WO1995001991A1

WO1995001991A1 - Allatostatins and their use

Info

Publication number: WO1995001991A1
Application number: PCT/GB1994/001440
Authority: WO
Inventors: John Patrick Edwards; Robert James Weaver
Original assignee: The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority date: 1993-07-05
Filing date: 1994-07-04
Publication date: 1995-01-19
Also published as: GB9313891D0; AU7078094A; GB2283974A; GB9501907D0

Abstract

The present invention relates to novel allatostatins, their use as pesticidal agents, compositions comprising them and methods for their preparation. The peptides may suitably be prepared by extraction from insects, by chemical synthesis or by recombinant DNA techniques.

Description

ALLATOSTATINS AND THEIR USE.

The present invention relates to novel peptides, compositions comprising them and their use as pesticidal agents. Further provided are methods for isolating and synthesising the peptides.

Juvenile hormones (JHs) , produced by the corpora allata (CA) , play a vital role in the regulation of insect development primarily in the control of adult sexual maturation, metamorphosis and reproduction (Engelmann, F, The Physiology of Insect Reproduction, Perga-mon, Oxford, 1970 ). The hormonal secretory activity of these endocrine glands is regulated by a variety of humoral and nervous factors originating, at least in part, in the insect brain (Scharrer, B, 1987, Annu Rev Entomol 32, 1-16) .

Peptidergic neurosecretory material from cells of the brain that project down to the CA have long been postulated to regulate the activity of these endocrine glands. These factors of cerebral origin may act either as stimulators or as inhibitors (allatotropins or allatostatins) of juvenile hormone biosynthesis and experimental evidence suggests that one of these may predominate, depending upon species at different times during development (Tobe, S.S. & Stay, B. 1 85 Adv Insect Physiol 18, 305-432).

Until quite recently little progress had been made towards isolating either an allatotropin or an allatostatin from any source. However, three different types of brain neuropeptide having an effect on the CA are now known. The first of these to be characterised was a 13-residue allatotropic peptide from heads of pharate adult Mandula sexta, the Tobacco Homworm moth (Kataoka, H et al, 1989. Science, 243, 1481-1483 ). A family of five allatostatins (ASTs) have been isolated from brains of the cockroach Diploptera punctata. and have been shown to be potent in vitro inhibitors of juvenile hormone biosynthesis by the CA in this species. All of the cockroach allatostatins are amidated neuropeptides varying from 8 to 18 amino acids in length (Woodhead A P et al,1989 Proc Natl Acad Sci USA, 86, 5997-6001; Stay, B et al, 1991. Insect Neuropeptides: Chemistry, Biology and Action, pp 164-176, Eds Menn, J J, Symp Series 453; Pratt, G E et al 1991, Proc Natl Acad Sci USA 8824l2-24l6 and Pratt, G E et al, 1989, Biochem Biophys Res Commun 163 1243-1247). They share carboxy-terminal amino acid sequence likeness, which shows some similarity to the enkephalin-related peptide [Met-enkephalin]- -Arg6-Gly7-Leu8 (met-8) , but are otherwise unique.

The third type of CA-active neuropeptide to be isolated and sequenced is a structurally unrelated allatostatin from heads of pharate adult moth, Manduca sexta (Kramer, S J et al, 1991. Proc Natl Acad Sci USA 889458-9462) . This compound is a cysteine-containing, amino terminal blocked (pGlu) 15-amino acid peptide which bears no resemblance either to the allatotropin of this same species or to any one of the Diploptera allatostatins. This compound causes rapid and potent inhibition of JH synthesis in CA from larvae and adult M.sexta but has no effect against CA from species of two other insect orders.

It has been established that at least two of the cockroach allatostatins (AST-1 and AST-2) have a weak capacity to inhibit JH synthesis by CA from adults of the more important pest species Periplaneta americana (Woodhead A P et al, 1989. Proc Natl Acad Sci USA, 86, 5997-6001). This species is related in terms of cockroach phylogeny but has a different emphasis to its mode of reproduction (Weaver R J et al, 1993. Insect Juvenile Hormone Research, eds Mauchamp B et al, I N R A Paris). However Diploptera allatostatins show at best rather weak activity spectra against the CA from different stages of developing P.americana.

The present invention provides novel peptides which show potent in vitro inhibition of JH synthesis in cockroach and provides methods for extraction and isolation of examples of these from the brains of adult females of P.americana. Specific peptides, designated Pea-AST-1 and Pea-AST-2, are amidated neuropeptides of 13 and 10 amino acids, respectively and each bear some structural similarity to members of a family of putative allatostatins identified from the cockroach D.punctata (Woodhead A P et al, 1989. Proc Natl Acad Sci USA, 86, 5997-6001). They share no sequence homology with either of the two known Lepidopteran allatoactive neuropeptides, Mas-AT and Mas-AS, isolated from pharate adults of the moth M.sexta.

Analogues of JH are currently being used for the control of a number of insect species in which the adult is the primary pest (Staal, G B et al, 1975, Annu Rev Entomol, 20, 417-460) but have not proved particularly useful for controlling the majority of crop-destroying insects. Those that have proved useful have been restricted in efficacy to a few species of Lepidoptera and have lacked sufficient potency for commercialisation.

The peptides provided by the present invention are suitable for control of insect development and thereby control of insect populations due to their ability to inhibit juvenile hormone production by CA from selected stages. Furthermore their activity, demonstrated herein on both P.americana and D.punctata provides a potential use in the control of insect populations of a range of orders of insect.

A first aspect of the present invention provides an isolated, enriched or purified peptide of Formula (I), equivalent to sequence ID No. 1 and 2 in the attached sequence listing

X-Arg-Leu-Tyr-Y-Phe-Gly-Z (I)

and conjugates thereof; wherein X is a sequence selected from S-Asp-Gly- and Q-Pro-Ser-Gly-R-Gln-; Y.Q.S and R are selected from single amino acid residues, and Z is an amidated leucine moiety; wherein Q and R are not both alanine when Y is glycine, and S is not glycine when Y is alanine.

Preferred peptides provided by the present invention are those of Formula I in which S and/or Y are selected from amino acids with hydrocarbon side chains and/or those in which R and Q are selected from sulphur containing amino acids. Particular peptides of the present invention are those in which Y is selected from glycine and alanine and/or R is an amino acid with a sidechain comprising a thioether eg. methionine and/or Q is an amino acid with a sidechain comprising a mercapto group eg. serine. Further particular peptides of the present invention are those in which Y and S are both alanine residues.

The present invention particularly provides an isolated, enriched or purified peptide of Formula (II), equivalent to sequence ID No. 3 and 4 in the attached sequence listings

X-Phe-Gly-Z (II)

and conjugates thereof; wherein X is a sequence selected from: Ala-Asp-Gly-Arg-Leu-Tyr-Ala- and Ser-Pro-Ser-Gly-Met-Gln-Arg- -Leu-Tyr-Gly- ; and Z is an amidated leucine moiety.

The term amidated leucine moiety will be understood to include sustituted leucine amide eg. N-methyl-leucine amide, substituted on the amide nitrogen, but preferred peptides of Formula (I) and Formula (II) are those in which the amidated leucine moiety is unsubstituted leucine amide.

It will be understood by those skilled in the art that some conjugates of the peptides of the present invention, having equivalent activity to the peptides, will also be useful as pesticides or as active agents in pesticidal compositions. Eg. the peptides may be conjugated to other peptides or to proteins. Particular conjugates that will have equivalent activity to the peptides of the present invention are their metabolic precursors.

A second aspect of the present invention provides the use of peptides for the control of insect development, particularly insects of the orders Periplaneta and/or Diploptera, more particularly insects of the species Periplaneta americana and/or Diploptera punctata.

A third aspect of the present invention provides compositions comprising:

i) A peptide of Formula (I) or a conjugate thereof as described above and

ii) an agriculturally or horticulturally acceptable carrier.

This aspect of the invention particularly provides compositions comprising:

i) A peptide of the Formula (II) or a conjugate thereof as described above and

ii) an agriculturally or horticulturally acceptable carrier.

Preferred compositions comprise peptides of Formula I or Formula II described as preferred above.

Preferred compositions contain carriers capable of holding peptides in homogenous form eg. in solution or suspension. Such carriers will be apparent to those skilled in the art and include compounds such as eg. dimethylsulphoxide (DMSO) which are capable of solubilising peptides. Alternatively vectors genetically manipulated to express the peptides or conjugates of the invention, may be used to enable plants to actively synthesise them for transfer to insect populations. Suitable vectors include viruses and plasmids but other examples will occur to those skilled in the art eg. bacteria or nematodes containing plasmids or viruses. The peptides of the present invention may also be formulated in an admixture with other pesticides, particularly insecticides, acaricides and/or nematocides.

A fourth aspect of the present invention is the preparation of peptides and conjugates described above. These may be prepared by extraction and purification from the brains of adult female pharate Periplaneta americana. by solid phase peptide synthesis or other known chemical techniques or alternatively by well known recombinant DNA techniques.

One suitable method for the preparation of the peptides comprises extraction from dissected, homogenised brains of adult female Periplaneta americana. using strongly acidic solvent such as 7 # ethanol in 0.2M HCl aqueous solution. The resulting crude extract may subsequently be separated into its components by fast liquid chromatography, to provide fractions with complex absorption spectra at 2l4nm. Providing a solvent concentration gradient during chromatography results in the peptides of the invention consistently eluting at particular solvent concentrations. Fractions containing the peptides of the invention may be determined by various methods, including the measurement of any Corpus allatum inhibitory activity and/or any allatostatin immunoreactivity. Once identified the fractions containing the peptides of the invention may be subjected to further chromatography in order to purify the peptides. Further details of suitable solvents and the fractions in which the peptides elute are provided in the examples.

Peptides and peptide conjugates of the present invention may also be produced using recombinant DNA technology, wherein naturally occurring, eg. P.americana. or synthetic DNA encoding for the active peptide or conjugate are identified, inserted into a suitable regulatory construct, eg. in a vector, and the construct is subsequently inserted into a host organism such as to enable that organism to express the peptide or conjugate.

Naturally occurring gene sequences may be identified by a number of techniques known to those skilled in the art, eg. Northern, Southern or Western Blotting using antibodies or probes targeted at the peptide or theoretically encoding sequence. For example, RACE PCR may be used to rapidly identify such genes ( Frohman et al (1988) Proc Nat Acad Sci USA Vol 85, 8998-9002) using degenerate primers. Synthetic or natural sequences may be identified as active peptide expressing by screening their host organisms, eg. E.coli transformed with them for production. Purified putative encoding DNA is ligated into a suitable vector,eg. pBluescript SK (Stratagene) and transformed into a host cell, eg. E.coli JM109 cells (Hanahan (1985) DNA cloning, A practical approach Vol 1 (Glover Ed) pp 109-135 IRL Press Oxford) . Expression may be identified by Northern blotting for RNA or by use of antibodies in Western blotting. Purification of the expressed protein or peptide may be carried out as is known in the art, eg. by affinity chromatography.

Suitable hosts for expression of the peptides, once the DNA sequence encoding them is determined, will be apparent to those skilled in the art, eg. yeast, bacteria, mammalian and fungal cells. DNA may be introduced into hosts by any suitable means eg. in the form of a plasmid or contained within a virus eg. a baculovirus. Transgenic plants comprising DNA encoding the peptides or conjugates of the invention may also be produced by inserting that DNA into a suitable . construct, and inserting that into an organism vector eg. Agrobacterium, (eg. by the methods described by Jones et al, Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in trangenic plants, Transgenic research 1, 285-297, (1992)). A further aspect of the present invention provides recombinant DNA encoding a peptide or peptide conjugate as described above and transgenic cells comprising such DNA. Transgenic cells may suitably be microbial cells, mammalian cells or plant cells. This aspect also provides transgenic plants comprising DNA encoding for a peptide or conjugate as discussed above, per se.

The peptides and methods of the present invention will now be illustrated by way of example only with reference to the following non-limiting examples. Further embodiments of the invention will occur to those skilled in the art in the light of these.

EXAMPLE 1: EXTRACTION AND PURIFICATION OF ALLATOSTATINS FROM ADULT FEMALE P.AMERICANA

The brains of adult female Periplaneta americana were dissected, homogenised and extracted with a strongly acidic solvent (75# ethanol in 0.2M HCl aqueous solution). The homogenates were centrifuged (4,000 x g, 20 min, 4°C) , the supernatants separated and the pellets re-extracted. After centrifugation the resultant acidic solution was diluted 6-fold with 0.1 TFA. An array of disposable reversed-phase extraction cartridges (Sep-Pak C_l8) was then used to remove fat from the crude extracts. Pilot studies revealed that up to 100 brain equivalents at a time could be applied to a single cartridge without significant washout or lack of retention of active materials.

All chromatographic separations were conducted using a Pharmacia"™ Fast Protein Liquid Chromatography (FPLC) system fitted with a pep-RPC 5/5 reversed-phase column. Gradient fractionation was achieved by means of a Pharmacia^R™ GP-250 programmer driving two P-500 pumps.

The eluted materials were detected by their absorbance at 214 nm. In order to prevent any oxidation of the peptide, 2-(methylthio)ethanol (0.1%) was added to the fractions at each purification step and the collection vessels were pre-loaded with 1 μg of HPLC purified oxidised insulin β-chain (Sigma) .

The first FPLC separation was performed under the following operating conditions: 100 solvent A for 5 min, a linear gradient to 10% B for 5 min, followed over the next 35 mins by a linear gradient (1%/min) from 10% B to 45% B; Solvent A, 0.1% trifluoroacetic acid (TFA) in water; Solvent B, 0.1% TFA in acetonitrile. Fractions were collected at one minute intervals and twenty six of these separations were required to process 4,400 brain equivalents of material. Allatostatin bioactivity had previously been shown to elute from such columns over a fairly broad range of increasing organic solvent concentrations (Weaver, R J et al, 1993. Insect Juvenile Hormone Research). However the increasing concentration of acetonitrile solvent of this separation eluted the peptides within a narrow region. The extraction was continued with the material eluting from the column at an acetonitrile concentration of between 28% and 30% (28-30 min) which showed biological activity when assayed against CA from day 4 virgin female P.americana.

The second FPLC separation was performed using a gradient of 1-propanol with 0.01% HCl. The pooled biologically active fractions from the first step were diluted 3^_fold with new solvent A, 0.01% HCl. Approximately 1000 brain equivalents at a time were pumped directly into the column, re-equilibrated with new solvent A.

Material was eluted from the column with a 35-min linear gradient of 0-30% 1-propanol in 0.01% HCl at a flow rate of 1 ml/min. A total of 16 peaks were observed and collected manually in each of these runs. Material eluting at 15% and 16% 1-propanol was found to possess demonstrable biological activity. The earlier eluting peak gave a weak positive response in the allatostatin RIA, whilst the later running peak gave a very strong positive response. The third FPLC separation utilised an identical reverse-phase column to that used in steps described above. The pooled, slower eluting (16% 1-propanol) active fractions from each of the previous runs were diluted 1:14 with 20 mM NH^OAc (pH 6.8) in 5% acetonitrile. This material was then carefully pumped into a column which had been previously pre-conditioned with the same solvent. The column was eluted with a 55-min linear gradient of 5~6θ% acetonitrile containing 20 mM NH_ή0Ac at a flow rate of 1 ml/min with the detector set at 0.1 AUFS. Periplaneta allatostatin 1 (Pea-AST-1) was recovered in the peak eluting at 29 min (31-7# acetonitrile). The carrier peptide B-insulin was the only major contaminant at this stage, at times almost co-running with Pea-AST-1. The presence of this "contaminant" was utilised in order to monitor the repetitive recovery by virtue of the fact the amount of added insulin was accurately known for each sample. It is to be assumed that the added insulin afforded some degree of loss prevention through otherwise unregulated and largely irreversible adsorptive processes.

An identical separation, using the pooled faster eluting (15% 1-propanol) fractions yielded three peptides one of which, eluting at 27 min (29.5% acetonitrile), was later characterised and designated Pea-AST 2.

The final FPLC separation step was a repeat of the first stage purification. Pooled active fractions from each the previous steps (ca.l ml) were diluted 1:10 with 0.1% aqueous TFA and pumped 2 mis at a time into the same FPLC column, re-equilibrated with 0.1% TFA. The column was eluted with the same solvent B (acetonitrile/0.1% TFA) and gradient conditions (10-45% B over 35 mi , 1 ml/min) as specified in the first step. In the first such run pure Pea-AST-1 was recovered in a single peak at 29 min. Two identical runs were required to purify Pea-AST-2, which finally eluted as a single peak at 28.5 min.

The β-insulin eluted separately from the peptide at this stage and the peptides obtained were of sufficient purity for further structural analysis. An overall yield of 2.3 nmol of each peptide was obtained from two large scale extractions from 4,500 adult female brains.

The peptides were sequenced with an Applied Biosystems model 477A pulsed liquid-phase protein sequencer coupled to an online phenylthiohydantoin analyser (Applied Biosystems model 120A) . Full sequence determination required 200-500 pmol of each peptide. Sequence analysis was undertaken at the Protein Sequencing Unit of the Department of Biochemistry at Royal Holloway and Bedford New College, Egham, Surrey. The molecular masses of the peptides were determined by M-Scan Ltd, Sunningdale, Ascot, Berkshire using positive ion fast atom bombardment mass spectrometry (FAB-MS) on a VG AutospecE double focusing mass spectrometer (VG Analytical, Manchester, UK). Amino acid analyses were conducted by M-Scan S.A., Geneva, Switzerland.

Two separate preparations of spectrally pure Pea AST-1, from 2000 and 2500 extracted brains, were used to elucidate the primary structure of this peptide. Automated Edman degradation of the first of these extracts indicated a thirteen amino acid peptide. The determined yields for selected, stable phenylthiohydantoin-amino acid derivatives versus sequencer cycle number were subjected to a linear regression analysis to determine the initial and average repetitive yield values for the analysis. These values were determined to be 150 pmol and 85% respectively. Positive ion FAB mass spectral analyses were subsequently conducted on small aliquots (15-200 pmol, respectively) of each of these two extracts using glycerol/thioglycerol as a FAB matrix. As a result of these analyses an average experimental [M+H]+ ion centred at m/z 1411.7 was obtained (the instrument being first optimised using a sample of vitamin B12 and calibrated with caesium iodide). This value is consistent with a molecular mass of 1410.7 and closely matches a theoretical value of 1410.67 obtained from analysis if amidation of the carboxy terminus is assumed. Peptide synthesis, liquid chromatography, Capillary Electrophoresis, and amino acid analyses were used to confirm purity and composition. The sequence was determined to be Ser-Pro-Ser-Gly-Met-Gln-Arg-Leu-Tyr-Gly-Phe- -Gly-Leu.

Similar techniques were used to analyse the peptide designated Pea- -AST-2 yielding the sequence Ala-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu. Amino acid content analysis confirmed the presence of the same amino acids: Ala (2), Arg (1), Asp (1), Gly (2), Leu (2), Phe (1), Tyr (1) and Fab-MS analysis showed a strong signal at m/z 1081.6. The molecular weight for the sequence indicated assuming the presence of an amidated carboxy terminus was calculated to be 1080.5- This measured [M+H]+ is thus consistent with the calculated mass for an amidated decapeptide of the sequence found and confirmed the structure of Pea-AST-2 to be Ala-Asp-Gly-Arg-Leu-Tyr-Ala-Phe-Gly-Leu-amide.

EXAMPLE 2: PEPTIDE SEQUENCE SYNTHESIS

A peptide having the sequence Ser-Pro-Ser-Gly-Met-Gln-Arg-Leu- -Tyr-Gly-Phe-Gly-Leu was synthesised with the carboxyl terminus in both amidated and free acid forms using standard automated solid-phase techniques (see eg. Barany, G et al, 1979. The peptides, eds Gross et al, Academic press, New York, Vol 2, pp 1-284). An Applied Biosystems model 430A automatic solid-phase peptide synthesiser (Charing Cross and Westminster Hospital Medical School Protein Synthesis Core Facility) was used and the synthetic peptides were purified by HPLC on preparative C_l8 reverse-phase columns.

Native Pea-AST-1 coeluted with the synthetic alpha-amidated form when co-injected in three different chromatographic systems. This was also shown to be the biologically active form of the peptide thus confirming the sequence analysis and proving that the carboxyl terminus is amidated. EXAMPLE : BIOLOGICAL ACTIVITY OF PEPTIDES

The biological activities of the synthetic amidated and free acid forms of synthetic Pea-AST-1 were compared with native Pea-AST-1 by measuring the degree of inhibition of juvenile hormone biosynthesis by CA from 4 day old adult female P.americana. The observed in vitro potency of the synthetic amidated form did not differ significantly from that of native Pea-AST-1 at three different inhibitor concentrations. By comparison, no detectable inhibition of JH synthesis was found when using the peptide lacking the carboxyl terminal amide, even at concentrations up to 1,000-fold higher than the highest concentration tested for the native peptide. Similar results were obtained with both native and synthetic Pea-AST-2.

The effects of Pea-AST-1 on juvenile hormone titres in P. mericana were tested using techniques described by Bergot, B J et al (198l, J Chromatog. , 204,, 231-244). Results show that the synthetic peptide is active when injected directly into the haemolymph of 4 day old virgin adult female P.americana and causes significant lowering of the measurable JH titre 12 hours postinjection. Juvenile Hormone III titre were found to be approximately 0.62 ng/g wet weight for those insects treated with lOOμg PeaASTl compared with 1.5 ng/g wet weight for those treated with the control or solvent alone. However no significant effect was observed when large quantities of this peptide were injected into the haemolymph of reproductively active mated adult female P.americana. a stage during which a very much higher basal level of JH titre than that found in 4 day old virgin females is routinely observed. In fact the average whole body titres of JH III in mid-cycle female P.americana are at least 18 times higher than equivalent titres in their younger counterparts. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: THE MINISTER OF AGRICULTURE FISHERIES AND

FOOD IN ETC

(B) STREET: WHITEHALL PLACE

(C) CITY: LONDON

(E) COUNTRY: UNITED KINGDOM

(F) POSTAL CODE (ZIP): SW1A 2HH

(A) NAME: EDWARDS John Patrick

(B) STREET: CENTRAL SCIENCE LABORATORY, LONDON ROAD

(C) CITY: SLOUGH

(D) STATE: BERKSHIRE

(E) COUNTRY: UNITED KINGDOM

(F) POSTAL CODE (ZIP): SL37HJ

(A) NAME: WEAVER Robert James

(B) STREET: CENTRAL SCIENCE LABORATORY, LONDON ROAD

(C) CITY: SLOUGH

(D) STATE: BERKSHIRE

(E) COUNTRY: UNITED KINGDOM

(F) POSTAL CODE (ZIP): SL37HJ

(ii) TITLE OF INVENTION: PESTCIDES (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release No. 1.0,Version No. 1.25(EP0) (vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: GB 9313891-5

(B) FILING DATE: 05-JUL-1993

2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Periplaneta americana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Xaa Asp Gly Arg Leu Tyr Xaa Phe Gly Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Periplaneta americana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Xaa Pro Ser Gly Xaa Gin Arg Leu Tyr Xaa Phe Gly Leu 1 10

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Periplaneta americana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Ala Asp Gly Arg Leu Tyr Ala Phe Gly Leu 1 5 10

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Periplaneta americana

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ser Pro Ser Gly Met Gin Arg Leu Tyr Gly Phe Gly Leu 1 10

Claims

1. An isolated, enriched or purified peptide of Formula (I)

X-Arg-Leu-Tyr-Y-Phe-Gly-Z (I)

and conjugates thereof; wherein X is a sequence selected from S-Asp-Gly- and Q-Pro-Ser-Gly-R-Gln-; Y,Q,S and R are selected from single amino acid residues, and Z is an amidated leucine moiety; wherein Q and R are not both alanine when Y is glycine, and S is not glycine when Y is alanine.

2. A peptide or conjugate as claimed in Claim 1 wherein Y is selected from glycine and alanine.

3. A peptide or conjugate as claimed in Claim 1 or Claim 2 wherein R is a methionine residue.

4. A peptide or conjugate as claimed in Claim 1 or Claim 2 wherein Q is a serine residue.

5. A peptide or conjugate as claimed in Claim 1 or Claim 2 wherein S is an alanine residue.

6. A peptide of the Formula (II)

X-Phe-Gly-Z (II)

and conjugates thereof; wherein Z is an amidated leucine moiety and X is a sequence selected from Ser-Pro-Ser-Gly-Met-Gln-Arg-Leu-Tyr-Gly- and Ala-Asp-Gly-Arg-Leu-Tyr-Ala-.

7. A peptide or conjugate as claimed in any one of Claims 1 to 6 wherein Z is leucine amide.

8. Use of a peptide or conjugate as claimed in any one of Claims 1 to 7 for the control of insect development.

9. Use of a peptide or conjugate as claimed in any one of Claims 1 to 7 for the control of populations of Periplaneta and/or Diploptera.

10. Use of a peptide or conjugate as claimed in any one of Claims 1 to 7 for the control of populations of Periplaneta americana and/or Diploptera punctata.

11. A composition comprising:

i) A peptide or conjugate as claimed in any of Claims 1 to 6 and

ii) an agriculturally or horticulturally acceptable carrier.

12. A composition as claimed in Claim 11 wherein the agriculturally or horticulturally acceptable carrier comprises dimethylsulphoxide.

13- A composition comprising a vector genetically modified to include DNA encoding for a peptide or peptide conjugate as claimed in any one of Claims 1 to 7 together with an agriculturally or horticulturally acceptable carrier.

14. A composition as claimed in Claim 13 wherein the gentically modifed vector is a virus or is contained within a bacteria or a nematode.

15- A composition as claimed in any one of Claims 11 to 14 further comprising at least one additional pesticide.

16. A composition as claimed in Claim 1 wherein the additional pesticide is selected from insecticides, acaricides and/or nematocides.

17. A method for the preparation of peptides or conjugates as claimed in any one of Claims 1 to 7 comprising extraction and purification from cockroach brains.

18. A method as claimed in Claim 17 wherein the insects are of the species Periplaneta americana.

19. A method as claimed in Claim 17 substantially as that described in Example 1.

20. A method for the preparation of peptides or conjugates claimed in any one of Claims 1 to 7 comprising solid phase peptide synthesis.

21. A method for the preparation of peptides or conjugates claimed in any one of Claims 1 to 7 comprising the insertion of naturally occurring or synthetic DNA encoding for the active peptide or conjugate into a suitable regulatory construct, and subsequent insertion of the construct into a host organism such as to enable that organism to express the peptide or conjugate.

22. A method as claimed in Claim 21 wherein the host organism is selected from yeast, bacteria, mammalian or fungal cells.

23. A transgenic cell comprising DNA encoding for a peptide or conjugate as claimed in any one of Claims 1 to 7-

24. A transgenic plant comprising DNA encoding for a peptide or conjugate as claimed in any one of Claims 1 to "J .

25. Recombinant DNA encoding a peptide or peptide conjugate as claimed in any one of Claims 1 to 7-