1 10s Blochemlcal SocietyTrans,jct~ons( 1 995) 23
Qualitative differences occur in phenoloxidase activity in
anopheles mosquitoes refractory to plasmodium.
TABLE1. Kinetic constants for parasite infectedhopheles
.amMae haemolvmDh Dhenoloxidase activitv.
L-DOPA Kinetic Constants
SAADIA AHMAD, COLIN J. LEAKE AND ALBERT J.
KETI-ERMAN.
Department of 'Medical Parasitology, London School of
Hygiene and Tropical Medicine, Keppel St, London WCIE
7HT
In insects, the enzyme phenoloxidase (PO; EC
1.14.18.1)forms an integral part of the cellular and humoral
defence system in response to foreign organisms. This
enzyme has been shown to have at least two functions.
Firstly, to synthesise melanin which encapsulates the
invaders and, secondly, as a recognition system for
generating other components of the host defence system. In
a study with European black flies Simulium damnosum s.I.,
infected with Onchocerca spp., it was demonstrated that
haemolymph phenoloxidase activities were lower than in
traumatised specimens of Simulium (1).It was proposed that
the PO system of black flies was not directly involved in
immune reactions contrary to other dipterans. Recent studies
on the encapsulation response of Anopheline mosquitoes to
simian or monkey malaria, Plasmodium cynomolgi, in a
selected line of Anopheles gambiae mosquitoes (2),showed
that the ookinetes or early oocysts were encapsulated
followed by melanisation. It was also shown that in the
refractory mosquitoes oocysts of avian, rodent, monkey and
human malaria were also encapsulated. These encapsulated
or melanised bodies were described as a rare event in the
Anopheles gambiae G3 (2),and it was concluded that this
system of refractoriness is interesting as a biological model
of insect immunity and host parasite interaction.
The present study was focused on the infection of
Anopheles gambiae mosquitoes with the rodent malaria
Plasmodium yoelii nigeriensis. As no quantitative data has
previously been available, a major part of the study has been
to develop a sensitive microtitre plate phenoloxidase enzyme
assay to reliably measure enzyme activity in haemolymph
samples from individual mosquitoes. The system has been
used to examine, in detail, temporal enzyme activities in the
selected strains and in response to parasite infection.
Four different strains of Anopheles gambiae S.S.were
selected for susceptibility (KIL and Zands S) and
refractoriness (REFMA and Zands R) to rodent malaria. The
parasite, N67 strain of Plasmodium yoelii nigeriensis, was
used from a large stock of infected mouse blood
cryopreserved in liquid nitrogen. Stock materialwas prepared
as previously described (3). The phenoloxidase activity was
determined by a modificationof a previously reported method
(4).
The encapsulation of parasites in the mosquitoes was
observed under the light microscope at 48 hours postinfection, this same time point was selected for the individual
assays. However, the assay data showed a wide variation
and did not show a clear trend among the strains. Therefore,
pooled haemolymph assays of the same strains and time
points were performed. Using haemolymph collected and
pooled from 225 individuals of each strains it was possible to
perform more complicated experiments such as,V and K,,,
determinations and inhibition kinetic studies.
Kinetic data of enzyme activities from 48 hours postinfected haemolymph showed that the susceptible strains had
Vmax
Km
PTU inhibition
IC50
pmol/min/mg
mM
PM
ZANDS R
1.79k0.082
0.707k0.132
23.2f2.63
ZANDS S
6.03f0.181
0.886fO.098
9.51 24.22
REFMA
2.8620.085
0.922kO.101
15.3+2.51
KIL
4.61 k0.108
0.918k0.079
22.4k2.57
higher phenoloxidase activity (TABLE
1).The,V for the LDOPA substrate was almost four times higher for the
susceptible ZANDS PO than that of the refractory ZANDS. A
similar situation existed for the susceptible KIL PO that had
almost double the , V
of that of the refractory REFMA.
Although the,V varied between the strains, the K,,, values
were found to be similar.
Studies with inhibition kinetics revealed different IC,
values for PTU for the different strains (TABLE1).The IC,
plots indicated there were multiple forms of phenoloxidase
present in the haemolymph (5).
The present study was conducted to establish the
quantitative and qualitative differences in phenoloxidase
activity in the encapsulation mechanism of the mosquitoes
response to the malaria parasite. Using enzyme kinetic data
we have demonstratedqualitativedifferences for haemolymptPO activity from Plasmodium refractory and susceptible
strains of adult mosauitoes. However, it remains to be showr.
just how enzyme activation confers immune competence to
the host (6).
Genetic analysis suggest that the ability of
refractoriness in mosquitoes is encoded for by at least two
genes, one of which is linked to an esterase loci (7,8).
Data from the present studies suggest that differences
in immune competence is not necessarily associated with the
amount of phenoloxidase present for encapsulation
processes, since virtually low levels of enzyme activity were
exhibited in refractory strains as was also observed for
Simulium (1).This inconsistency between the amounts of PO
activity and the susceptibility of the individual strains raises
the possibility that other mechanisms may also be operative
in the recognition/defence reaction in this system. Further
studies with purified enzyme should be carried out to resolve
the role of phenoloxidase in the melanisation or
encapsulation response of the mosquito to malaria parasites.
1. Hagen. H.E., Grunewald. J. and Ham, P.J. (1993) Ann. Trop. Med.
Parasitol. 87, 660
2. Cdlins, F.H., Sakai, R.K., Vernick, K.D., Paskewitz, S., Seeley, D.C..
Miller, L.H., Cdlins, W.E., Campbell. C.C.. and Gwadz. R.W. (1986)
Science 234, 607610
3. Ahmad, S. and Leake, C.J. (1993) Bangladesh J. Life Sci. 5, 13-21
4. Winder, A.J. and Harris, H. (1991) Eur. J. Biochem. 198, 317-326
5. Tahir. M.K. and Mannervik, B. (1986) J. Biol. Chem. 261, 1048-1051
6. Nappi, A.J., Christensen. B.M., Tracy, J.W. (1987) Insect Biochem. 17,
685-688
7. Vernick, K.D., Collins, F.H.. and Gwadz, R.W. (1989) Am. J. Trop. Med.
Hyg. 40,585-592
8. Vernick, K.D. and Cdlins. F.H. (1989) Am. J. Trop. Med. Hyg. 40,
593-597