Endopeptidase activity of larval Lacanobia oleracea corpus allatum: metabolism of Manduca sexta allatostatin and allatotropin

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT) by enzymes associated with the corpus allatum (CA) of larvae of the tomato moth, Lacanobia oleracea, was investigated using reversed-phase high performance liquid chromatography and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Manduca sexta allatostatin was metabolised by CA extract to Manse-AS5-15, Manse-AS6-15, and Manse-AS7-15, which indicates enzymic cleavage at the C-terminal side of arginine residues R3 and R5 and the N-terminal side of R5, suggesting this is due to a trypsin-like enzyme. In support of this, the same degradation products were identified after Manse-AS was incubated with trypsin, and CA enzymic activity could be inhibited up to 79% by aprotinin. Degradation of Manse-AT by CA extract was also trypsin-like, cleaving at the C-terminal side of the basic residues K3 and R11 to produce Manse-AT4-13 and Manse-AT1-11. Metabolism by trypsin produced the same deletion peptides, but the major product due to this enzyme was Manse-AT4-11. Hydrolysis of Manse-AT by CA could only be partially inhibited by high doses of aprotinin (36%), and the CA extract also cleaved Manse-AT between M8 and T9 to produce Manse-AT1-8. A trypsin-like peptidase appears to be the major enzyme present in the CA of larval L. oleracea that acts to metabolise Manse-AS and Manse-AT. In addition, an unidentified enzyme that cleaves between M and T residues degraded Manse-AT.     

Degradation of Manduca sexta allatostatin and allatotropin by proteases associated with the foregut of Lacanobia oleracea larvae

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT), by enzymes of the foregut of larvae of the tomato moth, Lacanobia oleracea was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Metabolism of 1nmol Manse-AS by foregut extract (1microg protein) was rapid, t(1/2) approximately 5min, with two major products produced. Mass spectrometry of HPLC fractions identified cleavage products Manse-AS-(4-15) and Manse-AS-(6-15), which indicates enzymatic cleavage at the C-terminal side of arginine residues (R(3) and R(5)). This degradation of Manse-AS could be inhibited by up to 80% by the serine protease inhibitor aprotinin, but not PMSF, pepstatin, E64, EDTA, or 1,10-phenanthroline.M. sexta allatotropin was also rapidly degraded when incubated with foregut extract, t(1/2) approximately 8min, producing two metabolic products, one of which was identified as Manse-AT-(1-11), showing enzymatic cleavage at the C-terminal side of arginine (R(11)). The second product was identified as Manse-AT-(1-8). Hydrolysis of Manse-AT could only be partially inhibited by high doses of aprotinin (30%).     

Alanine substitution and deletion analogues of Manduca sexta allatostatin: structure-activity relationship on the spontaneous contractions of the foregut of larval Lacanobia oleracea

Manduca sexta allatostatin (Manse-AS) is a 15-residue non-amidated peptide with a blocked N-terminus and a disulphide bridge between the cysteine residues at positions 7 and 14. Analogues of Manse-AS were used to examine the structural requirements of Manse-AS for inhibitory activity on spontaneous foregut contractions of larval tomato moth (Lacanobia oleracea). Breaking the disulphide bond between C(7) and C(14) by reduction reduced the potency of the peptide, suggesting that the conformation of Manse-AS is important for its biological activity. When either of the cysteine residues were replaced with alanine the Manse-AS analogue had no measurable bioactivity. Alanine substitution at Q(6) was as potent as Manse-AS, all other alanine substitution analogues (R(5), Y(8), F(9), N(10), P(11), I(12) and S(13)), were myoinhibitory but less potent than native Manse-AS to varying degrees. Analogues with alanine substitution at amino acids with aromatic side chains (Y(8) and F(9)) were the least active. Amino-terminal deletion analogues Manse-AS(6-15) and Manse-AS(7-15) were inactive whereas Manse-AS(5-15) was fully active but not as potent as Manse-AS. The results show that amino acid residues both inside and outside the disulphide ring are important for biological activity.     

In vitro and in vivo effects of myo-active peptides on larvae of the tomato moth Lacanobia oleracea and the cotton leaf worm Spodoptera littoralis (Lepidoptera; Noctuidae)

Neuropeptides from five different neuropeptide families [Manduca sexta allatostatin (Manse-AS), and Manse-AS deletion analogue(5-15), M. sexta allatotropin (Manse-AT), leucomyosuppressin, perisulfakinin, and myoinhibitory peptide I (MIP I)] were assayed for their ability to affect the development and food consumption of penultimate and last larval instars of two lepidopteran species, L. oleracea and S. littoralis. Injections of Manse-AS deletion analogue(5-15), Manse-AT, perisulfakinin, and MIP I had no observable effects on development, food consumption, or mortality compared to controls. Single injections of Manse-AS significantly reduced the weight gain and increased mortality of L. oleracea and S. littoralis larvae compared to controls. By contrast, feeding Manse-AS to L. oleracea had no such effects. These differences were probably due to the degradation of the peptide by digestive enzymes in the foregut of L. oleracea. In studies in vitro, perisulfakinin, and MIP I had no effect on the spontaneous foregut contractions of L. oleracea larvae. Leucomyosuppressin, however, had myoinhibitory effects on the foregut. Single injections of leucomyosuppressin significantly reduced the weight gain and food consumption of L. oleracea and S. littoralis larvae and increased mortality. These data suggest that the deleterious effects observed in vivo were due to the myoinhibition by Manse-AS and leucomyosuppressin of the normal peristaltic movements of the gut either by the intact peptide or by its cleavage products resulting from degradation in the haemolymph.     

Interactions between allatostatins and allatotropin on spontaneous contractions of the foregut of larval Lacanobia oleracea

The interactions between the activity of three neuropeptides, Manduca sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT) and cydiastatin 4, on the spontaneous foregut contractions of the tomato moth, Lacanobia oleracea, were investigated. Bioassays revealed that application of Manse-AS to the foregut at high concentrations (10(-7)M) stopped contractions completely, and this inhibition could not be reversed by Manse-AT. Conversely, Manse-AS could inhibit a Manse-AT stimulated tissue. In contrast, Manse-AT reversed the inhibition of foregut peristalsis by cydiastatin 4 (10(-7)M), and cydiastatin 4 counteracted the stimulation by Manse-AT. These results imply that the Manse-AS inhibitory effect is dominant over the stimulatory action of Manse-AT. However, when two peptides with opposing actions were added together, the overall effect on foregut peristalsis was determined by the relative concentrations of each peptide, suggesting that in these experiments, no peptide was dominant over the other. When Manse-AS and cydiastatin 4 were applied to foregut tissues simultaneously the overall effect was not significantly different to the individual peptides, i.e. there was no additive effect. This suggests that the individual activities of Manse-AS and cydiastatin 4 are suppressed by an undetermined mechanism in the presence of the other peptide. These results question the need for two structurally different allatostatins that have the same physiological effect on foregut peristalsis in L. oleracea larvae.     

Neuropeptides associated with the frontal ganglion of larval Lepidoptera

The occurrence of neuropeptides in the frontal ganglia of larvae of the tobacco hawkmoth, Manduca sexta, the tomato moth, Lacanobia oleracea and the cotton leafworm, Spodoptera littoralis was investigated using reversed-phase high performance liquid chromatography (RP-HPLC), matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) and enzyme-linked immunosorbent assay (ELISA). Only three types of peptides could be identified or assigned from frontal ganglion extracts; M. sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT), and F/YXFGL-NH2 allatostatins. The peptide profiles of frontal ganglion of L. oleracea and S. littoralis were similar, with ten identical [M+H]+ ions, seven of which could be assigned to known lepidopteran peptides (Manse-AT, cydiastatin 2, 3, 4 and helicostatin 1, 5, 9). In addition, mass ions corresponding to helicostatin 7 (which was confirmed by MALDI-post source decay analysis) and Manse-AS were present in frontal ganglia of L. oleracea and helicostatin 6 in frontal ganglia of S. littoralis. Only four mass ions from M. sexta frontal ganglia corresponded to known peptides, cydiastatin 3 and 4, helicostatin 1, and Manse-AT. The only difference between the profiles of frontal ganglia from different stages of L. oleracea were mass ions which could not be assigned, and no differences were observed in the allatoregulatory peptides present. In HPLC fractions of M. sexta frontal ganglia, F/YXFGL-NH2 allatostatin-like immunoreactivity was widespread suggesting that more allatostatins were present than were identified.     

In vitro transport of an allatostatin across the foregut of Manduca sexta larvae and metabolism by the gut and hemolymph

The degradation of synthetic cydiastatin 4 by enzymes of the foregut and hemolymph, and transport across the foregut of larvae of the tobacco hawkmoth moth, Manduca sexta, were investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). In the hemolymph in vitro, cydiastatin 4 had a half-life of ca. 30 min. Two degradation products were identified; cydiastatin 4(1-6), due to cleavage of the C-terminal di-peptide GL-amide, and cydiastatin 4(2-8), due to cleavage of the N-terminal A residue. This hydrolysis could be inhibited by up to 93% by 1,10-phenanthroline. Other protease inhibitors had lesser effects (<21% inhibition of degradation) including the aminopeptidase inhibitors amastatin and bestatin, and the chelator EDTA. When incubated with foregut extract in vitro, cydiastatin 4 had a half-life of 23 min, and the hydrolysis products detected were also cydiastatin 4(1-6) and cydiastatin 4(2-8). Similarly, 1-10 phenanthroline inhibited foregut enzyme degradation of cydiastatin 4 by ca. 80%, whereas amastatin, bestatin, and EDTA had very little effect (<10% inhibition). Cydiastatin 4 was transported, intact, from the lumen to the hemolymph side of foregut tissues that were mounted as flat sheets in modified Ussing chambers. This trans-epithelial flux of peptide was dose and time-dependent, but was <3% of the amount of cydiastatin 4 present in the lumen bathing saline. In contrast, no trans-epithelial transport of peptide was apparent across everted foregut sac preparations.     

In vivo effects of Manduca sexta allatostatin and allatotropin on larvae of the tomato moth, Lacanobia oleracea

Abstract. Injection of Manduca sexta allatotropin (Manse-AT) into fifth or sixth stadium larval Lacanobia oleracea had no significant effect on larval growth, development or food consumption, compared to control injected insects. In contrast, injection of M. sexta allatostatin (Manse-AS) into fifth stadium larvae resulted in a retardation of growth, reduction in feeding and increased mortality, compared to control injected insects, but had no effect on non-feeding (day 7) sixth instar larvae. Results suggest that Manse-AS is not acting on the corpora allata (CA) to inhibit Juvenile Hormone (JH) synthesis to produce the observed effects, but most likely by its myoinhibiting action on the foregut. Inhibition of foregut peristalsis by Manse-AS in vivo appears to suppress feeding, resulting in increased mortality. Foregut peristalsis may be inhibited by the intact peptide or a deletion peptide produced by cleavage of Manse-AS by haemolymph enzymes, because Manse-AS (5-15) also inhibits muscle contractions in the foregut in vitro .     

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

Fusion proteins containing neuropeptides as novel insect contol agents: snowdrop lectin delivers fused allatostatin to insect haemolymph following oral ingestion

The mannose-binding lectin from snowdrop (Galanthus nivalis agglutinin: GNA), when fed to insects, binds to the gut epithelium and passes into the haemolymph. The potential for GNA to act as a carrier protein to deliver an insect neuropeptide, Manduca sexta allatostatin (Manse-AS), to the haemolymph of lepidopteran larvae has been examined by expressing a GNA/Manse-AS fusion protein (FP) in Escherichia coli, and feeding purified FP to larvae of the tomato moth Lacanobia oleracea. FP, administered at 1.5 or 0.5% of dietary proteins, was found to strongly inhibit feeding and prevent growth of fifth stadium larvae, whereas neither GNA nor Manse-AS alone, nor a mixture of GNA and Manse-AS in control treatments, had deleterious effects at similar levels. Elevated levels of material reacting with anti-Manse-AS antibodies were detected in the haemolymph of insects fed diets containing FP, suggesting that transport of the peptide had occurred. Evidence for the delivery of intact FP to the haemolymph was provided by the co-elution of Manse-AS-like immunoreactivity with standard FP after size exclusion chromatography of haemolymph from FP-fed larvae. GNA/Manse-AS and similar fusion proteins offer a novel and effective strategy for delivering insect neuropeptides by oral administration, which could be used in conjunction with expression in transgenic plants to give crop protection in the field.     

Leucine aminopeptidase-like activity in Aplysia hemolymph rapidly degrades biologically active alpha-bag cell peptide fragments

We have investigated the role that proteolytic enzymes in Aplysia hemolymph play in the inactivation of the neurotransmitter alpha-bag cell peptide (alpha-BCP(1-9), Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu). alpha-BCP fragments containing Pro in positions 1 or 2, or Tyr in position 1, were degraded relatively slowly (half-life, t1/2 = 10-64 min), whereas fragments lacking these residues were degraded relatively rapidly (t1/2 = 0.5-2.7 min). Of 12 peptidase inhibitors tested, only bestatin, amastatin, and phenanthroline significantly inhibited alpha-BCP(3-9) degradation. alpha-BCP(3-9) yielded only four observable cleavage products (in order of decreasing abundance at early time points): alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), and alpha-BCP(7-9). Degradation of alpha-BCP(3-9), alpha-BCP(4-9), alpha-BCP(5-9), alpha-BCP(6-9), or alpha-BCP(7-9) was strongly inhibited by bestatin, moderately inhibited by amastatin, and not inhibited by arphramenine B. The rates of degradation of eight alpha-BCP fragments and three other peptides in plasma were well correlated with their rates of degradation in mammalian leucine aminopeptidase (LAP, EC 3.4.11.1). Collectively our data support the following ideas. 1) In hemolymph one or more LAP-like enzymes rapidly and sequentially cleave alpha-BCP(3-9) or other small peptides lacking Pro at positions 1 or 2 or Tyr at position 1. 2) LAP-like peptidases in hemolymph may act in concert with previously described ganglionic peptidases to degrade neurally released alpha-BCP(1-9) and alpha-BCP(1-8) into inactive fragments.     

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80-150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.     

Characterization of Adenosine deaminase (ADA) in hemolymph of Biomphalaria glabrata.

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 micromols.L(-1). Highest activity was found at 37 degrees C. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 microL of pure hemolymph per assay, pH 6.8, and 37 degrees C. This enzyme showed activities of 834 +/- 67 micromol.min(-1).L(-1) (25 degrees C) and 2029 +/- 74 micromol.min(-1).L(-1) (37 degrees C), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43 degres C or 70% at 50 degrees C for 15 minutes. The ADA lost from 26% to 78% of its activity when hemolymph was pre-incubated at 50 degrees C for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.     

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney

To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 μm) were incubated with 10-1,000 μmol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.     

Hemolymph and tissue-bound peptidase-resistant analogs of the insect allatostatins

While neuropeptides of the allatostatin family inhibit in vitro production of juvenile hormone, which modulates aspects of development and reproduction in the cockroach, Diploptera punctata, they are susceptible to inactivation by peptidases in the hemolymph, gut, and bound to internal tissues. Patterns of peptidase cleavage were investigated in two allatostatin analogs in which sterically bulky components were incorporated into the active core region to block peptidase attack. The results were used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both hemolymph and tissue-bound peptidases. This pseudotetrapeptide allatostatin mimetic analog represents a valuable tool to neuroendocrinologists studying mechanisms by which the natural peptides operate and the physiological consequences of challenging an insect with an allatostatin that is not readily degraded via peptidase enzymes. Disruption of critical physiological processes modulated by neuropeptides such as the allatostatins via peptidase-resistant mimetic analogs could form the basis for novel pest insect management strategies in the future.     

Interaction between Manduca sexta allatotropin and manduca sexta allatostatin in the fall armyworm Spodoptera frugiperda

A peptide that strongly stimulates juvenile hormone (JH) biosynthesis in vitro by the corpora allata (CA) was purified from methanolic brain extracts of adult Spodoptera frugiperda. Using HPLC separation followed by Edman degradation and mass spectrometry, the peptide was identified as Manduca sexta allatotropin (Mas-AT). Treating the CA from adult S. frugiperda with synthetic Mas-AT (at 10(-6) M) caused an up to sevenfold increase in JH biosynthesis. The stimulation of JH synthesis was dose-dependent and reversible. Synthetic M. sexta allatostatin (Mas-AS) (10(-6) M) did not affect the spontaneous rate of JH secretion from CA of adult S. frugiperda, nor did any of the allatostatins of the Phe-Gly-Leu-amide peptide family tested. However, when CA had been activated by Mas-AT (10(-6) M), addition of synthetic Mas-AS (10(-6) M) reduced JH synthesis by about 70%. This allatostatic effect of Mas-AS on allatotropin-activated glands was also reversible. When CA were incubated in the presence of both Mas-AT (10(-6) M) and various concentrations of Mas-AS (from 10(-8) to 10(-5) M), the stimulation of JH-biosynthesis observed was inhibited in a dose-dependent manner. The experiments demonstrate a novel mechanism of allatostatin action. In S. frugiperda JH synthesis was inhibited only in those glands which had previously been activated by an allatotropin.     

Allatostatins from the retrocerebral complex and antennal pulsatile organ of the American cockroach: structural elucidation aided by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

The occurrence of allatostatins in retrocerebral complexes and antennal pulsatile organs of the American cockroach, Periplaneta americana, was investigated. Previously, molecular cloning of the P. americana allatostatin gene had predicted 14 peptides of this family [Ding et al., Comparison of the allatostatin neuropeptide precursors in the distantly related cockroaches Periplaneta americana and Diploptera punctata. Eur J Biochem 1997;234:737-746], however, only two forms had been identified by peptide isolation procedures [Weaver et al., Identification of two allatostatins from the CNS of the cockroach Periplaneta americana: novel members of a family of neuropeptide inhibitors of insect juvenile hormone biosynthesis. Comp Biochem Physiol 1994;107(C):119-127]. Using an extract of only 200 corpora cardiaca/corpora allata, we have found that at least 11 allatostatins occur in the retrocerebral complex. These peptides were already separated from other substances of the crude extract in the first HPLC step with heptafluorobutyric acid as organic modifier, and subsequently identified by MALDI-TOF mass spectrometry. Moreover, we have demonstrated the occurrence of nearly all allatostatins, including the cleavage product of Pea-AST-2 (LPVYNFGL-NH2), in antennal pulsatile organs of males and females. Allatostatins are predominant neuropeptides in these organs. Additionally, only two other known peptides could be identified in these organs by mass screening: proctolin and leucomyosuppressin. The function of allatostatins in antennal pulsatile organs remains unclear. We assume a release into the hemolymph via the ampullac, which could act as neurohemal release sites. The method described for the identification of allatostatins is a very fast method for neuropeptide screening in neurohemal tissues.     

Determination of biodegradation products from sulfonated dyes by <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>using capillary electrophoresis coupled with mass spectrometry

Microbial treatment of environmental pollutants including dyes with white rot fungi has received wide attention as a potential alternative for conventional methods in wastewater treatment. The degradation products from dyes and mechanism underlying fungal degradation of dyes is desirable to be understood. Capillary electrophoresis coupled with mass spectrometry (CE-MS) was used in this study to determine biodegradation products of 4-[(4-hydroxyphenyl)azo]-benzenesulfonic acid, sodium salt (4HABA) and Acid Orange 7 (C.I. 15510), produced by a white rot fungus, Pleurotus ostreatus. Two major degradation products, benzenesulfonic acid and 4-hydroxy-benzenesulfonic acid, from both sulfonated compounds, were identified and their kinetic profiles in biodegradation were followed by CE-MS. Another product, 1,2-naphthoquinone, from Acid Orange 7 was identified using HPLC. Formation of these products in fungal degradation is discussed.Revisions requested 8 October 2004; Revision received 12 November 2004     

Allatoregulatory peptides in Lepidoptera, structures, distribution and functions

Allatoregulatory peptides either inhibit (allatostatins) or stimulate (allatotropins) juvenile hormone (JH) synthesis by the corpora allata (CA) of insects. However, these peptides are pleitropic, the regulation of JH biosynthesis is not their only function. There are currently three allatostatin families (A-, B-, and C-type allatostatins) that inhibit JH biosynthesis, and two structurally unrelated allatotropins. The C-type allatostatin, characterised by its blocked N-terminus and a disulphide bridge between its two cysteine residues, was originally isolated from Manduca sexta. This peptide exists only in a single from in Lepidoptera and is the only peptide that has been shown to inhibit JH synthesis by the CA in vitro in this group of insects. The C-type allatostatin also inhibits spontaneous contractions of the foregut. The A-type allatostatins, which exist in multiple forms in a single insect, have also been characterised from Lepidoptera. This family of peptides does not appear to have any regulatory effect on JH biosynthesis, but does inhibit foregut muscle contractions. Two structurally unrelated allatotropins stimulate JH biosynthesis in Lepidoptera. The first was identified in M. sexta (Manse-AT) and occurs in other moths. The second (Spofr AT2) has only been identified in Spodoptera frugiperda. Manduca sexta allatotropin also stimulates heart muscle contractions and gut peristalsis, and inhibits ion transport across the midgut of larval M. sexta. The C-terminal (amide) pentapeptide of Manse-AT is important for JH biosynthesis activity. The most active conformation of Manse-AS requires the disulphide bridge, although the aromatic residues also have a significant effect on biological activity. Both A- and C-type allatostatins and Manse-AT are localised in neurosecretory cells of the brain and are present in the corpora cardiaca, CA and ventral nerve cord, although variations in localisation exist in different moths and at different stages of development. The presence of Manse-AS and Manse-AT in the CA correlates with the biological activity of these peptides on JH biosynthesis. There is currently no explanation for the presence of A-type allatostatins in the CA. The three peptide types are also co-localised in neurosecretory cells of the frontal ganglion, and are present in the recurrent nerve that supplies the muscles of the gut, particularly the crop and stomodeal valve, in agreement with their role in the regulation of gut peristalsis. There is also evidence that they are expressed in the midgut and reproductive tissues.