Peptidergic control of the crop of the cabbage root fly, Delia radicum (L.) Diptera: Anthomyiidae): A role for myosuppressin.

There is much interest in targeting neuropeptide signaling for the development of new and environmentally friendly insect control chemicals. In this study we have focused attention on the peptidergic control of the adult crop of Delia radicum (cabbage root fly), an important pest of brassicas in European agriculture. The dipteran crop is a muscular organ formed from the foregut of the digestive tract and plays a vital role in the processing of food in adult flies. We have shown using direct tissue profiling by MALDI-TOF mass spectrometry that the decapeptide myosuppressin (TDVDHVFLRFamide) is present in the crop nerve bundle and that application of this peptide to the crop potently inhibits the spontaneous contractions of the muscular lobes with an IC50 of 4.4 × 10-8 M. The delivery of myosuppressin either by oral administration or by injection had no significant detrimental effect on the adult fly. This failure to elicit a response is possibly due to the susceptibility of the peptide to degradative peptidases that cleave the parent peptide to inactive fragments. Indeed, we show that the crop of D. radicum is a source of neuropeptide-degrading endo- and amino-peptidases. In contrast, feeding benzethonium chloride, a non-peptide agonist of myosuppressin, reduced feeding rate and increased the rate of mortality of adult D. radicum. Current results are indicative of a key role for myosuppressin in the regulation of crop physiology and the results achieved during this project provide the basis for subsequent studies aimed at developing insecticidal molecules targeting the peptidergic control of feeding and food digestion in this pest species.

Effects of Manduca sexta allatostatin and an analogue on the peach-potato aphid Myzus persicae (hemiptera: aphididae) and degradation by enzymes in the aphid gut.

The oral toxicity of the C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analogue δR³Î´R5Manse-AS, where R residues were replaced by their D-isomers, were tested against the peach-potato aphid Myzus persicae by incorporation into an artificial diet. Both peptides had significant dose-dependent effects on mortality, growth, and fecundity compared with control insects. The analogue, δR³Î´R5Manse-AS, had an estimated LC50 of 0.31 µg/µl diet and was more potent than Manse-AS (estimated LC50 of 0.58 µg/µl diet). At a dose of 0.35 µg δR³Î´R5Manse-AS/µl diet, 76% of the aphids were dead after 6 days and all were dead after 10 days. In comparison, three times the dose of Manse-AS was required to achieve 74% mortality after 8 days and 98% mortality after 16 days. The degradation of both peptides by extracts prepared from the gut of M. persicae was investigated. The estimated half-life of Manse-AS, when incubated with the gut extract from M. persicae, was 31 min. Degradation was due to a cathepsin L-like cysteine protease, carboxypeptidase-like activity, endoprotease activity with glutamine specificity, pyroglutamate aminopeptidase activity, and possibly trypsin-like proteases. The half-life of the δR³Î´R5 Manse-AS analogue was enhanced (73 min) with the D-isomers of R appearing to prevent cleavage around the R residues by cathepsin L-like cysteine proteases or from trypsin-like proteases. The greater stability of the analogue may explain its increased potency in M. persicae. This work demonstrates the potential use of Manse-AS and analogues, with greater resistance to enzymatic attack, in aphid control strategies.

Oral activity of FMRFamide-related peptides on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut.

Insect myosuppressins and myosuppressin analogues were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Acyrthosiphon pisum myosuppressin (Acypi-MS) and leucomyosuppressin (LMS) had significant dose-dependent effects (0.1-0.5µg peptide/µl diet) on feeding suppression, mortality, reduced growth and fecundity compared with control insects, but Acypi-MS was more potent than LMS. One hundred percent of aphids had died after 10days of feeding on 0.5µg Acypi-MS/µl diet whereas 40% of aphids feeding on 0.5µg LMS/µl diet were still alive after 13days. Myosuppressins were degraded by aphid gut enzymes; degradation was most likely due to a carboxypeptidase-like protease, an aminopeptidase and a cathepsin L cysteine protease. The estimated half-life of Acypi-MS in a gut extract was 30min, whereas LMS was degraded more slowly (t½=54min). No toxicity was observed when the analogues δR(9) LMS and citrolline(9) Acypi-MS or FMRFamide were fed to the pea aphid. These findings not only help to better understand the biological effects of myosuppressins in aphids but also demonstrate the potential use of myosuppressins in a strategy to control aphid pests.

Neuropeptides associated with the central nervous system of the cabbage root fly, Delia radicum (L).

The peptidome of the central nervous system of adult cabbage root fly, Delia radicum (L) was investigated using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Over twenty neuropeptides were identified from three different tissue sources, the combined brain/suboesophageal ganglion (SOG), the retrocerebral complex, and the thoracic-abdominal ganglion (TAG). A number of peptides were identified in all three tissues, including allatostatins, short neuropeptide F-like peptides, corazonin, a pyrokinin, and a myosuppressin. Adipokinetic hormone was restricted to the retrocerebral complex. Other peptides, including FMRFamides and sulfakinins were detected only in the brain/SOG and TAG. Some peptides, notably myoinhibitory peptides and tachykinins, which have been identified in other fly species, were not detected in any tissue sample. This study has structurally characterized for the first time, the neuropeptides from adult D. radicum.

Effects of Manduca sexta allatostatin and an analog on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut.

The C-type allatostatin, Manduca sexta allatostatin (Manse-AS) and the analog delta R(3)delta R(5)Manse-AS, where R residues were replaced by their d-isomers, were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Both peptides had significant dose-dependent feeding suppression effects, resulting in mortality, reduced growth and fecundity compared with control insects. The delta R(3)delta R(5)Manse-AS analog had an estimated LC(50) of 0.18 microg/microl diet, and was more potent than Manse-AS. At a dose of 0.35 microg delta R(3)delta R(5)Manse-AS/microl diet, 98% of aphids were dead within 3 days, at a rate similar to those aphids that had been starved (no diet controls). On comparison, it required 13 days and three times the dose of Manse-AS fed to aphids to attain 96% mortality. It is possible that the feeding suppression effects of Manse-AS on aphids are due to the inhibition of gut motility. The estimated half-life of Manse-AS when incubated with a gut extract from A. pisum was 54 min. Degradation was most likely due to cathepsin L cysteine and/or trypsin-like proteases, by an unidentified glutamine-specific protease and by a carboxypeptidase-like enzyme. The d-isomers of R in the Manse-AS analog appeared to prevent hydrolysis by cathepsin L cysteine and trypsin-like enzymes, and enhance its half-life (145 min). However delta R(3)delta R(5)Manse-AS was cleaved by enzymes with carboxypeptidase-like and chymotrypsin-like activity. The increased stability of the Manse-AS analog may explain its enhanced feeding suppression effects when continually fed to aphids, and demonstrates the potential use of Manse-AS in a strategy to control aphid pests.