Insect Biochemistry and Molecular Biology 31 2001 57–63 www.elsevier.comlocateibmb
A new form of arthropod phenoloxidase is abundant in venom of the parasitoid wasp Pimpla hypochondriaca
Neil Parkinson , Ian Smith, Robert Weaver, John P. Edwards
Central Science Laboratory, Sand Hutton, York YO41 1LZ, UK Received 13 March 2000; received in revised form 22 May 2000; accepted 25 May 2000
Abstract
We have recently identified phenoloxidase PO activity among several biologically active factors in venom from the parasitoid wasp Pimpla hypochondriaca. We have now isolated three genes, designated POI, POII and POIII, from a cDNA library made
from venom-producing glands and found that their products are related to pro-phenoloxidases PPOs, which are expressed as proenzymes in haemocytes and which mediate immune processes in arthropods. This is the first report of PO as a venom constituent.
Amino acid sequence comparisons between the three Pimpla POs and PPOs revealed several notable differences, including the absence of sequences which specify the site of proteolytic activation in insect PPOs and the unprecedented occurrence of signal
peptide sequences. NH
2
-terminal amino acid analysis of PO purified from venom yielded a peptide sequence matching the predicted mature NH
2
termini of POI and POII, confirming the authenticity of the signal peptide and indicating that proteolytic processing, other than to remove the signal peptide, does not occur in the wasp. Expression of POI, analysed by Northern hybridization, was
approximately uniform from the time of adult emergence to day 6 post-emergence, after which it declined. A novel means of host immune suppression, mediated by the unregulated activity of venom PO in the haemocoel, is proposed.
2001 Elsevier Science
Ltd. All rights reserved.
Keywords: Pimpla hypochondriaca ; hymenoptera; parasitoid; venom; phenoloxidase; cDNA
1. Introduction
Parasitoid wasps are important regulators of insect populations and comprise a large proportion of hymen-
opteran species Quicke, 1997. Their eggs are laid on or inside the host, which is subsequently used as a food
source by the developing parasitoid larva. Endoparasito- ids, whose eggs are deposited within the haemocoel,
must contend with host immune responses that are cap- able
of encapsulating
and destroying
artificially implanted eggs Feddersen et al., 1986. To circumvent
this response the female stings its host, injecting ovarian secretions which contain factors that neutralize host
immune function. These host conditioning factors reviewed by Vinson, 1990; Lavine and Beckage, 1995
include virus-like particles, which mask the parasitoid
Corresponding author. Tel.: +
44-1904-462000; fax: +
44-1904- 462252.
E-mail address: n.parkinsoncsl.gov.uk N. Parkinson.
0965-174801 - see front matter
2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 1 0 5 - 3
egg from the immune system; polydnaviruses, which infect and disrupt haemocytes; and ovarian proteins,
which also target haemocytes Feddersen et al., 1986; Vinson and Scott, 1974; Webb and Luckhart, 1994.
In addition to ovarian secretions, parasitoids also inject their hosts with venom. The venom of many ecto-
parasitoids induces host paralysis, preventing attack of the wasp’s eggs and larvae by biting, and is also
involved in the inhibition of moulting Weaver et al., 1997; Doury et al., 1995; Beard, 1963. In contrast, little
is known about the function of endoparasitoid venom, although a role in disabling haemocytes has been sug-
gested Tanaka, 1987; Osman, 1978; Kitano, 1982. Still less is known of the nature of venom components. While
two polypeptides occurring in venom of Chelonus sp., an endoparasitoid that oviposits into the egg of its host,
have recently been characterized Jones et al., 1992; Krishnan et al., 1994 the role of these proteins a chitin-
ase and a polypeptide of unknown function in the para- sitic process is not clear.
We are investigating venom from the pupal endopara-
58 N. Parkinson et al. Insect Biochemistry and Molecular Biology 31 2001 57–63
sitoid Pimpla hypochondriaca, and have previously identified several biologically active components includ-
ing an enzyme which oxidizes L-DOPA Parkinson and Weaver, 1999. Two enzymes known to catalyse oxi-
dation of L-DOPA are tyrosinase EC 1.14.18.1 mono- phenol
monooxygenase; L-DOPA:oxygen
oxidore- ductase and laccase EC 1.10.3.1 o-diphenol oxidase;
1,2-benzenediol:oxygen oxidoreductase. Tyrosinases are best known in catalysing synthesis of reactants
involved in early steps in the biosynthesis of melanin, a heteropolymer occurring widely in animals which serves
both as a pigment and to protect from ultraviolet light Mason, 1965. Laccases are found in insect integument
and catalyse the initial reactions leading to the synthesis of compounds that cross-link integument proteins to chi-
tin Sugumaran, 1988. This process is responsible for tanning the soft, newly synthesized cuticle which is ther-
eby converted into a hard and rigid structure.
Tyrosinase activity has been identified in arthropod haemolymph, and cDNAs encoding the enzyme have
been cloned from haemocytes of several insect and one crustacean species. Phylogenetic analysis has identified
the tyrosinase isolated from arthropod haemocytes as a distinct type Hall et al., 1995; Kawabata et al., 1995;
Fujimoto et al., 1995; Aspa´n et al., 1995; Jiang et al., 1997a,b; Park et al., 1997, which displays sequence
similarity to arthropod haemocyanins and is expressed as an inactive proenzyme Kawabata et al., 1995. The
term pro-phenoloxidase PPO or, following activation by proteolytic cleavage, phenoloxidase PO is com-
monly used to distinguish arthropod-specific tyrosinases, and this convention is followed here. PO is closely asso-
ciated with the arthropod immune system, and cleavage of PPO to yield PO occurs in response to microbial cell
wall products which activate PPO-specific proteases So¨derha¨ll and Hall, 1984; Ratcliffe and Rowley, 1987;
So¨derha¨ll and Smith, 1986; Ashida and Brey, 1997. A mechanism for PO-mediated clearance of microbial
infections has recently been proposed, in which quinones generated by PO activity cross-link bacteria to a protein
present on the haemocyte cytoplasmic membrane Marmaras et al., 1996.
We report here the analysis of Pimpla venom fractions containing L-DOPA-oxidizing activity. Genes encoding
the proteins in these fractions have been cloned and found to represent a new form of PO, with properties
that are strikingly different from those of all other arthro- pod POs which have been characterized to date.
2. Materials and methods
