert locust, Schistocerca gregaria

The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to compensate for PI present in their diet. To challenge this highly flexible physiological mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during experiments with a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non-plant PI (pacifastin-related inhibitors) were used to reduce the risk of a specific co-evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, allowing for a maximal inhibition of the proteolytic activity and (iii) digestive peptidase samples were taken during oral administration experiments to study compensatory mechanisms. Contrary to larvae fed on a diet containing plant-derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin-like PI. Nevertheless, the growth inhibition effect of this PI mixture attenuated after a few days, Moreover, a comprehensive study of the observed responses after oral administration of PI revealed that S. gregaria larvae can adjust their secreted digestive enzyme activities in two distinct ways depending on the composition/concentration of the PI-mixture.     

Comparative role of proteins in transport of HCH-isomers in desert locust, Schistocerca gregaria Forskal and bovines

The studies on binding of hexachlorocyclohexane (HCH) with carrier proteins were carried out to establish the role of proteins in the transport of insecticides in insects. Sephadex G-200 column chromatography resolved haemolymph of adult male desert locust, Schistocerca gregaria into three major protein peaks. There was significant binding of gamma-HCH with first protein peak (F1). Two classes of binding sites were observed on first protein peak for gamma-HCH. However low level of binding was observed with the third protein peak (F3) of the haemolymph. Bindings of HCH-isomers (alpha, beta and gamma) with bovine serum albumin (BSA) were not related to their water solubilities. Moderate to low affinities (1.4 -1.84 x 10(6) M(-1)) of HCH-isomers for BSA were observed. The present studies showed that more HCH binds to haemolymph lipoprotein of locust as compared to BSA. This indicates a significant role of haemolymph proteins in the transport of insecticides in insects.     

Subspecific taxonomy of the desert locust,Schistocerca gregaria (Orthoptera: Acrididae), based on molecular and morphological characters

We evaluated the validity of the subspecific designation for Schistocerca gregaria gregaria (Forskål) and Schistocerca gregaria flaviventris (Burmeister), isolated in distinct regions along the north–south axis of Africa. Towards this goal, we assessed the variation of multiple morphological and molecular traits within species. We first used elliptic Fourier and landmark‐based relative warps analyses to compare the size and shape of two internal and two external structures of male genitalia. We provide a discriminant function which classified the specimens with 100% accuracy and selected shape elements of the external structures only (cercus and epiproct). We also tested eight molecular markers, and because of either absence of variation or contamination by mitochondrial DNA (mtDNA)‐like sequences, we used a clone‐and‐sequence analysis of the standard cytochrome c oxidase subunit I mitochondrial DNA barcode only. We differentiated 185 true mitochondrial sequences from 66 mitochondrial DNA‐like sequences, most of which were from S. g. gregaria specimens. On the dataset of mitochondrial origin, we identified three characteristic point mutations that diagnosed the two allopatric subspecies with 94% accuracy. Minimum spanning network and parsimony tree analyses identified S. g. flaviventris as a monophyletic lineage distinct from the nominate subspecies. Accordingly, microsatellite data indicate rarely occurring admixture events only, showing that independent evolutionary history is the norm.     

Effect of phase status on responses to AKHI in the desert locust,Schistocerca gregaria gregaria

Hyperlipaemic response to adipokinetic hormone (AKH I) was demonstrated in both solitary and gregarious phases of the desert locust, Schistocerca gregaria gregaria. Time-course studies showed that the gregarious locusts had a faster response to the hormone than their solitary counterparts. At peak response time (90 min), the gregarious locusts were more sensitive to AKH I doses below 2 pmol while the solitary locusts had a higher response above this dose. Upon injection of the hormone, lipoprotein conversion occurred, resulting in the formation of the low density lipoprotein (LDLp). The LDLp formed in the gregarious locusts was much larger than that of the solitary locusts. The fat body lipid reserve (expressed as % fat body dry weight) was significantly (P < 0.01) higher in the gregarious (79.02 ± 2.77%) than in the solitary locusts (65.23 ± 2.55%). Triacylglycerol was the major lipid class representing 83.9 and 73.9% of the total lipids in gregarious and solitary locusts, respectively. The higher fat body lipid reserves and efficient LDLp formation in response to AKH in gregarious locusts compared to solitary locusts suggests a physiological adaptation for prolonged flights. © 1996 Wiley-Liss, Inc.     

Racemization free coupling of peptide segments. Synthesis of an insect neuropeptide.

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH2 has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed alpha-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt/NMM always rendered highly optically impure products containing 10-20% of the D-epimer.     

The dark-colour-inducing neurohormone of locusts in relation to an albino mutant of Schistocerca gregaria

In the albino mutant of an Okinawa strain of Locusta migratoria (L.) (Orthoptera: Acrididae), albinism is caused by the absence of the dark‐colour‐inducing neurohormone (DCIN), which is present in the corpora cardiaca (CC) of normally coloured phenotypes. This study tests whether the absence of DCIN is responsible for albinism in an albino mutant of another locust, Schistocerca gregaria (Forsk.) (Orthoptera: Acrididae). This seemed feasible because a single Mendelian unit controls albinism in both species. However, implantation of CC, or injection of an extract of CC, from albino donors of S. gregaria, induce dark coloration in crowded nymph recipients of the Okinawa albino mutant of L. migratoria, as effectively as do implanted CC, or injections of extract of CC, from normal phenotype donors of S. gregaria. Therefore, DCIN is present in the albino mutant of S. gregaria, and consequently, the albinism in this mutant is not caused by its absence. Implantation of CC, or injection of extracts of CC, from albino donors of S. gregaria to conspecific albino nymphs does not induce darkening. Only extremely high doses of synthetic DCIN injected into albino nymphs of S. gregaria are effective, inducing some darkening. The dose to induce such darkening in albino nymphs of S. gregaria is 50 nmol, ≈ 5 × 10⁶ times higher than that (10 femtomol) needed to induce equivalent darkening in nymphs of the Okinawa albinos of L. migratoria. The results are discussed and some possible explanations of the observed effects outlined.