The phenoloxidase from the hemolymph of Diplopoda

10 Pages·1996·1.7 MB·English

by  Willi E R Xylander

The Phenoloxidase from the Hemolymph of Diplopoda
Willi E. R. XYLANDER
Institut fur Allgemeine und Spezielle Zoologie, Justus-Liebig-Universitat Giessen
Siephanstr. 24, D - 35390 Giessen, Germany
ABSTRACT
The phenoloxidases of the diplopods Chicobolus sp. and Rhapidostreptus virgator occur in the hemolymph as
proenzyme, prophenoloxidase (proPO). It can be activated in vitro by incubation with ethanol or methanol whereas
chymotrypsin only activates the proPO of Rhapidostreptus. Microbial substances have little effect on the proPO of both
species. Dopa showed to be a good substrate, dopamine and pyrogallol were less, tyrosine and others hardly converted at
all. Phenylthiourea is a potent inhibitor. The pH optimum was about pH7 in both species. In SDS-PAGE the proPO had a
molecular weight of about 230 kDa in both species and in Lithobius forficatus. The proPO is located in the grana of the
two types of granular hemocytes but not in the plasmatocytes. Microbial substances and other material (glass, sephadex.
latex beads) did not induce exocytosis and activation of the proPO but it is activated during wound clot formation (as
shown by mclanisation of the clot). Thus the activation mechanism of this defence system is somewhat different from
that of insects and crustaceans and may be involved in antimicrobial defence at wound margins.
RESUME
La phenoloxydase de l'hemolymphe des diplopodes.
La phenoloxydase des diplopodes Chicobolus sp. el Rhapidostreptus virgator existe dans 1 hemolymphe en tant que
proenzyme de la phenoloxydase (proPO). Elle peut etre activee in vitro par I'&hanol et le methanol, tandis que la
chymotrypsine active uniquement celle de Rhapidostreptus. Les substances microbiennes ont tres peu deffet sur les
proPO des deux especes. La dopa s'av^re etre un bon substrat. tandis que la dopamine et le pyrogallol le sont moins. En
revanche, la tyrosine et d'autres substances ne sont pour ainsi dire pas metabolisees. La phenylthiouree convient
parfaitement commc substance inhibitrice. La valeur optimale du pH est pH7. Dans le SDS-PAGE, la proPO des deux
esp&ces, de memc que celle de Lithobius forficatus, a un poids moleculaire d’environ 230 kDa. Elle est surtout localisee
dans les grana des deux types d'hemocytes granulaires. mais elle manque dans les plasmocytes. Des substances
microbiennes el d’autres mat£riaux (verre, grains de sephadex et de latex), ne provoquent pas 1 exocytose des grana
contenant la proPO mais sont actives pendant la cicatrisation (comme on peut le depister dans la melanisation).
Apparemment, les mecanismes d'activation de ce systfcme immunologique sont differents de ceux des insectes et des
crustacSs ; ils sont probablement impliques dans la defense antimicrobienne qui se developpe au bords des plaies.
INTRODUCTION
The phenoloxidase (PO) from the hemolymph of arthropods and the enzymes involved in
its activation play an important role in immune defence responses. In many arthropods, they are
stored in the hemocytes as inactive zymogen, the prophenoloxidase (proPO). Once set free by
exocytosis the enzyme becomes “sticky” and attaches to the surface of foreign particles where
the activation of the proPO and subsequent formation of melanin occurs (SODERHALL, 1%2).
XYLANDER. W. E. R., 1996. — The Phenoloxidase from the hemolymph of Diplopoda. In: Geoffroy, J.-J
MAURifes, J.-P. & Nguyen Duy - Jacquemin, M„ (eds), Acta Myriapodologica. Mem. Mus. natn. Hist, not., 169 : 411
420. Paris ISBN : 2-85653-502-X.
412 WILLI E. R. XYLANDER
The immune defence reactions in which the hemolymph PO is involved comprise synthesis of
bacteriostatic and fungicide intermediate products of melanin formation, opsonization, melanin
deposition on the surfaces of metazoan and protozoan parasites, and wound closure (ASHIDA &
Yamazaki. 1990; Chadwick & ASTON, 1978; Gotz& Vey, 1974; Pye, 1974; RATCLIFFE et
al., 1984; Vey & GOTZ. 1975). This paper presents the results of investigations on the
hemolymph PO of two diplopods (Rhapidostreptus virgator and Chicobolus sp.).
MATERIAL AND METHODS
Two diplopods (Chicobolus sp. and Rhapidostreptus virgator) were reared and hemolymph was obtained
following the description given by Xylander & Nevermann (1990), The methods of PO investigations in the in-vitro-
system (see Fig. 1). the calculation of PO-activity and the preparation of SDS-PAGE under non-reducing conditions were
described in detail by Xylander & Bogusch (1992). The procedure of demonstration of intracellular proPO in the
hemocytes was presented by Xylander & Nevermann (1993).
measurement of extinction
at *190 nm for
30 min or lh
Fig. I. — Procedure of in-vitro-measurement of PO activity.
RESULTS
In-vitro-activity
n ac rTt]e in'vitr°'actiyity °f the phenoloxidase (PO) without any activator is comparatively low
0.65 Lml-1 min-i] in Rhapidostreptus (XYLANDER & BOGUSCH, 1992) and 0.133 [ml-i min-il
in Chicobolus (Fig. 2). This means that the PO occurs in the hemolymph as inactive zymogen,
the piophenoloxidase (proPO). The level of PO-activity in the hemolymph depended oifthe
season; it was generally higher in summer (during the main period of activity) than in winter
Source: MNHN, Paris
PHENOLOXIDASE FROM THE HEMOLYMPH OF DIPLOPODA 413
File proPO can be activated by incubation of hemolymph with organic solvents (e. g.
ethanol, methanol, SDS) and proteases (a-chymotrypsin) for 10 min; maximum activity (the
highest activity measured) was generally higher in Rhapidostreptus than in Chicobolus,
however, the relative increase in activity after activation (activity without activation = 1) was
higher in Chicobolus (compare Figs 2 & 3). Ethanol is the best activator tested and raises the
PO-activity 20times (in Rhapidostreptus) to 35times (in Chicobolus; A = 7.42; see Figs 2 & 3).
Methanol is a less efficient activator than ethanol in both species, a-chymotrypsin is a good
activator in Rhapidostreptus but shows only little effect in Chicobolus (Figs 2 & 3).
moximum activity
Fig. 2. — Absolute in-vitro-activity of the
hemolymph of Rhapidostreptus
virgator and Chicobolus sp. without
and with application of various
potential activators.
Rhapidostreptus Chicobolus
without activ. ethanol Wilt methanol .-1 chymotrypsin
(WVW
t.'-ii-J zymosan bact. LPS
relotive activity
Fig. 3. — Relative in-vitro-activity of the
hemolymph of Rhapidostreptus
virgator and Chicobolus sp. without
and with application of various
potential activators, (activity
without activator = 1). Note that the
relative activity increase in
Chicobolus is higher than in
Rhapidostreptus although the latter
has a higher absolute activity.
Rhapidostreptus Chicobolus
without activator ethanol methonol
!_I chymotrypsin zymosan
The activity after chymotrypsin activation increases with elongation of incubation time in
both species (Fig. 4); the highest activity is reached after 60 min of incubation. After activation
with ethanol the molecular weight (MW) of the PO-active band in SDS-PAGE does not differ
from unactivated hemolymph whereas after activation with a-chymotrypsin two different bands
occur with lower MW (XYLANDER & BOGUSCH, 1992), indicating that during ethanol
activation - in contrast to a-chymotrypsin - a conformational change of the proPO leads to its
activity rather than a protein cleavage.
414
WILLI E. R. XYLANDER
maximum activity
Fig. 4. — PO activity after preincubation of
hemolymph samples [min-i ml
hemolymph-i], from
Rhapidostreptus virgator and
Chicobolus sp. with bovine o-
chymotrypsin for 1, 10, 30 and 60.
The activity of the PO increases in
both species with duration of
10 30 chymotrypsin incubation.
incubation time I min
Chicobolus Rhapidostreptus
FlG. 5. — Activity of ethanol and of o-
chymotrypsin activated and
unactivated hemolymph samples
[min-i ml hemolymph-i] from
Rhapidostreptus and Chicobolus
with or without potential PO-
inhibitors.
Rhopidostreptus Chicobolus
(cid:9633)
ethanol chymotrypsin chymotrypsin + PTU
(cid:9633)
ethanol + ED'A ethanol (cid:9830) EGTA without activator
Fig. 6. — Activity of PO [min-1 ml
hemolymph-1] from the hemolymph
of Chicobolus at different pH.
Source: MNHN, Paris
PHENOLOXIDASE FROM TI IE HEMOLYMPH OF DIPLOPODA 415
Li Rh -Ch-
7. — Polyacrylamid-gel (7.5% tH tH Hz tH +X X
acrylamid) of hemolymph (tH) «* N
m
and hemocyte lysate (Hz) of P 53
Lithobius forficatus ( .'•) .
Rhapidostreptus (:<h) and
Ml
Chicobolus (Ch), with
subsequent reactivation of 205
—-
enzymes by incubation in a 2%
Triton-X 100 aquaeous solution
for 30 min. washing in buffer
and incubation in an 0.02 M
dopa solution in 0.01 M 116 (cid:9658)
cacodylate buffer (pH 7.0). At
the position of reactivated PO 97 — ^
dopachrom and melanin is
formed resulting in pinkish and
later brownish or black band. 68-
Molecular standard (SDS-6H.
Sigma. Munich) run in the same
gel indicates the MW of the PO
(corresponding at about 230 kD
in all three species).
46
-
— PAGE of hemocyte lysate, total hemolymph and plasma of Rhapidostreptus (run under the same conditions and
with subsequent procedure described for Fig. 7). The left part of the gel was stained with Coomassie Brillant
Blue, the right treated as described for Fig. 7). No difference in MW is visible.
Source
416 WILLI E. R. XYLANDER
octivity
1
5 I
Fig. 9. — PO-activity of Chicobolus in the
hemocyte lysate (obtained after
moderate centrifugation at about 60-
100 g at 4°C for 10 min) and
hemolymph after ethanol
activation.
hemolymph hemocyte lysate
Fig. 10. — Staining capabilities of
hemocyte monolayers (in % of each
hemocyte type) on glass slides of
Chicobolus after ethanol activation
and dopa incubation.
i n o staining weak reaction strong reaction
Microbial activators (zymosan, murein, bacterial lipopolysaccharides) which are potent
inductors of PO-reaction in other arthropods have little effect neither on total hemolymph nor on
hemocyte lysate (Figs 2 & 3 and unpubl. results); therefore, another activation mechanism may
occur in Diplopoda than in Decapoda and Insecta.
Dopa is the substrate used best by the PO of EtOH-activated hemolymph from
Rhapidostreptus and Chicobolus as dopamin and pyrrogallol are less effectively used and
tyrosin, pyrocatechol and norephedrin hardly at all (see also XYLANDER & BOGUSCH, 1992).
The PO can be inhibited in the two diplopods and Lithobius by phenylthiourea and at least
in the diplopods by EDTA and EGTA (Fig. 5) indicating that the PO is of the tyrosinase-type
and Ca + dependend. The pH-optimum of the PO in both diplopods is pH 7.0, although the
2
activity is rather high between pH 6.0 and 8.0 (Fig. 6; see also XYLANDER & BOGUSCH, 1992
for results on Rhapidostreptus).
SDS-PAGE under non-reducing conditions
After SDS-PAGE under non-reducing conditions, reactivation of enzymes by incubation in
Triton-X 100 and incubation in dopa brownish to black bands occur at the position in the gel of
the PO and proPO, respectively. The proPO has corresponding molecular weights (MW) of
Source: MNHN, Paris
PHF.NOLOXIDASF. FROM THE HEMOLYMPH OF DIPLOPODA 417
about 230 kD in Lithobius, Rhapidostreptus and Chicobolus (Fig. 7). After incubation with
a-chymotrypsin the MW is reduced to about 200 and 180 kD whereas EtOFI has no such effect
(XYLANDER & BOGUSCH, 1992). PO-active bands have the same MW in hemocyte lysate, total
hemolymph and plasma of Rhapidostreptus (Fig. 8).
Localization of the proPO
After moderate centrifugation of hemolymph of Rhapidostreptus virgator at 4°C at about
60-100 g, resuspension and sonification of the hemocyte pellet the activity was mainly (about
80%) found in the hemocyte lysate whereas only little (about 20%) occurred in the plasma
(XYLANDER & BOGUSCH, in prep.); hemolymph samples from the same pool not centrifuged
had about the sum of plasma and hemocyte lysate activity. Investigations with Chicobolus with
the same procedure led to degranulation of hemocytes and artificially high activity in the plasma
(Fig. 9); if hemolymph was poured into the same amounts of ice-cold hemocyte stabilizing
buffer (after SODERHALL et al., 1979) about half of the activity occured in the hemocytes.
Investigations of glutaraldehyd-fixed hemocyte monolayers of Rhapidostreptus and
Chicobolus after activation with ethanol and subsequent dopa-overlay showed that PO-activity
occured mainly in the granular hemocytes (with different intensities which could be designed to
two types of granular hemocytes with different spreading capabilities; FIG. 10). Plasmatocytes
remained unstained whereas the prohemocytes showed high variability in their PO-reaction
(Figs 10 & 11). PTU in control hemocyte monolayers inhibits intracellular PQ reaction in both
species.
Exocytosis of the PO system in Rhapidostreptus cannot be initiated by addition of
solutions of various microbial cell wall components as it has been found in insects and
crustaceans (JOHANSSON & SODERHALL, 1985. 1989a, b, c).
A P £ B
*1
« " #
4
FiG. II. — Granular hemocytes and plasmatocytes of Chicobolus in a hemocyte monolayer. A. Phase contrast. B. Bright
lield. Only the stained granular hemocytes are visible under these conditions. P: plasmatocytes; G; Granular
hemocyte; U: Prohemocyte.
418 WILLI E. R. XYLANDER
DISCUSSION AND CONCLUSION
As in other arthropods the PO of diplopods occurs in the heinolymph as inactive proPO.
Activators found to be efficient in the diplopods also have been reported to activate the proPO of
insects and crustaceans (cf. ASHIDA & YAMAZAKI. 1990; GOTZ, 1988; JOHANSSON &
SODERHALL, 1989b). However, microbial cell wall components (murein, 13-1,3-glucans,
lipopolysaccharides from outer cell membrane of Gram-negative bacteria) which activate the
proPO in insects and crustaceans (ASHIDA et a!., 1983; ASHIDA & SODERHALL, 1984; ASHIDA
& Yoshida. 1988; Johansson & Soderhall, 1989b; Smith & Soderhall, 1983;
SODERHALL. & HALL. 1984; SODERHALL & UNESTAM. 1979; SODERHALL et al., 1988) and
have been considered to be the inducers of in vivo PO-reactions did not have any effect on
diplopods with the system used (XYLANDER. 1992; XYLANDER & BOGUSCH, 1992, this
paper). Regarding other capabilities (PTU-inhibition, pH-optimum. Ca + dependence,
2
preference for dopa as substrate) the proPO and PO of diplopods correspond to that of insects,
crustaceans and the few chilopods investigated.
The MW of the two diplopods and Lithobius ranges at about 230 kD. This is higher than
most data found in most insects and crustaceans the PO and proPO of which have a MW of 60-
80 kD; however, corresponding MW (higher than 200 kD) have also been reported for various
crustaceans and insects (NELLAIAPPAN et al., 1989; YAMAURA et al, 1980). ASPAN &
SODERHALL (1991) and GILLESPIE et al. (1991) reported that the proPO tends to aggregate to
form polymeres indicating that the bands found could be a distinctive polymere (tetramere?) of
the proPO (cf. ASPAN & SODERHALL, 1991). Own investigations using gel filtration
chromatography of hemolymph of Rhapidostreptus (data not shown), however, also led to MW
of more than 230 kD of the proPO.
Whether the proPO of arthropods occurs mainly free in the hemolymph or in the
hemocytes depends on the taxon investigated. In most insects and all crustaceans investigated,
the proPO is predominantly located in the hemocytes; it is discharged and activated after
infections (cf. JOHANSSON& SODERHALL, 1989b; ASHIDA & YAMAZAKI, 1990). In some
insects, however, the proPO is reported to occur free in the plasma (GOTZ et al., 1987. SAUL et
al, 1987). In Rhapidostreptus the majority of the proPO is also found intracellularly
(XYLANDER & BOGUSCH, in prep.), whereas in Chicobolus (probably as a preparation artifact
due to unsufficient stabilization of hemocytes in a crayfish saline) only about 50% are found in
the hemocyte lysate.
In all diplopods and chilopods investigated the granular hemocytes are the site of proPO
localization (Bowen, 1968; Krishnan & Ravindranath, 1973; Nevermann et al. 1991;
XYLANDER & NEVERMANN, 1993), whereas only few plasmatocytes show a faint staining after
dopa incubation; the spherulocytes of chilopods are PO-negative (NEVERMANN et al, 1991;
XYLANDER & NEVERMANN, 1993). In chilopods staining is rather slow in comparison to
diplopods (XYLANDER & NEVERMANN, 1993).
Although microbial cell wall components do not initiate exocytosis of the PO cascade as in
crustaceans and insects (cf. reviews in JOHANSSON & SODERHALL, 1989b; ASHIDA &
YAMAZAKI, 1990) rather strong melanization can be found at wound margins of diplopods. This
indicates that the PO is activated after an injury or infection and is involved in subsequent
immune response: one function of this enzyme may be to support rapid wound closure and to
kill bacteria and fungi before they enter the hemocoel and may lead to dangerous infections.
Furthermore, an intracellular activation has been found in hemocytic aggregates around foreign
material in vitro and in vivo (NEVERMANN, 1989; NEVERMANN & XYLANDER, this volume).
As in other arthropods where intracellular activation has been reported (VOLKMANN, 1991;
NAYAR et al, 1992) the “melanized hemocytes” in the capsule may constitute an efficient
barriere for metabolic waste from potential parasites and invaders on one hand and for nutrients
from the hemolymph necessary for their survival on the other hand. Thus melanized hemocytes
Source: MNHN, Paris
PHENOLOXIDASE FROM THE HEMOLYMPH OF DIPLOPODA 419
may support the function of a multilayered hemocyte capsule formed around parasites as a
typical cellular defence reaction of arthropods.
ACKNOWLEDGEMENTS.
I would like to thank O. BOGUSCH, H -U. Jahn. L. Nevermann. A. Hudel, and Prof. Dr. P. Gotz for their support.
Investigations and participation in the CIM congress were supported by the President of the Justus-Liebig-University
Giessen.
REFERENCES
Ashida. M., Ishizaki Y. & Iw ah an a H., 1983. — Activation of pro-phenoloxidase by bacterial cell walls or B-1,3
glucans in plasma of the silkworm. Bombyx mori. Biochem. Biophys. Res. Comm. 113 : 562-568.
Ashida, M. & SOderhaall. K., 1984. — The prophenoloxidase activating system in crayfish. Comp Biochem.
Physiol., 77B : 21-26.
Ashida. M. & Yamazaki H. I.. 1990. — Biochemistry of the phenoloxidase system in insects: with special reference to
its activation. In : E. Ohnishi & H. Ishizaki. Moiling and metamorphosis. Tokyo. Japan Sci. Soc. Press, Berlin.
Springer-Verlag : 239-265.
Ashida, M. & Yoshida H., 1988. — Limited proteolysis of prophenoloxidase during activation by microbial products in
insect plasma and effect of phenoloxidase on electrophoretic mobilities of plasma proteins. Insect Biochem.. 18 :
1 1-19.
Aspan, A. & SOderhall. K., 1991. — Purification of prophenoloxidase from crayfish blood cells, and its activation by
an endogenous serine proteinase. Insect Biochem., 21 : 363-373.
Bowen, R. C., 1968. — Tyrosinase in millipede hemocytes. Trans. Amer. Microsc. Soc., HI : 390-392.
Chadwick. J. M. & Aston. W. P.. 1978. — An overview of insect immunity. In : Gershwin. M. E. & E. L. Cooper.
Animal models of comparative and developmental aspects of immunity and diseases. Oxford. Pergamon Press : 1-14.
Gillespie, J. P.. Bidochka, M. J. & Khachatourians. G. G., 1991. — Separation and characterization of grasshopper
hemolymph phenoloxidases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comp. Biochem.
Physiol., 98C : 351-358.
GOTZ. P.. 1988. — Immunreaktionen bei Wirbellosen, insbesondere Insekten. Verb. Dtsch. Zool. Ges.. 81 : 113-129.
Gotz, P., Enderlein, G. & ROETTGEN, I., 1987. — Immune reactions of Chironomus larvae (Insecta: Diptera) against
bacteria. J. Insect Physiol., 33 : 993-1004.
Gotz, P. & Vey, A.. 1974. — Humoral encapsulation in Diptera (Insecta) - defense reactions of Chironomus larvae
against fungi. Parasitology, 68 : 193-205.
Johansson, M. W. & SOderhall, K., 1985. — Exocytosis of the prophenoloxidase activating system from crayfish
haemocytes. J. Comp. Physiol., B156 ; 175-181.
Johansson, M. W. & SOderhall, K., 1989a. — A peptide containing the cell adhesion sequence RGD can mediate
degranulation and cell adhesion of crayfish granular haemocytes in vitro. Insect Biochem., 19 : 573-579.
Johansson, M. W. & SOderhall. K., 1989b. — Cellular immunity in crustaceans and the proPO system. Parasitology
Today, 5 : 171-176.
Johansson, M. W. & SOderhall, K.. 1989c. — A cell adhesion factor from crayfish haemocytes has degranulating
activity towards crayfish granular cells. Insect Biochem.. 19 : 183-190.
Krishnan, G. & Ravindranath. M. H.. 1973. — Phenoloxidase in the blood cells of millipedes. J. Insect Physiol.,
19 : 647-653.
Nayar, J. K., Mikarts, L. L., Knight, J. W. & Bradley. T. J.. 1992. — Characterization of the intracellular
melanization response in Anopheles quadrimaculatus against subperiodic Brugia malaxi larvae. J. Parasitol.. 78 :
876-880.
Nellaiappan. K., Vinayagam, A. & Kalyani. R.. 1989. — Electrophoretic pattern of blood and cuticular phenoloxidase
of different crustaceans. Exp. Biol.. 48 . 177-179.
Nevermann, L., 1989. — Licht- und elektronenmikroskopische Untersuchungen der Haemocyten von Lithobius
forficatus und ihre Bedeutung fur zellulare Abwehrreaktionen. Diploma-thesis. University of Giessen, 69 pp.
Nevermann, L., Xylander, W. E. R. & Seifert, G.. 1991. — The hemocytes of the centipede Lithobius forficatus
(Chilopoda, Lithobiomorpha) - Light and electron microscopic studies using in-vitro techniques. Zoomorphology,
1 10 : 317-327.
Pye. A. E., 1974. — Microbial activation of prophenoloxidase from immune insect larvae. Nature, 251 : 610-613.
Ratcliffe, N. A., Leonard. C. & Rowley. A. F., 1984. — Prophenoloxidase activation: Nonself recognition and cell
cooperation in insect immunity. Science, 226 : 557-559.
420 WILLI E. R. XYLANDER
Saul, S. J., Bin, L. & Sugumaran. M., 1987. — The majority of prophenoloxidase in the hemolymph of Manduca sexta
is present in the plasma and not in the hemocytes. Dev. Comp. Immunol., 11 : 479-485.
Smith. V. & SOderhAll. K.. 1983. — B-1,3 Glucan activation of crustaceans hemocytes in vitro and in vivo. Biol.
Bull., 164 : 299-314.
SOderhAll, K.. 1982. — Prophenoloxidase activating system and melanization - a recognition mechanism of
arthropods? A review. Dev. Comp. Immunol.. 6 : 601-611.
SOderhAll. K. & HALL, L.. 1984. — Lipopolysaccharide-induced activation of prophenoloxidase activating system in
crayfish haemocyte lysate. Biochim. Biophys. Acta. 797 : 99-104.
SOderhAll. K.. HAll. L.. Unestam. T. & Nyhlen, L., 1979. — Attachment of phcnoloxidase to fungal cell walls in
arthropod immunity. J. Inv. Pathol., 34 : 285-294.
SOderhAll. K.. Rogener. W.. SOderhAll, I., Newton, R. P. & Ratcliffe, N. A., 1988. — The properties and
purification of a Blaberus craniifer plasma protein which enhances the activation of haemocyte prophenoloxidase by
a 8-1.3-glucan. Insect Biochem., 18 : 323-330.
SOderhAll. K. & Unestam. T., 1979. — Activation of serum prophenoloxidase in arthropod immunity. The specificity
of cell wall glucan activation and activation by purified fungal glycoproteins of crayfish phenoloxidase. Can. J.
Microbiol.. 25 : 406-414.
Vey, A. & GOtz. P.. 1975. — Humoral encapsulation in Diptera (Insecta) - comparative studies in vitro. Parasitology,
70 : 77-86.
VOLKMANN, A., 1991. — Localization of phenoloxidase in the midgut of Periplaneta americana parasitized by larvae of
Moniliformis moniliformis (Acanthocephala). Parasitol. Res., 77 : 616-621.
Xylander, W. E. R., 1992. — Immune defense reactions of Myriapoda - A brief presentation of recent results. [/// : E.
MEYER. K. Thaler & W. SCHEDL, Advances in Myriapodology.] Ber. nat.-med. Verein Innsbruck, Suppl. 10 : 101-
1 10.
XYLANDER, W. E. R. & BOGUSCH, O., 1992. — Investigations on the phenoloxidase of Rhapidostreptus virgator
(Arthropoda. Diplopoda). Zool. Jb. Physiol., 96 : 309-321.
Xylander, W. E. R. & BOGUSCH. O., (in prep.) — The Prophenoloxidase of the millipede Rhapidostreptus virgator
(Diplopoda, Spirostreptidae) is mainly located in the granular hemocytes.
Xylander. W. E. R. & Nevermann, L.. 1990. — Antibacterial activity in the hemolymph of Myriapoda (Arthropoda). J.
Inv. Pathol., 56 . 206-214.
Xylander, W. E. R. & Nevermann, L.. 1993. — Phenoloxidase-active hemocytes in Lithobius forficatus, Scolopendra
cingulata (Chilopoda) and Chicobolus sp. (Diplopoda). Verb. Dtsch. Zool. Ges., 86 : 197.
Yamaura, I., YONEKURA, M.. Katsura, Y., Ishiguro. M. & Funatusu, M.. 1980. — Purification and some physico¬
chemical properties of phenoloxidase from the larvae of housefly. Agric. Biol. Client., 44 : 55.
Source: MNHN, Paris