Resistance of Moniliformis dubius to the defense reactions of the American cockroach, Periplaneta americana

JOURNAL OF INVERTEBRATE PATHOLOGY

26, 65-73 (1975)

Resistance of Moniliformis dubius to the Defense Reactions of the American Cockroach, Periplaneta americana 1.2 BARRY M. BRENNAN Department ofEntomology, University ofHawaii, Honolulu, Hawaii 96822 AND

THOMAS C. CHENG Institute for Pathobiology, Center for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015 Received September 30, 1974 The successful development of Moniliformis dubius is apparently dependent upon its ability to inhibit the phenoloxidase system of its host, Periplaneta americana. Inhibition of the phenoloxidase system may be directly associated with the polyanionic mucins found in the trilaminate envelope surrounding the developing acanthocephalan larva or cystacanth. It is suggested that these polyanionic mucins complex with the divalent cation, Cu 2+, needed for activation of tyrosinase. Injection of an immunogen prepared from M. dubius acanthella and cystacanths apparently decreases the titer of tyrosinase but does not lead to the production of new hemolymph proteins. Changes in the female hemolymph protein pattern after saline or immunogen injection and bleeding are described. A naturally occuring agglutinin may also be present.

that the host immune system did not react or that the parasite was able to overcome the reaction, or more probably, a combination of both phenomena. Since acanthocephalans lack an alimentary tract in both larval and adult stages, chemical interchange must take place at the body surface. If the body surface becomes covered with host hemocytes for an extended period of time, the developing parasite dies, presumably because cellular encapsulation restricts the availability of both nutrients and oxygen (Salt, 1970). Robinson and Strickland (1969) and Rotheram and Crompton (1972) have demonstrated that a hemocytic reaction always occurs immediately after the acanthor penetrates the cockroach gut, although the host may not always encapsulate and destroy the parasite. Survival of the parasite must be contingent on blocking or preventing a continuous defense reaction of the host. Lackie (1972a), after studying the effects

INTRODUCTION Moniliformis dubius (= M. moniliformisi is a common acanthocephalan parasite of the American cockroach, Periplaneta americana, in Honolulu, Hawaii (Schaeffer, 1970). Fully embryonated eggs are passed in the feces of the definitive host, usually Rattus spp. The acanthor stage hatches from the egg and actively penetrates the midgut of the cockroach. There are six acanthella stages, which are denoted by various structural differentiations, and an infective cystacanth stage (King and Robinson, 1967). The life cycle is completed when the intermediate host is ingested by a suitable definitivehost. The occurrence of a live parasite in the hemocoel of the cockroach is an indication 'Published with approval of the Director of the Hawaii Agricultural Experiment Station as Journal Series No. 1808. 2This research was carried out at the University of Hawaii and its publication is supported by a Grant (FD00416-04) to T. C. Cheng. Requests for reprints should be directed to T. C. Cheng.

65 Copyright e 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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of temperature on the development of M. dubius, stated that the survival of the parasite depended more upon its own metabolic activities than on passive mechanisms. This active resistance is further supported by the observations of Rotheram and Crompton (1972) who noted that although the acanthor is initially encapsulated by the host's hemocytes, subsequent activity by the parasite results in the repulsion of these hemocytes. Robinson and Strickland (1969) and Wanson and Nickol (1973) also noted that the number of hemocytes found on the acanthor decreased with the parasite's growth and development. Inasmuch as they found that no hemocytic reaction occurred after the parasite began to develop, it seems likely that successful parasitization is dependent upon properties associated with the parasites epicuticle, or glycocalyx. Wright and Lumsden (1970) described the acanthor integument as a trilaminate membrane with an amorphous external component. Rotheram and Crompton (1972) stated that the outermost membrane showed both tubular and vesicular profiles and that the vesicular layer appeared to be composed of microvilli. This membranous layer persists throughout the development of the parasite, eventually giving rise to the envelope surrounding the cystacanth (Lackie and Rotheram, 1972). Robinson and Strickland (1969) reported that removal of the envelope resulted in encapsulation and melanization. Periplaneta americana apparently has some capacity to either prevent the penetration of the acanthor and/or successfully encapsulate and destroy all stages of the parasite. Lackie (1972b) found that at low levels of infection only 20-30% of acanthors given orally as "mature" eggs succeeded in developing to the cystacanth stage. A proportion of acanthors perished shortly after penetration and were found as melanized, encapsulated objects loosely attached to the hemocoelic side of the lower midgut. At higher dosages, the major losses occurred before or during penetration of the midgut.

Schaeffer (1970) found that 16% of the naturally infected cockroaches contained remains of cystacanths or slightly shriveled and melanized cystacanths. Robinson and Strickland (1969) noted that in some cockroaches no melanized larvae were observed, while in others, up to 40% were melanized. The lack of a consistently observable host reaction may be due to some factor(s) produced by the parasite which inhibits or blocks a host reaction. The purpose of this study was to further examine the ability of and the method by which M. dubius blocks the defense reactions of P. americana. MATERIALS AND METHODS

Agglutination and Melanization Larval stages of M. dubius were obtained from naturally infected P. americana. Acanthellas and cystacanths were washed five times with sterile saline and sonicated with a Biosonik II (Brownwill, VWR Scientific, San Francisco, Cal.). The stock preparation (immunogen) consisted of approximately four acanthocephalans per milliliter of Yeager's physiological cockroach saline (YPCS). The immunogen was kept frozen at - 20°C. Observations on agglutination and melanization were made on the following preparations: (1) nonimmunized plasma plus YPCS, (2) nonimmunized plasma plus immunogen, and (3) immunized plasma plus immunogen. Only adult, female cockroaches were used. Twenty CO 2-anesthetized cockroaches were actively immunized with 10 III of the immunogen. The immunogen was injected with a microinjector fitted with a 27-gauge needle. All injections were made between the left hind coxa and the first abdominal sternite. Twenty-four hours later, Will of hemolymph were collected in a capillary tube from each cockroach by making a small incision between the right-hand coxa and the first abdominal sternite. The hemolymph from each cockroach was centrifuged to remove cellular components. The 20 samples of plasma were transferred individually to clean microtest tubes and

RESISTANCE OF

Moniliformis

an equal amount of the homologous immunogen was added . As controls, 20 nonimmunized cockroaches were bled as described above. The hemolymph was processed in the same manner and an equal amount of the immunogen was added. The hemolymph from a third group of 20 cockroaches was also collected, centrifuged, and transferred as outlined above. An equal amount ofYPCS was added to each of these microtest tubes in place of the immunogen. All 60 microtest tubes were stoppered and kept at 4· C. These tubes were checked every 24 hr for 7 days for both agglutination and melanization. The degree of melanization was determined by comparing with a previously prepared color chart consisting of four colors (light yellow to dark brown). In an attempt to quantify the data, each tube was given a discrete value based on its degree of melanization, i.e., 0, no melanization, to 4, heavy melanization. There was an inherent weakness in this method of evaluation; some plasma appeared to be intermediate between two colors. When this occurred the higher value was given. The degree of agglutination was visually determined and semiquantitatively graded as follows: 0, · no agglutination; + I, light or indefinite clumping; + 2, moderate clumping; and +3, heavy or complete agglutination . Chi-square and covariance analyses were employed to detect differences between each group and to indicate what relationship might exist between agglutination and melanization within each group.

Electrophoresis Approximately 5 }ll of hemolymph were collected from each of 23 randomly selected male cockroaches (Group A) in the manner described. Group A was then injected with 10 }ll of YPCS . Daily hemolymph samples were collected for a period of 10 days. Dead or moribund cockroaches were removed daily, and the healthy cockroaches were reinjected with 1O}l1 of the saline on the fourth

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67

and eighth days. The hemolymph samples were pooled and stored at - 20· C until ready for electrophoretic separation. A second group of 25 male cockroaches (Group B) was bled and then injected with 10 }ll of the immunogen. Their bleeding and injection schedule paralleled that of Group A. The hemolymph from 10 additional cockroaches (Control) was collected on a daily basis to determine what effect daily bleeding and sham injections with a sterilized 27guage needle might have on protein metabolism. Hemolymph samples were centrifuged at 2260 rpm for 25 min to remove hemocytes and other cellular components. Protein separation was achieved with disc electrophoresis (Canalco, using 0.4 M Tris buffer, pH 8.5, at 24± I· C. Five microliter samples were applied directly to the 7.5 % acrylamide gels. Electrophoresis was conducted atpH 9.5 and 5mA per column until the bromophenol blue tracking dye reached the surface of the lower buffer bath . Gels were stained with 0.5% Amido-Schwarz in 7% acetic acid for a minimum of I hr, and then destained in 7% acetic acid at )2.5 rnx for 45 min. The relative proportion of each protein fraction was determined with a recording densitometer (Photovolt Densicord) coupled to an automatic intergrator (Photovolt). Student's t test (Langley, 1970) was used to detect significant changes in the relative proportions of each protein fraction. RESULTS

Agglutination and Melanization The quantitative values for the degrees of agglutination and melanization in the 60 microtest tubes were recorded at 24-hr intervals for a period of 7 days. The total values for each group for each day are presented in Table l. Very little agglutination or melanization occurred in the tubes containing the normal plasma plus YPCS . There were no significant differences between the control plasma agglutination and melanization regression lines when investigated with covariance analysis (Table 2).

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BRENNAN AND CHENG

TABLE 1 Total Degree of Agglutination and Melanization Observed over a 7-Day Period in Three Groups of 20 Micro-test Tubes of American Cockroach Plasma. a Agglutination Day

A

B

1 2 3 4 5 6 7

0 0 0 0 0 1 1

20 22 22 22 23 24 25

Total

2

158

TABLE 2 Covariance Analysis of the Relationship Between the Rate of Agglutination and Melanization Observed Over a 7-Day Period in Three Groups of 20 Micro-test Tubes of American Cockroach Plasma."

Melanization C

A

B

19 20 21 23 26 26 36

0 0 0 0 1 1 2

1 9 17 27 32 40 44

C

15 17 19 20

171

4

170

89

1 7

11

a A =normal plasma plus saline; B =normal plasma plus acanthocephalan immunogen; C =Immunized plasma plus acanthocephalan immunogen.

Agglutination and melanization both occurred in the tubes containing the normal plasma immunogen. A comparison of the regression lines indicated that the slopes of the lines were significantly different, suggesting that the two processes proceed at different rates. Agglutination and melanization were also present in the immunized plasma plus immunogen. The slopes of the two regression lines were not significantly different. When the rates of melanization and agglutination of the three groups were compared with each other there appeared to be several significant differences. The rate of melanization was significantly faster in the normal plasma plus immunogen than in either of the two other groups. There was no significant difference between the rate of agglutination in the normal plasma plus saline and the normal plasma plus immunogen. However, the rate at which agglutination occurred in the immunized plasma was significantly greater than the rate of agglutination in the normal plasma plus immunogen. Nearly two-thirds of the total agglutination observed occurred during the first 24 he. The total amount of agglutination which occurred in the normal plasma plus immunogen did not differ significantly from

Within group Melanization vs agglutination

Group(s)

F

A

C

2.06 109.33 b 0.03

A and B BandC A and B BandC

410.19 b 66.19 b 0.06 10.84 b

B Between groups Melanization Agglutination

I Significant at the 0.01 level of probability with 1, 10 df. aA = normal plasma plus saline; B =normal plasma plus acanthocephalan immunogen; C =immunized plasma plus acanthocephalan immunogen.

that which occurred in the immunized plasma plus immunogen. However, the total degree of melanization which occurred in these two plasmas was significantly different at the 0.01 level of probability (x 2 = 39.03, with 6dJ). Electrophoresis Preliminary experiments indicated that variations occurred in the concentration of each protein fraction even among cockroaches of the same sex, which were identically reared and treated. Therefore, the hemolymph from all the cockroaches in each group (A, B. or Control) was pooled and each of the protein fractions was expressed as a percentage of the total protein concentration within the pooled sample. Electrophoretic separation revealed nine clearly distinguishable protein fractions. These fractions were designated fractions 1IX, with fraction I having the greatest mobility. In addition, two diffuse protein fractions, which were not quantified, occurred between fractions II and III. On the second and third days a tenth protein fraction with slightly greater mobility than fraction III occurred in all three groups. This fraction, IIIb, persisted until the fourth day in the control group and reappeared on the fifth

RESISTANCE OF

Moniliformis

TABLE 3 Relative Proportions (%) of Protein Fractions Occurring in Periplanetaamericana Hemolymph (Day 0) Compared with Relative Proportions of Protein Fractions Occurring on the Ninth Day After Sham Injection (Control) , or After Injection with Saline (A), or Acanthocephalan Immunogen (B)

Day 0 Fraction I II lIla IlIb IV V VI VIla VUb VIII

Day 9

Mean ± SEa

Control

14.7 ± 6.5 16.0 ± 3.0 6.7 ± 5.8 0 5.7 ± 3.5 20.3 ± 10.8 8.3 ± 2.1 13.9 ± 8.3 11.9 ± 4.5 3.4 ± 2.2

6.0b

32.4c 4.9 0 4.4 18.1 0.5c 12.1 17.6b 4.4

A

B

6.7b

6.4 b

33.6c 5.4

25 .7 c

o

o

5.4 13.4

5.7 12.71

10.7 20.1b 4.7

15.7 22.1b 4.0

OC

3.6

Oc

aMean and standard error are based on 3 pooled samples from 58 American cockroaches. bSignificant at the 0.05 level. CSignificant at the 0.01 level.

day in the immunized group. However, it never constituted more than 2.6% of the total protein. During the course of the experiment, the relative concentrations of several of the protein fractions changed. However, there were no consistently significant group differences between corresponding protein fractions collected on the same day . Within each group over the 10-day period, both hypoproteinemia (Fractions I and VI) and hyperproteinemia (Fractions II and VIIb) were evident (Table 3). In all four of these fractions (I, II, VI, and VIIb), the changes which occurred between preinjection and ninth day postinjection samples were significant. Although the remaining five fractions fluctuated during the course of the experiment, they did not differ significantly from samples taken prior to injection. No enhanced or anamnestic response as evidenced by an increased production of specific proteins occurred after the second or third injections. DISCUSSION The use of the terms "agglutinin" and "agglutination" occurs throughout much of

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the literature of invertebrate immunology and for this reason will be used to describe an entity and process which might more properly be referred to as a coagulant and coagulation. Yeager and Knight (1933) stated that the apparent coagulum observed in the American cockroach, P. americana. was merely a clump of hemocytes with filamentous pseudopodia. These pseudopodia become sticky, agglutinate, and finally disintegrate to yield a cellular coagulum . They observed no obvious coagulation in the plasma and did not know to what extent the plasma contributed to the coagulum formation. However, Ermin (1939) reported the formation of "meshworks" independent of lymphocytes in the same insect. The results obtained in this study would appear to support the observations of Ermin. It is possible that some hemocytes taken in the samples may have released a substance or factor prior to being removed by centrifugation. If such was the case, the coagulation or agglutination observed may be due as much to hemocytes as to plasma. Agglutination may also be due to an imbalance in the colloidal system (Paillot, 1921) following addition of the immunogen, or to a naturally occurring agglutinin found in the plasma and independent of the hemocytes. Siakotos (1960) found that the clotting reaction in the cockroach may be associated with a lipoprotein fraction of the plasma. A peak (fraction VI) corresponding to his lipoprotein fraction was present in the plasma of all three groups investigated in this study. This fraction decreased from an average of 8.3% (preinjection samples) to 0.3% (samples collected on the ninth day postinjection) of the total protein. Since there were no significant differences between corresponding protein fractions collected on the same day, the hypoproteinemia (fractions I and VI) and the hyperproteinemia (fraction II) are believed to be the result of injury metabolism, including coagulation, rather than with an immunological reaction . Seaman and Robert (1968) showed that

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BRENNAN AND CHENG

adult male P. americana are capable of immobilizing a protozoan, Tetrahymera pyriformis. Immobilization was associated with a preformed hemolymph protein sample which became altered in response to immunization. Inasmuch as the relative mobilities of fractions I and II are similar, it is suggested that a similar alteration phenomenon may have occurred after hemolymph samples were taken, i.e., the hyperproteinemia of fraction II may have been at the expense of fraction I. Protein depletion, particularly in one or two fractions, appears to be a common abnormality in infected insects or in melanized hemolymph (McKinstry and Steinhauer, 1970). They noted that this abnormality may indicate a relationship between melanization and host defense reactions to pathogens. The defense reaction of an insect to a large foreign body often involves melanization which may occur in conjunction with or shortly after cellular encapsulation. Since melanization often occurs following autolysis of the innermost hemocytes comprising capsules, Salt (1970) was of the opinion that it could be considered a secondary reaction, but not a humoral one. However, in mosquitoes infected with nematodes, melanization occurs in the absence of an observable hemocytic response and therefore might be considered to be a humoral reaction (Poinar and Leutenegger, 1971; Bronskill, 1962; Esslinger, 1962). It therefore appears that the relative importance of melanization and encapsulation as defense reactions in and of themselves varies with the organisms involved in the host-parasite relationship. However, the interdependence of the two reactions may be related to the number and types of hemocytes. Nappi and Stoffalano (1972) noted changes in the number and type of hemocytes before evidence of either melanization or encapsulation of the nematode Heterotylenchus autumnalis by the face fly, Musca autumnalis. Nappi (1970), after investigating the changes in the hemocytes of Drosophila euronotus, suggested the possi-

bility that abortive development of the hymenopterous parasite Pseudeucoila bochei was due to the production of humoral substances that have a cellular basis. These humoral substances were believed to be part of or closely related to the phenoloxidases found in oenocytoids. Lysis of the oenocytoids is thought to bring about melanization of the cellular elements of the capsule (Nappi and Streams, 1969). The sequence of reactions leading to the formation of melanin is initiated by a phenoloxidase, tyrosinase. Sekeris and Mergenhagen (1964) studied the phenoloxidase system of the blowfly, Calliphora erythrocephala, and found that the inactive precursor of phenoloxidase is present in the hemolymph while the activator enzyme was located in the cuticle. Activation of the enzyme precursor, in order to yield the active phenoloxidase, can occur after injury or at the onset of puparium formation. In the presence of mitochondria, the phenoloxidase showed equal mono- and diphenoloxidase activity. The importance Ofhemocytes as a source of phenoloxidases or prephenoloxidases and the hemolymph as a transport medium for hormones and activator enzymes is thus evident in wound healing, puparium formation, tanning, and melanization of parasites and parasitoids. Lackie (l972b) reported that only 20-30% of the eggs capable of hatching in vitro gave rise to cystacanths when fed to cockroaches. The wastage occurred very early in the infection process, during or immediately after penetration of the gut. A periodic acidSchiff-positive, polyanionic, amorphous material is present between the innermost egg envelope and the acanthor (Wright, 1971). This amorphous material is present on the hatched acanthor only as a thin discontinuous layer (Wright and Lumsden, 1970). The high degree of acanthor mortality may be associated with this discontinuity, or conversely, the ability of the acanthor to avoid or inhibit encapsulation may be associated with this layer. Discontinuity in

RESISTANCE OF

Moniliformis

the amorphous layer could easily occur when the acanthor hatches. Since two of the embryonic envelopes contain either keratin or protein-chitin complex (Wright, 1971), the surface of the acanthor could become abraded when it rapidly propels itself from the egg. The glycocalyx, the so-called "epicuticle" of the acanthor and cystacanth, is composed of weak acidic mucopolysaccharides, neutral polysaccharides and/or glycoproteins (Wright and Lumsden, 1968, 1970). Morine (1959) proposed that the surface mucins act to protect endoparasitic helminths from digestion by inhibiting the interaction of enzymes with polyelectrolytes of opposite charge. The catalytic activity of the phenolases is based on the valency change of the copper found in the prosthetic group from a cuprous to a cupric state (Kertesz and Zito, 1962). Phenoloxidases normally consist of an inactive precursor, an activator enzyme, and possibly other components (Harowitz and Fling, 1955). The results seem to indicate that the polyanionic mucins associated with the glycocalyx apparently inhibit the tyrosinase by complexing the Cu2+. Blocking the action of tyrosinase on its normal substrate would prevent the production of toxic substances (quinones) and ultimately melanization. This inhibition would limit the cockroaches ability to overcome the developing acanthocephalan. The importance of the divalent actions to the host defense mechanism is suggested by the reduced encapsulation of the hymenopterous parasite Cardiochiles nigriceps in Heliothis zea following the injection of EDT A (Brewer and Vinson, 1971). Chelating agents are also able to dissociate hemocytes involved in encapsulation (Reik, 1968). Whether the polyanionic mucins could act as a chelating agent or merely complex with the divalent cations is unknown. Moniliformis dubius acanthors are normally surrounded by several layers of hemocytes immediately after penetration of the gut. These layers may persist for 4-6

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71

days (Robinson and Strickland, 1969), although Mercer and Nicholas (1967) reported the presence of hemocytes on the acanthor 30 days postinfection. Since melanization occurs when the tyrosinase is released following autolysis of the innermost blood cells of the capsule (Salt, 1970), the survival of the acanthor would be dependent upon preventing the lysis of the hemocytes or blocking the action of phenoloxidases. If autolysis of the innermost cells is dependent upon mechanical pressure exerted throughout the capsule, it would appear that the acanthor could do little to prevent lysis. However, it seems reasonable to assume that the acanthor may be able to inhibit the action or activation of tyrosinase. There would be two crucial periods during the development of the larval acanthocephalan: (1) during hatching when its protective amorphous material may be at least partially removed, and (2) during ecdysis when the phenoloxidases titer in the hemocoel is high. This last factor may help explain the 16% melanized cystacanths recovered from naturally infected cockroaches in Hawaii (Schaeffer, 1970). Injection of saline or immunogen into the cockroach may have provoked two responses; a wound healing response and the release of diuretic hormone to restore blood volume. Both responses might increase hemocyte permeability to tyrosine or lysis of hemocytes containing phenolosidases (Mills and Whitehead, 1970). The presence of cystacanth envelope particles in the immunogen would inhibit the activation of the tyrosinase. This would account for the decreased melanization of the blood which occurred in vitro. Additional reduction in melanization would be expected after the immunogen was added to the centrifuged plasma. Hemolymph from cockroaches injected with saline would have a high tyrosinase titer because no inhibitory substances would be present. This suggests that some substance found in the immunogen is responsible for the inactivation of tyrosinase, and thus, ultimately for the

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ability of the acanthocephalan to withstand the normal defense reaction of the cockroach. It is also possible that the cations in Yeager's physiological cockroach saline may have contributed to the lysis of the hemocytes by altering the ionic charges of the hemocyte membrane. In the shamoperated controls, the integrity of the hemocyte membranes would be unaffected by either the ions present in the saline or the effects of an increased titer of the diuretic hormone. These factors may be responsible for the lack of melanization in hemolymph collected from sham-operated cockroaches. ACKNOWLEDGMENTS The assistance of Dr. Erik Rifkin and Messrs. Herbert Vee and Peter Galloway with the electrophoretic procedures is gratefully acknowledged. Appreciation is also extended to Drs . M. T am ashiro, W. C. Mitchell , and F. C. Chang for their suggestions during the preparat ion of the manuscript. The senior author was supported in part by an NDEA Fellowship (Title IV).

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Moniliformis

intermediate host of Moniliformis moni/iformis (Bremser) in Honolulu, Hawaii. Proc. He/mintho/. Soc. Wash., 37, 204-207. SEAMAN, G. R. AND ROBERT, N. L. 1968. Immunological response of male cockroaches to injection of Tetrahymena pyriformis. Science, 161, 1359-1361. SEKERIS, C. E. AND MERGENHAGEN, D. 1964. Phenoloxidase system of the blowfly, Calliphora erythrocepha/a. Science, 145,68-69. SIAKOTOS, A. N. 1960. The conjugated plasma proteins of the American cockroach. II. Changes during the molting and clotting processes. J. Gen. Physiol., 43, 1015-1030. WANSON, W. W. AND NICKOL, B. B. 1973. Origin of the

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envelope surrounding larval acanthocephalans. J. Parasito/.,59,1147. WRIGHT, R. D. 1971. The egg envelopes of Moniliformis dubius. J. Parasito/., 57,122-131. WRIGHT, R. D. AND LUMSDEN, R. D. 1968. Ultrastructural and histochemical properties of the acanthocephalan epicuticle. J. Parasitol., 54, 11111123. WRIGHT, R. D. AND LUMSDEN, R. D. 1970. The acanthor tegument of Moni/iformis dubius. J. Parasito/ .. 56, 727-35. YEAGER, J. F. AND KNIGHT, H.·H. 1933. Microscopic observations on blood coagulation in several different species of insects. Ann. Entomo/. Soc. Amer.. 26, 591-602.