WO2006027571A2

WO2006027571A2 - Insect sucrase

Info

Publication number: WO2006027571A2
Application number: PCT/GB2005/003435
Authority: WO
Inventors: Angela Douglas; David Ashford
Original assignee: University of York
Current assignee: University of York
Priority date: 2004-09-08
Filing date: 2005-09-06
Publication date: 2006-03-16
Anticipated expiration: 2007-03-08
Also published as: WO2006027571A3

Abstract

We describe an isolated nucleic acid molecule that encodes a polypeptide with sucrase activity and the use of this polypeptide as a target to identify agents with sucrase inhibitory activity.

Description

Nucleic Acid Molecule

The invention relates to a nucleic acid molecule that encodes a polypeptide with sucrase activity and the use of said nucleic acid and/or polypeptide in screening assays for the identification of agents, typically insecticidal agents, which inhibit sucrase activity.

The Hemiptera or "true bugs" are divided into two sub-orders, the Homoptera and Heteroptera. Members of this order are all characterised by having piercing and sucking mouth parts that are adapted for feeding on the juices of plants and animals. Many of these insects are important agricultural and horticultural crop pests. These include a wide range of homopteran bugs, such as many of the whiteflies (Aleyrodoidea), the aphids (Aphidoidea), the mealybugs and scale insects (Coccoidea), the jumping plant lice (Psylloidea), and some of the froghoppers or spittlebugs, treehoppers (Cercopoidea), planthoppers (Fulgoroidea) and leafhoppers (Cicadelloidea), cicadas (Cicadoidea); as well as several heteropteran species, particularly among the Cimicimorpha, e.g. squash bugs (Coreidae), the capsid bugs (Miridae), and Pentatomomorpha, e.g. the shield bugs or stink-bugs (Pentatomidae) .

The aphids are abundant pests and cause severe damage to plants,. Aphids have natural predators, the best known of which is the ladybird. However, the primary control of aphid infestation is through the use of aphicides, for example pyrethroid insecticides, such as α-cypermethrin or deltamethrin that have a rapid knockdown effect on aphids.

For many phloem-feeding Homopteran insects, including aphids, whitefly and psyllids, the high sugar content of phloem sap in most plants provides an abundant source of carbon but simultaneously presents an osmotic challenge. The total solute composition, and therefore phloem osmotic potential, is high (-0.6 to -3.0 MPa) and can vary considerably between and within plants (Downing, 1978; Wilkinson et al., 1997; Ponder et al., 2000; Fisher, 2000). The osmotic potential of the imbibed phloem sap could be up to three-fold higher than the aphid haemolymph, which is remarkably stable over a wide concentration range of dietary sucrose (e.g. approx. - 1.0 MPa for Myzus persicae and Acyrthosiphon pisum: Downing, 1978; Fisher et al., 1984; Wilkinson et al., 1997). Furthermore, honeydew tends to be isosmotic with the haemolymph (Downing, 1978; Fisher et al., 1984; Wilkinson et al., 1997) suggesting that the osmotic potential of the phloem sap is modified as it travels through the gut. Efficient metabolism of phloem sugars, mainly sucrose, in the insect gut is considered key to this regulation for aphids and other insects feeding on phloem.

Although the osmotic potential of aphid honeydew is relatively constant, its carbohydrate composition is dependent upon the concentration of sugar ingested, and frequently includes sugars of higher molecular weight than the imbibed sucrose (Bacon and Dickinson, 1957; Baron and Guthrie, 1960; Fisher et al., 1984; Walters and Mullin, 1988; Rhodes et al., 1997; Wilkinson et al., 1997). The presence of tri- and oligosaccharides in the honeydew indicates a degree of sucrose modification in the insect gut, and suggests that regulation of the osmotic potential difference between the aphid gut contents and body fluids is associated with the lower osmotic potential per-unit-weight of oligosaccharides compared to sucrose.

The presence of sucrose hydrolysis and transglucosidase activities in the insect gut has been determined for pea aphids {Acyrthosiphon pisum: Ashford et al., 2000; Cristofoletti et al., 2003) and potato aphids (Macrosiphwn euphorbiae: Walter and Mullins, 1988), but the role of these enzyme activities in aphid osmoregulation on high osmolality diets requires proof.

We have isolated and cloned a novel nucleic acid molecule that encodes a polypeptide with sucrase activity.

The use of a specific α-glucosidase inhibitor to manipulate enzyme activity in the gut of feeding aphids has shown that inhibition of this gut sucrase inhibits oligosaccharide synthesis and that this imposes an increased osmotic stress on feeding aphids resulting in aphid death. The sucrase represents a target for the screening of agents that inhibit gut sucrase enzyme activity and therefore agents identified through this screen will have utility as insecticides and, in particular, aphicides.

According to an aspect of the invention there is provided an isolated nucleic acid molecule comprising a DNA sequence selected from the group consisting of: (i) a nucleic acid molecule consisting of the DNA sequence as represented in Figure 5; (ii) a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above and which encode a polypeptide with sucrase enzyme activity; and

(iii) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA, sequence defined in (i) and (ii).

In a preferred embodiment of the invention there is provided an isolated nucleic acid molecule that anneals under stringent hybridisation conditions to the sequences described in (i), (ii) and (iii) above.

Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours

Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: Ix SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize) Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55⁰C for 20-30 minutes each.

In a preferred embodiment of the invention said nucleic acid molecules are isolated from insect nucleic acid.

Preferably said insect nucleic acid is isolated from insects of the order Hemiptera.

Preferably said nucleic acid is isolated from nucleic acid of the sub-order Homoptera or Heteroptera.

hi a preferred embodiment of the invention said insect nucleic acid is isolated from the genus: Aleyrodoidea; Aphidoidea; Coccoidea; Psylloidea; Cicadoidea; Fulgoroidea; Cicadelloidea; Cercopoidea; Cimicomorpha; Pentatomomorpha

hi a preferred embodiment of the invention said nucleic acid is cDNA. In an alternative preferred embodiment of the invention said nucleic acid is genomic DNA.

According to a further aspect of the invention there is provided a polypeptide encoded by a nucleic acid molecule according to the invention.

In a preferred embodiment of the invention said polypeptide is a variant polypeptide and comprises the amino acid sequence represented in Figure 5, which sequence has been modified by deletion, addition or substitution of at least one amino acid residue wherein said modification retains or enhances the enzyme activity of said polypeptide.

A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies.

hi addition, the invention features polypeptide sequences having at least 75% identity with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof, hi one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequences illustrated herein. According to an aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the invention.

In a preferred embodiment of the invention said vector is adapted for the recombinant expression of said nucleic acid molecule.

A vector including nucleic acid (s) according to the invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome for stable transfection.

Preferably the nucleic acid in the vector is operably linked to an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi- functional expression vector which functions in multiple hosts.

By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter.

hi a preferred embodiment the promoter is a constitutive, an inducible or regulatable promoter. According to a further aspect of the invention there is provided a cell transfected or transformed with a nucleic acid molecule or vector according to the invention.

Preferably said cell is a eukaryotic cell. Alternatively said cell is a prokaryotic cell.

Li a preferred embodiment of the invention said cell is selected from the group consisting of; a fungal cell (e.g. Pichia spp, Saccharomyces spp, Neurospora spp); insect cell (e.g. Spodoptera spp); a mammalian cell (e.g. COS cell, CHO cell); a plant cell.

In a preferred embodiment of the invention said cell is a plant cell. Preferably said plant cell is part of a plant or seed.

According to a further aspect of the invention there is provided a method to manufacture a polypeptide according to the invention comprising: i) providing a cell according to the invention; ii) incubating said cell under conditions conducive to the production of said polypeptide; and optionally iii) isolating said polypeptide from said cell or the growth media surrounding said cell.

hi a preferred method of the invention said polypeptide is provided with an amino acid affinity tag to facilitate the isolation of said polypeptide.

Affinity tags are known in the art and include, maltose binding protein, glutathione S transferase, calmodulin binding protein and the engineering of polyhistidine tracks into proteins that are then purified by affinity purification on nickel containing matrices. Li many cases commercially available vectors and/or kits can be used to fuse a protein of interest to a suitable affinity tag that is subsequently transfected into a host cell for expression and subsequent extraction and purification on an affinity matrix. In our co-pending PCT application, PCT/GB2004/002432 (currently unpublished, the content of which is incorporated by reference in its entirety), we describe an affinity tag isolated from a plant lipase that has affinity for lipid membranes, for example oil bodies. This tag may be added to the polypeptide of the invention to facilitate its purification from cells recombinantly expressing the polypeptide of the invention.

According to an aspect of the invention there is provided the use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of the DNA sequence as represented in Figure 5 ; ii) a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above and which encode a polypeptide with sucrase enzyme activity; and iii) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in (i) and (ii), as a target for the identification of agents with sucrase enzyme inhibitory activity.

According to a further aspect of the invention there is provided a screening method for the identification of an agent that has sucrase enzyme inhibitory activity comprising the steps of: i) providing a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule consisting of the DNA sequence as represented in Figure 5 ; b) a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above and which encode a polypeptide with sucrase enzyme activity; and c) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in (i) and (ii). ii) providing at least one candidate agent to be tested; iii) forming a preparation that is a combination of (i) and (ii) above; and iv) testing the effect of said agent on the enzyme activity of said sucrase.

In a preferred method of the invention said method includes the additional step of testing the insecticidal activity of said candidate agent.

In a further preferred method of the invention said insecticidal activity is aphicidal activity.

In a preferred method of the invention said polypeptide comprises the amino acid sequence as shown in Figure 5, or sequence variant thereof, wherein said variant is modified by deletion, addition or substitution of at least one amino acid residue.

Preferably said polypeptide consists of the amino acid sequence shown in Figure 5.

In a preferred method of the invention said agent is a sugar analogue.

According to a further aspect of the invention there is provided a method to determine the ability of a molecule to associate with a sucrase polypeptide comprising the steps of: i) providing computational means to perform a fitting operation between said molecule and a polypeptide defined by the amino acid sequence in Figure 5; and ii) analysing the results of said fitting operation to quantify the association between the molecule and the sucrase polypeptide.

The rational design of binding entities for proteins is known in the art and there are a large number of computer programs that can be utilised in the modelling of 3- dimensional protein structures to determine the binding of chemical entities to functional regions of proteins and also to determine the effects of mutation on protein structure. This may be applied to binding entities and also to the binding sites for such entities. The computational design of proteins and/or protein ligands demands various computational analyses which are necessary to determine whether a molecule is sufficiently similar to the target protein or polypeptide. Such analyses may be carried out in current software applications, such as the Molecular Similarity application of QUANTA (Molecular Simulations Inc., Waltham, Mass.) version 3.3, and as described in the accompanying User's Guide, Volume 3 pages. 134-135. The Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e., moving structures). When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure.

The person skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target. The screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within that binding pocket.

Useful programs to aid the person skilled in the art in connecting the individual chemical entities or fragments include: CAVEAT (P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules". In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)). CAVEAT is available from the University of California, Berkeley, California. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, California). This is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2145-2154 (1992); and HOOK (available from Molecular Simulations, Burlington, Mass.). These citations are incorporated by reference. Once the ligand has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. The computational analysis and design of molecules, as well as software and computer systems therefor are described in US Patent No 5,978,740 which is included herein by reference.

In a preferred method of the invention said sucrase polypeptide is a modified sucrase polypeptide wherein said modification is to the binding site of said molecule and wherein said modification is the addition, deletion or substitution of at least one amino acid residue such that the binding affinity and/or specificity of said molecule for said binding site is altered.

In a further preferred method of the invention said molecule is modified to alter its binding affinity and/or specificity for said sucrase polypeptide.

In a preferred method of the invention said molecule is an antagonist for said sucrase polypeptide.

In a further preferred method of the invention said sucrase polypeptide or modified sucrase polypeptide is encoded by a nucleic acid molecule as represented in Figure 5, or a nucleic acid molecule that hybridises under stringent hybridisation conditions to said nucleic acid molecule and encodes a polypeptide with sucrase activity.

hi a preferred method of the invention said modified sucrase is modified in a sugar binding site.

In a further preferred method of the invention said molecule is a sugar analogue. According to a further aspect of the invention there is provided a method for the rational design of mutations in sucrase polypeptides comprising the steps of: i) providing a 3D model of a first polypeptide as represented by the amino acid sequence in Figure 5; ii) providing a 3D model of a variant polypeptide wherein said variant polypeptide is a modified sequence variant of said first polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue in Figure 5; iii) comparing the effect on the 3D model of said second polypeptide when compared to the 3D model of said first polypeptide; optionally iv) testing the effect of said modification on the enzyme activity of said second polypeptide when compared to said first polypeptide.

In a preferred method of the invention said modified second polypeptide is modified in a sugar binding site.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following Figures:

Figure 1 illustrates the inhibition of sucrase activity in aphid gut homogenates. (A) Effect of acarbose, deoxynojirimycin and deoxygalactonojirimycin concentrations. (B) Effect of acarbose on the activity of disaccharidase. Values represent the mean+s.e. of n=3. Curves indicate non-linear regression fits (see text) and broken lines indicate 50% inhibition of enzyme activity;

Figure 2 illustrates the impact of dietary acarbose on aphid survival. Two dietary sucrose concentrations, (A) 0.2 M sucrose and (B) 0.75 M sucrose, were used and the starting number of aphids was identical for each treatment (n=9);

Figure 3 illustrates the impact of dietary acarbose on the carbohydrate composition of aphid honeydew. Aphids fed for 48 h on (A, B) 0.2 M or (C, D) 0.75 M dietary sucrose and in the presence of 0-5 μM acarbose. (A, C) Profile of ¹⁴C-radioactivity

(dpm) eluted showing position of standards (G=glucose, F=fructose, S=sucrose,

(^oligosaccharides. (B, D) Carbohydrate composition of honeydew as a percentage of total radioactivity present. Values represent the mean ± s.e. (n=3, except for 0.5 μM data where n=2) of the percentage of dpm recovered from dietary [U-¹⁴C]- sucrose as monosaccharides, disaccharides and oligosaccharides;

Figure 4 illustrates the impact of dietary acarbose on the haemolymph osmotic potential of aphids. Aphids fed for 48 h on 0.2 M or 0.75 M dietary sucrose. Values represent the mean ± s.e. of n=5-8 replicates apart from the datapoint at 5 μM acarbose on 0.75 M sucrose for which n=2; and

Figure 5 is the nucleic acid and amino acid sequence of Acyrthosiphon pisum sucrase. Materials and methods

Cloning and Expression of a Putative Sucrase from the Aphid Acythrosiphon pisum

The formation of glucose polymers from sucrose is an ability shared by a number of bacterial species. Certain bacteria, notably Leuconostoc mesenteroides, Streptococcus spp. and lactic acid bacteria contain glucansucrases (EC 2.4.5.1) which catalyse the formation of various polymers of D-glucose (both straight-chain and branched, with varying linkages present) from sucrose (Monchois et al, 1999), a process similar to that occurring in the aphid gut, although the degree of polymerisation observed is much higher (up to hundreds of residues, vs. up to 10 residues in the insect). These enzymes contain two domains, a catalytic domain of approx. 900 amino acids, and a C-terminal glucan binding domain of approx. 500 amino acids. The catalytic domain is a member of family 70 of glycosyl hydrolases. No glycosyl hydrolases of this family have been found in insects (or other eukaryotes) to date, and these enzymes were not considered as possible orthologues of aphid sucrase.

A more promising candidate for similarity to aphid sucrase is the amylosucrase of Neisseria polysaccharea (with similar enzymes occurring in other bacterial spp.).

Amylosucrase (EC 2.4.1.4) produces a linear polymer of a-l,4-linked glucose from sucrose, with concomitant release of fructose (Skov et al, 2000). It can also act as a sucrose hydrolase, and can produce maltose and maltotriose (Potocki de Montalk et al, 2000). This protein contains 636 amino acids, and contains a single alpha- amylase type catalytic domain (IPR006047). The protein is a member of glycohydrolase family 13; this family is represented in all kingdoms, with many examples in insects.

The predicted proteins from the genome of Drosophila melanogaster were compared with the amino acid sequence of the amylosucrase of Neisseria polysaccharea

(accession number AAM51153) using BLAST software. There was significant similarity between the amylosucrase and a distinct set of Drosophila proteins. E values for this sub-set of proteins were ≤K)^"17, whereas E values for other predicted proteins were >10^"2. These proteins were: CG8690, CG8693, CG8694, CG8695, CG8696, CGl 1669, CG14934, CG14935-PA/PB, CG30359, CG30360-PA/PB; of these CG14935 was the most similar, with an E-value of 3xlO^~26. A phylogenetic tree produced by comparison of the amino acid sequences of these proteins using the Clustal method is shown in fig. 1(A). All the D. melanogaster proteins are annotated as α-glucosidases or maltases, but in no case has the function been experimentally verified. It was therefore hypothesised that a family 13 glycohydrolase present in the aphid gut could well have been adapted to produce glucose polymers, since the family 13 glycohydrolase in bacteria like N. saccharea clearly has this capacity. Regions of strong sequence conservation in the D. melanogaster proteins similar to amylosucrase, and the amylosucrase itself, were identified using a sequence alignment (Clustal method, Megalign software). Results are summarised in Table 1.

Preparation of probes for putative A. pisum sucrase cDNA clones using PCR

(i) Primers based on amylosucrase/maltase sequences (glycohydrolase family 13) The regions of conserved amino acid sequence between Drosophila maltase sequences and amylosucrase identified by multiple sequence alignment were used to predict the sequences of primers for PCR (taking codon usage in the Drosophila genes into account); primers are listed in Table 1.

(U) Preparation of RNA templates

RT-PCR experiments used total RNA extracted from dissected aphid guts, or whole insects, as a template. Whole insects were collected from plants, frozen in liquid nitrogen and ground while frozen prior to extraction, using Tri reagent (Sigma Genosys) or an RNAeasy kit (Qiagen) according to the suppliers' instructions. Dissected guts could not be treated in the same way, as severe degradation of RNA took place due to difficulties in handling. Instead guts were collected into "RNAlater" solution (Ambion Inc.; www.ambion.com), frozen, and RNA was extracted using the RNAeasy kit as before. RNA from both guts and whole insects was of good quality, with minimal degradation, as assessed by formaldehyde- formamide agarose gel electrophoresis (see fig. 2A). For some experiments, polyA+ RNA was used as a template; this was prepared from total RNA using the PolyATract system (Promega) according to the protocols supplied.

(Ui) RT-PCR of A. piswa RNA using amylosucrase/'maltase primers In initial experiments a single gene specific primer (forward) was used in combination with an oligo-dT primer (reverse) with an extra base at the 3' end (A,C or G in separate primers). A one-step RT-PCR protocol was followed (Access RT- PCR system; Promega). Amplification of both total and polyA+ RNA from whole insects and guts (40 cycles) using either primer 1 or primer 2 (based on amino acids 204-212 and 224-229 of the final A. pisum putative sucrase sequence respectively; see Table 1 and fig. 3) gave disperse products; reamplifϊcation with specific combinations of gene-specific and oligo-dT primers gave no specific products. This approach was not pursued further.

A second set of experiments was carried out using total RNA and polyA+ RNA from whole insects. In these experiments, a reverse gene-specific primer (primer 5, Table 1; based on amino acids 206-212 of the final A. pisum putative sucrase sequence; see fig. 4) was used in combinantion with one of two forward gene specific primers. Primer 3 (amino acids 92-97) was used for the initial amplification, and primer 4 (amino acids 130-135) was a nested primer, used for re-amplification of any initial product. Total RNA extracted from Drosophila (whole insects) was used as a positive control, hi these experiments, first strand cDNA was synthesised from RNA, using an oligo-dT primer, and reverse transcriptase. The cDNA was purified, and used as a template in the PCR reactions. Amplification of Drosophila total RNA, A. pisum total RNA and A. pisum polyA+ RNA (25 cycles) with primers 3 and 5 under these conditions resulted in the production of a specific product of approx. 400 bp. This product could be reamplified, after excision from gel and purification, with primers 4 and 5, to give a product of approx. 300 bp (fig. 2B). The 400 bp PCR product was cloned using the TOPO-TA cloning method (mvitrogen), and the resulting plasmids were characterised by DNA sequencing. The amplified sequence had a high level of sequence similarity to the appropriate region of the D. melanogaster "maltase" gene CGl 4935, and thus encoded part of a putative A. pisum sucrase. The sequence of this PCR prooduct is incorporated in the composite sequence figure (fig. 3).

(iv) RACE to obtain putative A. pisum sucrase cDNA

The sequence of the cDNA encoding A. pisum sucrase was completed using the

Rapid Amplification of cDNA Ends (RACE) procedure. 5' and 3' RACE ready cDNAs were prepared from total RNA from Drosophila (whole insect), A. pisum (whole insect) and A. pisum (dissected guts), using a Clontech SMART RACE cDNA amplification kit (www. BDbiosciences.com), according to the protocols supplied. The cDNAs were amplified using gene-specific primers 6 and 7 (Table 1) for A. pisum sucrase, and the generic primers supplied for 5' and 3' RACE. A. pisum templates gave RACE products of the expected size (fig. 2C); the 5'RACE product was approx. lOOObp, and the 3' RACE product was approx. 1600 bp. The RACE products were cloned and characterised by DNA sequencing. The sequences matched the earlier PCR product, and could be assembled into a complete sequence for the sucrase cDNA (see fig. 3).

Preparation and screening of A. pisum cDNA libraries

(i) Preparation of RNA from aphid guts and whole insects

The preparation of total and polyA+ RNA from whole aphids and dissected aphid guts is described above (section 2iii).

(U) Preparation and verification of cDNA libraries Initial attempts to produce a cDNA library used a SMART cDNA synthesis and cloning kit (Clontech; www.Bdbiosciences.com), following the manufacturer's protocols. The resulting double-stranded cDNA was cloned into the vector used by this system, λ Triplex2. Total RNA from dissected guts was used as the template for library construction, as purification of polyA÷ RNA resulted in a template that gave poor yields of low molecular weight first strand cDNA. However, although this method gave libraries containing large numbers of recombinant phage, the inserts cloned were predominantly <500bp. A number of randomly selected clones were subjected to DNA sequencing as a quality check, and were predominantly sequences derived from mitochondria or bacterial symbionts, or A-T rich random sequence. It was concluded that either gut RNA is highly deficient in polyA+ RNA, or is highly unstable under the extraction conditions, and, after several attempts, preparation of a gut-specific library was not pursued further.

PoIyA+ RNA purified from total RNA extracted from whole aphids was a good template for first strand cDNA synthesis, and gave a product with a size distribution from approx. 200-5000 bases (maximum staining intensity approx. 700 bases). This cDNA was made double stranded and checked by cloning into a plasmid vector (pCR 2.1, TOPO TA cloning method). Random clones were sequenced, and showed a high proportion of coding sequences with similarity to Drosophila gene products. Cloning into the SMART system vector, λ Triplex2, did not occur at high efficiency. The procedure was thus repeated using the Stratagene ZAP-cDNA synthesis kit (www.stratagene.com), according to the manufacturer's protocols. The double- stranded cDNA was cloned into the λ ZAP II vector, resulting in a library of 5.2 x 10⁶ independent clones prior to amplification. Quality control was carried out by random selection of clones, excision, and sequencing of inserts. Average insert size was >1000bp, and most sequences contained good open reading frames with similarity to database sequences. The λ ZAP II cDNA library was used for subsequent work.

(Ui) Screening cDNA libraries for clones encoding sucrase(s) The whole insect cDNA library was screened by hybridisation (final wash conditions; IxSSC, where SSC is 0.15M NaCl, 0.015 M sodium citrate buffer, pH 7.0, 0.1% SDS, 65⁰C) to phage plated on four 20x20cm plates in duplicate. Plugs containing positive plaques were re-screened after plating out at appropriate dilutions, and repeated until single plaques could be picked off unambiguously. In an initial screen, the 400bp PCR product encoding a section of a putative A. pisum sucrase (section 2iv) was used as a probe. This probe showed strong hybridisation to clones encoding a protein with homology to Drosophila cuticular proteins, and did not select the A. pisum "sucrase" cDNA. The screening was therefore repeated using the complete coding sequence of the putative A. pisum sucrase (as obtained by RACE; section 2v) as a probe. After plaque purification, 5 positive clones were obtained. These contained inserts varying in size from 1200 bp to 2000 bp. All the cDNA clones agreed with the composite sequence assembled by 5' and 3' RACE, and shown in fig. 3. This sequence contains a complete coding sequence (1773 nt), 102 nt of 5'UTR, 147 nt of 3' UTR (including a polyA signal sequence at nt 1994-1999) and a poly(A) "tail".

Analysis of the protein encoded by the putative A. pisum sucrase cDNA

The predicted A. pisum putative sucrase protein has 590 amino acids; the first 21 amino acids are predicted to comprise a signal peptide (SignalP v. 3.0 software, www.cbs.dta.dk/semces/SignalP/), giving a mature protein of 569 amino acids, predicted Mr 66,372. There are 4 predicted N-glycosylation sites, at residues 18, 134, 169 and 454, but the first of these is in the signal peptide and thus cannot be used in the mature protein. The protein contains two distinct domains. The N-terminal domain is relatively hydrophilic, and comprises residues 12 - 476 of the mature protein (33 - 497 in fig. 3). This corresponds to the glycohydrolase family 13 alpha amylase catalytic domain (TPR006047 / SSF51445; Interpro numbering; www.ebi.ac.uk). The residues necessary for catalytic activity are present (Asp catalytic nucleophile, residue 236; GIu catalytic proton donor, residue 304). The C- terminal domain is relatively hydrophobic, and comprises residues 478-562 of the mature protein (499 - 583 in fig. 3). This corresponds to an alpha amylase C-terminal beta sheet domain (SSF51011; Interpro numbering). The two domains are illustrated graphically by a hydrophobicity plot in fig. 4. The mosquito Culex pipiens contains an α-glucosidase which is similar in sequence to the putative A. pisum sucrase, and which is normally anchored to the gut membrane by a C-terminal glycosylphosphatidylinositol (GPI) moiety (Silva-Filha et ah, 1999; Darboux et ah, 2001); removal of the C-terminal region results in the protein being secreted in soluble form (Darboux et ah, 2002). The sequence similarity between C. pipiens α-glucosidase and the putative A. pisum sucrase is not as strong in the C-terminal domain as in the N-terminal catalytic domain, and the sucrase is not predicted to contain a GPI-anchor site (big-PI predictor; mendel.imp.univie.ac.at/mendeljsp/index.jsp); however, it is possible that the relatively hydrophobic C-terminal domain of the putative sucrase may result in the enzyme being associated with membranes.

hi order to determine whether A. pisum contained multiple genes encoding proteins similar to the putative sucrase, genomic DNA was prepared from whole aphids, using a Sigma GenElute DNA extraction kit (www.sigma-aldritch.com) according to the manufacturer's protocols. Approx. 2 μg portions of the resulting DNA were digested with different restriction enzymes, and the resulting fragments were separated by agarose gel electrophoresis and subjected to Southern blotting. The resulting blot was hybridised with a probe prepared by labelling a DNA fragment corresponding to the complete coding sequence of the putative sucrase, and washed to a stringency of 0. IxSSC at 65°C. The resulting blot (fig. 5) gave single bands for 3 of the restriction enzymes tested (BamH I, EcoR I and Xba I) and three bands for the fourth (Hind IE); none of these enzymes have sites in the sequence determined for the putative sucrase cDNA. Given the likelihood of introns being present in the corresponding gene, the results are consistent with a single "sucrase" gene being present.

Assembly of expression constructs for putative A. pisum sucrase

The complete predicted mature coding sequence for the putative A. pisum sucrase (residues 22-590 in fig. 3) was amplified using primers containing additional 5' Pst I and 3' Xho I restriction sites (primers 8 and 9, Table 1). The resulting PCR product was cloned in to pCR 2.1 and checked to ensure that no sequencing errors were present. The insert was excised by digestion with Pst I and Xho I, purified by agarose gel electrophoresis, and ligated into the yeast expression vector pGAPZαB (Invitrogen), which had been digested with Pst I and Sal I. The ligation was transformed into E. coli, and a resulting clone was rechecked by sequencing to ensure the correct construct was present. The recombinant pGAPZ plasmid containing the sucrase coding sequence was linearised by digestion with Bin I, and putified linear DNA was used to transform chemically competent cells of Pichia pastoris strain X33 (Invitrogen; manufacturer's protocols for transformatiuon followed). Transformed yeast cells were plated on zeocin-containing media (50 μg/ml) for selection.

Putative transformants were screened by colony PCR, and positives from the PCR assay were grown as small-scale cultures and assayed by immunodot-blot assay of culture supernatant. Ten clones were assayed, of which 8 gave apsitive signal with anti-(his tag) antibodies. The best expressers from this assay were selected for further study.

Expression and purification of putative A. pisum sucrase

A selected clone of Pichia pastoris expressing the recombinant putative sucrase was grown for four days in shake flask culture (in rich medium), and the culture supernatant was precipitated with ammonium sulphate to 90% saturation. The resulting pellet was collected, redissolved in 2M NaCl and purified by chromatography on phenyl-Sepharose (2M - OM gradient in NaCl), collecting the peak of protein that eluted in water, follwed by nickel affinity chromatography. Fractions were analysed by SDS-PAGE. The peak from the phenyl-Sepharose column contained a polypeptide of mol. wt. approx. 60 kDa as the major product, but this product did not bind to the nickel affinity column, despite giving a reaction with anti-(his tag) antibodies on Western blot.

This procedure was scaled up by growing the Pichia clone expressing the putative sucrase in a 2 litre laboratory fermenter, in a minimal medium. Under these conditions, the phenyl-Sepharose purification step led to very poor recovery of protein, with most of the recombinant protein from the culture supernatant binding irreversibly to the column. Small amounts of 60 kDa product, which reacted with anti-(his tag) antibodies as before were eluted in the water wash. As an alternative, the culture supernatant was diluted four-fold, brought to pH 3.6, and purified by ion exchange chromatography on a column of S-Sepharose equilibrated with 5OmM sodium-acetate buffer, pH 3.6. Under these conditions the putative sucrase was retained on the column, and was eluted in the concentration range 0. IM - 0.4M NaCl by a salt gradient, as a broad peak. Analysis by SDS-PAGE showed a single band of protein at approx. 60 kDa in all peak fractions (fig. 6). This method gave a much higher yield of protein, estimated as approx. 2.5mg per litre of culture.

An attempt to purify the recombinant protein by ion-exchange chromatography at pH 7.4 in the presence of 0.1% Triton X-100 was unsuccessful; the column gave very poor resolution, and all fractions were contaminated with trehalase, a known extracellular protein in yeasts. The insects

A clonal culture, UY2, of the pea aphid Acyrthosiphon pisum was derived from a single alate female collected in 1993 from Pisum sativum. Parthenogenetic cultures were maintained on Vicia faba cv. The Sutton with 16 h L: 8 h D at 16 ⁰C for gut preparations and at 20 ⁰C for performance experiments; aphids raised at the lower temperature are larger and thus yield more gut material for enzyme assay.

Sucrase assay of gut preparations

Alimentary tracts from oesophagus to anus were dissected from adult apterous aphids, in the presence of 0.9% NaCl, with the aid of fine needles. The guts were disrupted in ice-cold 0.9% NaCl using a small hand-held plastic pestle, and the homogenates were stored at -20 ⁰C prior to use. Sucrase activity of gut homogenates was assayed as described previously in (Ashford et al., 2000). Sucrase and other disaccharidase activities were assayed essentially as described by Dahlqvist (1984) using reagents from a Sigma Diagnostics glucose assay kit. The recommended concentration of the chromogen, o-dianisidine, (40 μg/ml) was found to be limiting in some experiments and was therefore increased to 125 μg/ml. One unit of sucrase is defined as the amount of enzyme that releases lμmol glucose from the relevant substrate per minute at 37⁰C. For inhibition assays, a constant amount of homogenate containing 0.65 mU sucrase was assayed in the presence of 500 pM to 5 mM inhibitor. The compounds tested were two known α— glucosidase inhibitors, acarbose and deoxynojirimycin (DNJ) and, as a control, deoxygalactonojirimycin (DGJ), an inhibitor of α-galactosidase.

The honevdew and haemolymph of feeding aphids

Experimental material was 7 d old final instar nymphs raised from day 2 on chemically-defined diet of formulation A (Prosser and Douglas, 1992), containing 0.5 M sucrose, 0.15 M amino acids, vitamins, minerals and organic acids, and buffered with KH₂PO₄ to give a final pH of 7.5. Seven-day-old nymphs were then transferred to test diets containing either 0.2 or 0.75 M sucrose, and acarbose inhibitor at a concentration of 0, 0.1, 0.5, 1 or 5 μM. Diets were administered in sachets stretched over a Perspex ring (2.5 cm diameter, 0.7 cm height), according to the experimental details described below.

The radiolabeled inulin technique (Wright et al., 1985; Wilkinson and Ishikawa, 1999) was used to quantify aphid feeding rate; ³H-inulin (8 μCi ml^'1: Calbiochem) was administered to 7 d old aphids orally via chemically-defined diets as described above. Honeydew was collected on 25 mm GF/A Whatmann filters was dissolved by placing the filters in vials containing 4 ml Ultima Gold™ XR scintillation fluid

(Packard Bioscience B. V., Grδningen, The Netherlands). Radioactivity was determined by scintillation counting in a Packard Tri-Carb Liquid Scintillation

Analyzer using a pre-set ³H window. Feeding rate was quantified by comparison with the specific activity of the radiolabeled diet, with aphids feeding from replicate non-radioactive sachets included as controls. Diet sachets containing only water and radiolabeled inulin were included as a further control. Aphid survival on the test diets was monitored daily and feeding was assessed every 2 days throughout the experimental period.

For the analysis of honeydew composition, radiolabeled [U-¹⁴C]-sucrose (Amersham, UK) was checked for purity by thin layer chromatography and was included in the diets (specific activity of 8 μCi ml^"1). Aphids feeding from replicate non-radioactive sachets were included as controls. Honeydew collected over the 24- 48 h period of aphid feeding was dissolved in sterile de-ionised water and stored at - 20⁰C prior to analysis.

Aphid haemolymph was collected from aphids that had been feeding for 48 h from the test diets described above. Aphids were submerged in water-saturated paraffin oil and a single middle leg was removed from the aphid using forceps. Exuding haemolymph was collected under the oil using silanised microcapillaries and samples were stored in the capillaries at -20 ⁰C until analysis.

Chemical analyses

Honeydew sugars from aphids fed on 14C-labelled sucrose diets were separated as described in Ashford et al (2000) by normal phase HPLC on a GlycoSep N column (Oxford Glycosciences, UK) using an Alliance HPLC system with Millennium software (Waters). The column was equilibrated with 20% water, 80% acetonitrile at 6O⁰C and flow rate 1 ml/min. On injection, these conditions were maintained for 12 min. The proportion of water was then increased in a single step to 30% and a linear gradient from 30% to 80% water was applied over 25 min. [U-14C]-glucose (Amersham, UK), [U- 14C] -sucrose and the products of [U-14C]-sucrose hydrolysed at 70 ⁰C in the presence of 0.5 M HCl for 1 h were used as standards. During HPLC, 0.38 ml fractions of the column effluent were collected in a 96 well Lumaplate (Packard) and, following drying under vacuum, the radioactivity in each fraction was determined by scintillation counting on a Packard TopCount NXT microplate scintillation counter.

The osmotic potential of 0.05-0.5 nl samples of aphid haemolymph was determined by freezing-point depression (Malone and Tomos, 1992) calibrated against 0-0.6 mol I^"1 NaCl standard.

Statistical analyses

The IC₅₀ values for inhibition of in vitro disaccharidase activity were obtained from four-parameter logistic curve fits to the data using SPSS SigmaPlot v8.02 (Novell Inc., US). Parametric statistical tests were applied to datasets confirmed to be normally-distributed (Ryan- Joiner one-sample test) with homogeneous variances (Bartlett's test), following logarithmic or arcsin-square root transformation where indicated. The impact of diet composition on aphid feeding rate, honeydew composition and haemolymph osmotic potential was tested using ANOVA or, where non-parametric analyses were required, using Kruskall-Wallis and Mann- Whitney tests. Post-hoc analysis was by Tukey's pairwise comparison.

The survival (number of days lived) of aphids on test diets containing 0.2 M or 0.75 M sucrose and 0.5, 1 and 5 μM acarbose was assessed using generalised linear modelling available in the GLHvI statistical package (survival analysis: see Crawley, 1993). The influence of diet composition was explored using a Weibull model, which assumes an age-dependent mortality risk and can incorporate a linear combination of explanatory variables (in this case, the presence of a range of acarbose concentrations in the diet). The Weibull model was fitted firstly without incorporating the explanatory variable, then a second model fitting including the explanatory variable. The change in deviance (which approximates χ²) when the explanatory variable was added to the model was used to assess the significance of the effect of acarbose treatment on survival. The percentage change in the deviance provides a measure of the explanatory power of the model

EXAMPLE 1

Impact of α-glycosidase inhibitors on gut sucrase activity in vitro

Two of the 3 tested compounds were effective at inhibiting glucose release by the aphid gut sucrase (Fig. IA). Acarbose was the most potent inhibitor (IC₅₀ = 5.9xlO^"8 M) followed by deoxynojirimycin (DNJ: IC₅₀ = 3.4xlO^"5 M); inhibition by deoxygalactonojirimycin (DGJ) was low even at millimolar concentrations (IC₅₀ = 7.1IxIO^"2 M). Between 50% and 100% inhibition was achieved by acarbose in the nano-to-micro-molar range of concentrations. Both sucrase activity (0.035+0.003 μmol min^"1 mg^"1 protein) and maltase activity (0.017+0.0002 μmol min^"1 mg^"1 protein) were present in the gut extracts (Fig. IB), and acarbose was equally effective at inhibiting the gut maltase (IC₅₀ = 4.7x10^"8 M). Gut trehalase activity was not inhibited by acarbose (Fig. IB).

EXAMPLE 2

Impact of dietary acarbose on aphid survival and feeding

The 7 d old aphids settled and initially fed on all diets, as indicated by honeydew production. Aphid feeding on the water control diet was extremely low and aphid death due to starvation on this diet took between 4 and 7 days (Table 1). By contrast, survival was high for aphids feeding on the 0.2 M and 0.75 M sucrose diets (Fig. 2). However, inclusion of acarbose in the diet increased aphid mortality in a concentration-dependent manner (Table 1; Fig. 2A, B). The most severe effects were observed at the highest acarbose concentration (5 μM), which significantly reduced aphid survival at both sucrose concentrations, but particularly on the 0.75 M sucrose diet (Table 1; Fig. 2B). Aphid mortality data for 0 and 0.1 μM acarbose treatments could not be included in the survival analysis due to low aphid mortality on these treatments during the experimental period.

After 2 d of exposure to the test diets, aphid feeding rate on the 0.75 M sucrose diet was less than half the rate on the 0.2 M diet (for all acarbose treatments combined, median of 0.40 and 0.97 μl aphid^"1 d^"1, respectively: Mann Whitney U-test of In- transformed data: W_45;4s =2563.0, pO.OOl). Inclusion of acarbose had no significant effect on feeding rate on the 0.2 M sucrose diet (Table 1). By contrast, post-hoc analysis indicated that feeding rate was significantly reduced by 1-5 μM acarbose on the 0.75 M sucrose diet (Table 1).

EXAMPLE 3

Impact of dietary acarbose on honeydew composition

Dietary [U-¹⁴C]-sucrose was incorporated into honeydew monosaccharides (glucose and fructose), disaccharides, oligosaccharides and an unknown peak that eluted before fructose (Fig. 3A, C). Consistent with previous studies (Wilkinson et al., 1997; Ashford et al., 2000), in the absence of dietary acarbose, aphids on diet containing 0.2 M sucrose produced honeydew composed mainly of monosaccharides (53-80%; Fig. 3A, B), while oligosaccharides (80-85%) dominated the honeydew of aphids feeding on 0.75 M sucrose (Fig. 3C, D).

The sugar composition of honeydew was altered dramatically by dietary acarbose (Fig. 3B, D). At both dietary sucrose concentrations, honeydew of aphids ingesting acarbose contained elevated amounts of disaccharides (Kruskal-Wallis analysis of arcsin-square root transformed data for 0.2 M sucrose diet: H₄=8.86, pO.l; Kruskal- Wallis analysis of arcsin-square root transformed data for 0.75 M sucrose diet: H₄=8.43, p<0.1), and this was at the expense of monosaccharides in the 0.2 M sucrose diet (Kruskal-Wallis test of arcsin-square root transformed data: H₄=8.32, p<0.1: Fig. 3B), and of oligosaccharides in the 0.75 M sucrose diet (Kruskal-Wallis test of arcsin-square root transformed data: H₄=7.94, p<0.1: Fig. 3D). In addition, a small increase in oligosaccharide production was noted at intermediate acarbose concentrations on the 0.2 M sucrose diet (Fig. 3B). EXAMPLE 4

Impact of dietary acarbose on haemolymph osmotic potential

The osmotic potential of the two diets reflected the difference in sucrose concentration (average values of -0.7 and -2.6 MPa for 0.2 M and 0.75 M sucrose diets, respectively), hi the absence of acarbose, haemolymph osmotic potential after 48 h of feeding was similar on the 0.2 M and 0.75 M sucrose (2-sample t-test: T₁₂ =- 1.27, p>0.1; Fig. 4). Haemolymph osmotic potential of aphids feeding on 0.2 M sucrose diet was not significantly altered by the presence of acarbose (ANOVA of In- transformed data: F₃₎₂₇ =1.23, p>0.1; Fig. 4). By contrast, the osmotic potential on 0.75 M sucrose diet became significantly more negative on diets containing >0.1 μM acarbose (Tukey's post-hoc analysis following ANOVA of In-transformed data: F_3j28 =6.85, p<0.001: Fig. 4). Note that aphids feeding on 5 μM acarbose on the 0.75 M sucrose diet were dehydrated and pure haemolymph samples were difficult to extract; hence the number of replicates was small (n=2) and the measurements at this acarbose concentration were excluded from the statistical analysis.

References

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Baron, R. L., Guthrie, F. E., 1960. A quantitative and qualitative study of sugars found in tobacco as affected by the green peach aphid, Myzus persicae, and its honeydew. Annals of the Entomological Society of America 53, 220-228. Byrne, D. N., Miller, W. B., 1990. Carbohydrate and amino acid composition of phloem sap and honeydew produced by Bemisia tabaci. Journal of Insect Physiology 36, 433-439. Cristofoletti, P. T., Ribeiro, A. F., Deraison, C, Rhabe, Y., Terra, W. R., 2003. Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum. Journal of Insect Physiology 49, 11-24.

Darboux, I. , Nielsen-LeRoux, C. , Charles, J.-F. & Pauron, D. (2001) The receptor of Bacillus sphaericus binary toxin in Culexpipiens (Diptera: Culicidae) midgut: molecular cloning and expression. Insect Biochem. MoI. Biol. 31: 981-990 Darboux, L, Pauchet, Y., Castella, C, Silva-Filha, M.H., Nielsen-LeRoux, C,

Charles, J.-F. and Pauron, D. (2002). Loss of the membrane anchor of the target receptor is a mechanism of bioinsecticide resistance. Proc. Natl. Acad.

ScL USA 99: 5830-5835.

Downing, N, 1978. Measurements of the osmotic concentrations of stylet sap, haemolymph and honeydew from an aphid under osmotic stress. Journal of Experimental Biology 77, 247-250. Duspiva, F., 1955. Enzymatische Prozesse bei der Honingtaubuldung der Aphiden. Verleichende Deutsche Zoologie Gesellschaft 18, 40-447. Fisher, D. B., 2000. Long distance transport. In: Buchanan, B. B., Gruissem, W., Jones, R. L., (Eds), Biochemistry and Molecular Biology of Plants. American Society of Plant Physiologists, Rockville, MD., pp. 730-784.

Fisher, D. B., Wright, J. P., Mittler, T. E., 1984. Osmoregulation by the aphid Myzus persicae: a physiological role for honeydew oligosaccharides. Journal of

Insect Physiology 30, 387-393.

Monchois, V., Willemot, R. -M. and Monsan, P. (1999). Glucansucrases: mechanism of action and structure-function relationships. FEMS Microbiology Rev. 23: 131-151. Ponder, K. L., Pritchard, J., Harrington, R., Bale, J. S., 2000. Difficulties in location and acceptance of phloem sap combined with reduced concentration of phloem amino acids explain lowered performance of the aphid Rhopalosiphum padi on nitrogen deficient barley (Hordeum vulgare) seedlings. Entomologia Experimentalis et Applicata 97, 203-210. Potocki de Montalk, G., Remaud-Simeon, M., Willemot, R.-M., Sarcabal, P., Planchot, V. and Monsan, P. (2000). Amylosucrase from Neisseria polysaccharea: novel catalytic properties. FEBS Letts. 471: 219-223.

Prosser, W. A., Douglas, A. E., 1992. A test of the hypotheses that nitrogen is recycled and upgraded in an aphid (Acyrthosiphon pisum) symbiosis. Journal of Insect Physiology 38, 93-99.

Rhodes, J. D., Croghan, P. C, Dixon, A. F. G., 1997. Dietary sucrose and oligosaccharide synthesis in relation to osmoregulation in the pea aphid, Acyrthosiphon pisum. Physiological Entomology 22, 373-379.

Silva-Filha, M. H. , Nielsen-LeRoux, C. & Charles, J.-F. (1999) Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae). Insect Biochem. MoI. Biol. 29: 711-721

Skov, L.K., Mirza, O., Henriksen, A., Potocki de Montalk, G., Remaud-Simeon, M.,

Sarcabal, P., Willemot, R.-M., Monsan, P. and Gajhede, M. (2000). Amylosucrase, a glucan-synthesising enzyme from the α-amylase family. J.

Biol. Chem. 276: 25273-25278. Walter, F. S., Mullin, C. A., 1988. Sucrose-dependent increase in the oligosaccharide production and associated glycosidase activities in the potato aphid Macrosiphum euphorbiae (Thomas). Archives of Insect Biochemistry and Physiology 9, 35-46.

Wilkinson, T. L., Ashford, D. A., Pritchard, J., Douglas, A. E. 1997. Honeydew sugars and osmoregulation in the pea aphid Acyrthosiphon pisum. Journal for Experimental Biology 200, 2137-2143.

Wilkinson, T. L., Ishikawa, H., 1999. The assimilation and allocation of nutrients by symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum. Entomologia

Experimentalis et Applicata 91, 195-201.

Winter, H., Lohaus, G., Heldt, H. W., 1992. Phloem transport of amino acids in relation to their cytosolic levels in barley leaves. Plant Physiology 99, 996- 1004. Wright, J. P., Fisher, D. B., Mittler, T. E., 1985. Measurement of aphid feeding rates on artifical diets using H-3-inulin. Entomologia Experimentalis et Applicata 37, 9-11.

Table l:Impact of dietary acarbose on aphid feeding and survival. Measurements were taken after 48 h of feeding from 0.2 M or 0.75 M sucrose diets. Values represent mean (±s.e.) for each test diet (n=9) or from a water control (n=7). Mean age at death of aphids is shown for treatments that were amenable to survival analysis (see text for explanation).

Feeding rate Survival (d)

(μl aphid^"1 d^"1)

Acarbose 0.2 M sucrose 0.75 M sucrose 0.2 M 0.75 M

(μM) sucrose sucrose

(Water) 0 ^a0.03 (+0.02) ^a0.03 (±0.02) 6.6 (±0.4) 6.6 (±0.4)

(Sucrose diet) 0.77 (±0.27) 0.56 (±0.06) >10^a >10^a n

0.1 0.91 (±0.17) 0.48 (±0.06) >10^a >10^a

0.5 0.59 (±0.17) 0.54 (±0.05) 8.2 (±0.5) 9.4 (±0.5)

1.0 1.09 (±0.10) 0.19 (±0.09) 7.2 (±0.5) 4.1 (±0.5)

5.0 0.85 (±0.15) 0.22 (±0.04) 4.6 (±0.5) 2.8 (±0.5)

Kruskal- ANOVA: Deviance = Deviance = Wallis test: F₄₎₄₄ =8.90 36.8 (3 df) 70.5 (3 df) H₄=4.41 p>0.1 pO.OOl pO.OOl pO.OOl %Dev = 28.4 %Dev = 45.4

^aData not included in statistical analysis.

Claims

1. An isolated nucleic acid molecule comprising a DNA sequence selected from the group consisting of: i) a nucleic acid molecule consisting of the DNA sequence as represented in Figure 5; ii) a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above under stringent hybridisation conditions and which encode a polypeptide with sucrase enzyme activity; and iii) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in (i) and (ii).

2. A nucleic acid molecule according to Claim 1 wherein said nucleic acid is isolated from insect nucleic acid.

3. A nucleic acid molecule according to Claim 2 wherein said insect nucleic acid is isolated from an insect of the order Hemiptera.

4. A nucleic acid molecule according to Claim 3 wherein said nucleic acid is isolated from an insect of the sub-order Homoptera or Heteroptera.

5. A nucleic acid according to Claim 4 wherein said nucleic acid is isolated from an insect of the taxa Aleyrodoidea; Aphidoidea; Coccoidea; Psylloidea;

Cicadoidea; Fulgoroidea; Cicadelloidea; Cercopoidea; Cimicomorpha; Pentatomomorpha.

6. A polypeptide encoded by a nucleic acid molecule according to any of Claims 1-5.

7. A polypeptide according to Claim 6 wherein said polypeptide is a variant polypeptide and comprises the amino acid sequence represented in Figure 5, which sequence has been modified by deletion, addition or substitution of at least one amino acid residue wherein said modification retains or enhances the enzyme activity of said polypeptide.

8. A vector comprising a nucleic acid molecule according to any of Claims 1-5.

9. A vector according to Claim 8 wherein said vector is adapted for the recombinant expression of said nucleic acid molecule.

10. A cell transfected or transformed with a nucleic acid molecule or vector according to any of Claims 1-5 or 8 or 9.

11. A cell according to Claim 10 wherein said cell is a eukaryotic cell.

12. A cell according to Claim 10 wherein said cell is a prokaryotic cell.

13. A cell according to Claim 11 wherein said cell is a plant cell.

14. A plant comprising a cell according to Claim 13.

15. A seed comprising a cell according to Claim 13.

16. A method to manufacture a polypeptide comprising: i) providing a cell according to any of Claims 10-13; ii) incubating the cell under conditions conducive to the production of the polypeptide; and optionally iii) isolating the polypeptide from the cell or the growth media surrounding the cell.

17. A method according to Claim 16 wherein polypeptide is provided with an amino acid affinity tag to facilitate the isolation of the polypeptide.

18. The use of a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule consisting of the DNA sequence as represented in Figure 5; ii) a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above and which encode a polypeptide with sucrase enzyme activity; and iii) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in (i) and (ii), as a target for the identification of agents with sucrase inhibitory activity.

19. A screening method for the identification of an agent that has sucrase inhibitory activity comprising the steps of: i) providing a polypeptide encoded by a nucleic acid molecule selected from the group consisting of: a. a nucleic acid molecule consisting of the DNA sequence as represented in Figure 5; b. a nucleic acid molecule comprising DNA sequences that hybridise to the sequence identified in (i) above and which encode a polypeptide with sucrase enzyme activity; and c) a nucleic acid molecule comprising DNA sequences that are degenerate as a result of the genetic code to the DNA sequence defined in (i) and (ii). ii) providing at least one candidate agent to be tested; iii) forming a preparation that is a combination of (i) and (ii) above; and iv) testing the effect of said agent on the enzyme activity of said sucrase.

20. A method according to Claim 19 wherein the method includes the additional step of testing the insecticidal activity of said candidate agent.

21. A method according to Claim 20 wherein said insecticidal activity is aphicidal activity. '

22. A method according to any of Claims 19-21 wherein said polypeptide comprises the amino acid sequence as shown in Figure 5, or sequence variant thereof wherein said variant is modified by deletion, addition or substitution of at least one amino acid residue.

23. A method according to Claim 22 wherein said polypeptide consists of the amino acid sequence shown in Figure 5.

24. A method to determine the ability of a molecule to associate with a sucrase polypeptide comprising the steps of: i) providing computational means to perform a fitting operation between said molecule and a polypeptide defined by the amino acid sequence in Figure 5; and iii) analysing the results of said fitting operation to quantify the association between the molecule and the sucrase polypeptide.

25. A method according to Claim 24 wherein said sucrase polypeptide is a modified sucrase polypeptide wherein said modification is to the binding site of said molecule and wherein said modification is the addition, deletion or substitution of at least one amino acid residue such that the binding affinity and/or specificity of said molecule for said binding site is altered.

26. A method according to Claim 24 or 25 wherein said molecule is modified to alter its binding affinity and/or specificity for said sucrase polypeptide.

27. A method according to any of Claims 24-26 wherein said molecule is an antagonist for said sucrase polypeptide.

28. A method according to any of Claims 24-27 wherein said sucrase polypeptide or modified sucrase polypeptide is encoded by a nucleic acid molecule as represented in Figure 5, or a nucleic acid molecule that hybridises under stringent hybridisation conditions to said nucleic acid molecule and encodes a polypeptide with sucrase activity.

29. A method according to any of Claims 25-28 wherein said modified sucrase polypeptide is modified in a sugar binding site.

30. A method according to any of Claims 24-29 wherein said molecule is a sugar analogue.

31. A method for the rational design of mutations in sucrase polypeptides comprising the steps of: i) providing a 3D model of a first polypeptide as represented by the amino acid sequence in Figure 5; ii) providing a 3D model of a variant polypeptide wherein said variant polypeptide is a modified sequence variant of said first polypeptide which is modified by addition, deletion or substitution of at least one amino acid residue in Figure 5; iii) comparing the effect on the 3D model of said second polypeptide when compared to the 3D model of said first polypeptide; optionally iv) testing the effect of said modification on the enzyme activity of said second polypeptide when compared to said first polypeptide.

32. A method according to Claim 31 wherein said modified second polypeptide is modified in a sugar binding site.