Journal of Insect Physiology 46 2000 1249–1257 www.elsevier.comlocatejinsphys
Acid phosphatases in the haemolymph of the desert locust, Schistocerca gregaria, infected with the entomopathogenic fungus
Metarhizium anisopliae
Y. Xia, P. Dean, A.J. Judge
1
, J.P. Gillespie
2
, J.M. Clarkson, A.K. Charnley
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK Received 13 August 1999; accepted 19 January 2000
Abstract
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
β -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.
2000 Elsevier
Science Ltd. All rights reserved.
Keywords: Metarhizium anisopliae; Schistocerca gregaria; Haemocytes; Acid phosphatase; Insect immunity; Fungal enzymes
1. Introduction
Insect fungal pathogens invade their hosts through the external skeleton. A battery of extracellular cuticle
degrading proteases and chitinases facilitate passage through the integument and provide nutrition for the
fungus Charnley and St Leger, 1991; St Leger, 1995. Once in the insect, Deuteromycete pathogens like Met-
arhizium anisopliae proliferate to a greater or lesser extent in the haemolymph, where they may be confined
Corresponding author: Tel.: +
44-1225-323011; fax: +
44-1225- 826779.
E-mail address: bssakcbath.ac.uk A.K. Charnley.
1
Present address: Department of Immunology, University of Birmingham Medical School, Edgbaston, Birmingham B15 2TT, UK.
2
Present address: Department of Biochemistry, University of Bris- tol, Bristol BS8 1TD, UK.
0022-191000 - see front matter
2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 2 - 1 9 1 0 0 0 0 0 0 4 5 - 7
prior to host death. Efficient use of nutrients in the hae- molymph while combating the insect’s blood-borne
defences will be critical to successful parasitism. Good evidence implicates pathogen hydrolases in penetration
of the exoskeleton, but the role of extracellular fungal enzymes in haemolymph has received little attention.
Cuticle degrading subtilisin fungal proteases such as PR1 have powerful general proteolytic activity St Leger
et al., 1986a. Unfortunately for the fungus they are gen- erally absent from the haemolymph due to repression by
low molecular weight compounds.
Simple sugars principally the diasaccharide trehalose and phosphorylated sugars e.g. glucose-1-phosphate
are found at high concentration in the haemolymph. While repressive for fungal proteases they are a readily
available source of carbon for the fungus, though utilis- ation may depend on secretion of the appropriate hydro-
lase. The necessary enzymes,
α glucosidasetrehalase
1250 Y. Xia et al. Journal of Insect Physiology 46 2000 1249–1257
and acid phosphatase, are secreted in culture by M. ani- sopliae St Leger et al., 1986b; Xia et al., unpublished.
Attempts to identify fungal enzymes in insect haemo- lymph must take into account the fact that defence
related proteins, including enzymes, may be produced by the host in response to the fungal invasion. The insect
immune system is comprised of a battery of humoral and cellular defences that can interact in the destruction of
pathogens reviewed by Gillespie et al., 1997. The first line of defence in the haemolymph is often phagocytosis.
This is carried out primarily by the plasmatocytes. Co- operative attack by species of insect haemocytes can lead
to immobilisation of groups of pathogens within granu- lomas nodules and the encapsulation of larger invaders.
So called humoral defences such as production of mel- anin and antimicrobial peptides also play their part.
The study of invertebrate haemocyte cell killing mechanisms is fragmentary; lysozyme has been well
studied but there is little information on other hydrolases Anderson and Cook, 1979; Zachary and Hoffmann,
1984. Recently there have been a number of reports on the cytochemistry of the blood of oysters and mussels
Alvarez et al., 1995; Anderson et al., 1992; Anderson, 1994. The few studies on insects have concentrated on
the characterisation of naı¨ve haemolymph Chain and Anderson, 1983; Miranpuri et al., 1991. Phagocytosis
is known to stimulate production of lysosomal enzymes of which acid phosphatase AcP is a key component.
AcP has been found in insect haemocytes and shown to be released into the plasma Lai-Fook, 1973; Rowley
and Ratcliffe, 1979. Cheng 1983 reported hypersynth- esis of acid phosphatase by haemocytes of the mollusc
Biomphalaria
glabrata during
phagocytosis. The
enzyme was subsequently released into the plasma where its role is unknown though alteration of surface
properties of foreign particles by the enzyme may aid immunological recognition though a direct role of AcP
in cell killing can not be ruled out.
We showed recently that mycosis of the desert locust with M. anisopliae var acridum resulted in changes in
the properties of the haemolymph that occurred in two stages Gillespie et al., 2000. During the first stage, in
the first 2 days after inoculation, there was an increase in total haemocyte count which was due in large part
to an increase in coagulocytes, the number of nodules and prophenoloxisase pPO activity. All of these para-
meters were shown to change with respect to the control values when there was no or only a minor presence of
fungus in the haemolymph. This suggests that there is a “signal” which is either a host derived molecule
released from the integument during fungal penetration or a soluble fungal metabolite that activates the immune
system. It is apparent that, whilst the immune system is stimulated by the fungus, the impact of the cellular host
defenses on M. anisopliae var acridum is minimal since the haemocytes remain unattached to fungal particles
and there is no indication that nodules incorporate fungus.
The second stage of the infection process occurred when the fungus had entered the haemocoel and repli-
cated extensively 3–4 days after inoculation. At this time, all parameters measured apart from pPO were at
levels significantly below those of controls and mycosed locusts in stage 1 of infection. This may be because the
immune system has now been overcome by the fungus or fungally-derived metabolites.
The object of the present study was to determine the effect of fungal infection on haemolymph acid phospha-
tase in the desert locust. It was hoped to be able to dis- tinguish any effect of infection on host derived acid
phosphatase, which would be part of the immune response, and enzyme secreted by the fungus to promote
host invasion.
2. Materials and methods
