采用再生几丁质亲和层析和两性电解质等电聚焦电泳，纯化了扁豆荚几丁酶，其分子量30kD，等电点为9.1，主要呈内切酶活性。扁豆荚中不同组织几丁酶比活力有很大差异。在豆荚发育过程中，其酶活性变化呈单峰曲线，而比活力则持续上升，表明扁豆几丁酶活性变化与发育相关。经HgCl2处理后，扁豆荚壳和种子的几丁酶活性均明显提高。在扁豆荚发育的不同阶段，几丁酶的诱导特性也有明显差异，幼嫩豆荚几丁酶诱导活性的增加更为明显。

Trehalose is the main haemolymph sugar in most insects including the tobacco hornworm, Manduca sexta, and is potentially a prime target for an invading pathogenic fungus. There was considerably more trehalose-hydrolysing activity in the haemolymph of caterpillars infected with Metarhizium anisopliae than in controls. This appeared to be due primarily to additional isoforms; one of which could also hydrolyse maltose and was designated an a-glucosidase. A comparable isoform was identified in in vitro culture of the fungus, supporting a fungal origin for the in vivo enzyme. The in vitro fungal enzyme, a-glucosidase-1 (a-gluc-1), was purified to homogeneity and partially characterised. A study with the trehalase inhibitor trehazolin and C14 trehalose suggested that extracellular hydrolysis is important for fungal mobilisation of trehalose. Haemolymph glucose increases significantly during mycosis of tobacco hornworm larvae by M. anisopliae, consistent with the hydrolysis of trehalose by extracellular fungal enzymes. The implications for the host insect are discussed.

Aneutral trehalase gene (NTH1) was isolated from a kEMBL3 genomic library of the insect pathogenic fungus Metarhizium anisopliae. Sequencing of the gene revealed extensive homology with other fungal neutral trehalases. The NTH1 gene exists as a single copy in the genome. Two STREs exist in the 50UTR of NTH1, which may mediate transcriptional activation of the NTH1 gene in response to various stresses. The NTH1 gene encodes a protein of 737 amino acids with a calculated Mr of 83.1kDa. Acyclic adenosine 30,50-monophosphate-dependent phosphorylation consensus site and a putative calcium binding site were found in the amino-terminal domain of NTH1, consistent with a regulatory enzyme. Expression of the trehalase cDNAwas achieved in Saccharomyces cerevisiae. Southern blot analysis of RT-PCR products indicated that the neutral trehalase gene is transcribed in vitro in cell-free haemolymph of the tobacco hornworm Manduca sexta and in vivo in the early stage of infection.

A comparison has been made between the effects of wounding, chemical stimulation of the immune system and fungal infection on acid phosphatase (AcP) activity in the haemolymph of the desert locust, Schistocerca gregaria. Untreated control locusts had constitutive levels of AcP. As a lysosomal enzyme, AcP may have a role in autophagy and cell turn over as well as defence. Injection of saline and b-1,3-glucan caused significant increases in haemocyte and plasma AcP. AcP activity also increased in the haemolymph on the 3rd day after inoculation with the entomopathogenic fungus M. anisopliae var acridum. This coincided with a decline in the total haemocyte count and a marked reduction in the proportion of plasmatocytes and coagulocytes that stained positive for AcP. Therefore a priori it seemed unlikely that the extra AcP in infected insects came from the host. A fungal origin for the enzyme was suggested by the identification of AcP isoforms from haemolymph of different treatments. Control inoculated (oil only) insects had an AcP at a pI of 4.3 that was stimulated further by the injection of laminarin. Additional isoforms appeared at around 7.3–7.5 in the laminarin treatment. However, the 4.3 isoform appeared to be suppressed in the insects infected with M. anisopliae var acridum. The band intensity was more like that of the control than the laminarin-injected insects. Two new isoforms appeared later on in infection. These enzymes had pIs that corresponded to some of the AcPs produced in vitro by the fungus. The results are discussed in the light of the possible benefits of secreted fungal acid phosphatases to the pathogen.