- Email: [email protected]
Defense reactions of mosquitoes to filarial worms: Role of the microfilarial sheath in the response of mosquitoes to inoculated Brugia pahangi microfilariae
IOURNAL
OF INVERTEBRATE
PATHOLOGY
4,
275-281 (1984)
Defense Reactions of Mosquitoes to Filarial Worms: Role of the Microfilarial Sheath in the Response of Mosquitoes to Inoculated Brugia pahangi Microfilariae DANIEL R. SUTHERLAND,
BRUCE M. CHRISTENSEN,
AND KEITH
E FORTON
Department of Veterinary Science, 1655 Linden Drive, University of Wisconsin, Madison, Wisconsin 53706 Received January 24, 1984; accepted April 23, 1984 The melanization response of Aedes trivittatus and A. aegypti (black-eyed Liverpool strain) against intrathoracically inoculated sheathed and chemically exsheathed Brugia pahangi microfilariae (mff) was assessed daily through 5 days postinoculation (PI). Response of A. aegypti against exsheathed mff was significantly reduced on all days compared with the response against sheathed mff, and a significantly greater percentage of exsheathed mff were alive through 4 days PI than were sheathed mff. The melanization response of A. trivittatus was nearly 100% effective against either sheathed or exsheathed mff by Day 2 PI. When mff were allowed to migrate through A. aegypti midguts in vitro before inoculation into intact A. aegypti, nearly 94% (120/128) of the parasites recovered had avoided the response and were developing. Penetration of A. trivittatus midguts in vitro by mff before inoculation into intact A. trivittatus did not prevent a melanization response. Inoculation of mff into A. trivittatus following A. aegypti midgut penetration, however, resulted in almost 60% (98/171) of the mff avoiding the response and developing as normal L, larvae after 5 days PI. The possibility of mff acquiring host antigens during midgut penetration and therefore avoiding recognition as nonself by mosquitoes, and (or) the possibility of the midgut environment modifying or stimulating mff to inhibit the response of mosquitoes are discussed. 0 1984 Academic
Press, Inc.
Brugia pahangi; Aedes aegypti; Aedes trivittatus; exsheathment, chemical; encapsulation; melanization; response, defense; sheath: microtilaria; papaya extract; mosquitoes; Filarioidea. KEY WORDS:
INTRODUCTION
The ability of mosquitoes (either susceptible or refractory strains) to encapsulate and melanize Dirofiluria immitis microfilariae (mff) after intrathoracic inoculation was easily explained by the abnormal environment in which mff were placed (Christensen et al., 1984). The response observed against Brugia pahangi mff inoculated into the hemocoel of normally susceptible mosquitoes, however, was not expected. This filarial worm normally is exposed to the hemocoel environment during its migration from the midgut to the thoracic musculature, where development occurs. Because B. puhungi mff have been reported to exsheath within the midgut prior to penetration (Esslinger, 1962; Ewert, 1965), it was postulated that perhaps the sheath present on inoculated mff stimulated a response in
mosquitoes we examined (Christensen et al., 1984). The present study therefore was designed to determine the role the microtilarial sheath might play in the defense response of mosquitoes to intrathoracically inoculated B. puhungi mff. MATERIALS
AND METHODS
Two mosquito species (Aedes trivittutus and A. uegypti black-eyed Liverpool strain), normally susceptible to B. puhangi infections, were used in this study. Source of mosquitoes and B. puhangi mff, and methods for rearing and inoculating mosquitoes were as previously described (Christensen et al., 1984). In order to determine if exsheathed mff could avoid the mosquito response, B. puhangi mff were isolated and concentrated from jird blood as described by Christensen
275 0022-201 l/84 $1.50 Copyright .,. r.
6 1984 by Academic Press. Inc. .c .-. A.._. :__ :_ “_.. ‘Ye... -^r^r,,aA
276
SUTHERLAND,
CHRISTENSEN,
et al. (1984). Isolated mff were then chemically exsheathed using methods of Devaney and Howells (1979). Briefly, mff were incubated for 30 min in Hank’s balanced salt solution (HBSS) containing 3 active units/ml protease (Type III crude papaya extract, Sigma) at 37°C and a pH of 7.0. Following incubation, mff were washed twice in Aedes saline (Hayes, 1953) by mixing and centrifuging before inoculation into mosquitoes. Control mosquitoes received mff treated in the same manner except that they were incubated in HBSS without enzyme. Four- to 6-day-old adult female mosquitoes were individually inoculated with 510 exsheathed or sheathed mff, and individuals were dissected daily through 5 days postinoculation (PI). Number of mff recovered, percentage melanized, and percentage living were determined for each mosquito dissected. One replicate of experiments with A. trivittutus and two replicate experiments with A. uegypti were conducted. Results of replicate experiments were not significantly different (x’ analysis); therefore, data were combined. To ascertain if our methods of chemical exsheathment altered mff as regards the mosquito response, we allowed mosquitoes to fully engorge by blood feeding on a microfilaremic jird. Immediately after feeding the midgut was removed (with foregut, hindgut, and Malpighian tubules attached) and placed in Aedes saline in a depression slide. Microfilariae that penetrated through the midgut were aspirated into a micropipette, and 5-10 were inoculated into individual 4- to 6-day-old adult mosquitoes. Inoculated mosquitoes were held for 5 days PI and then were dissected, and the response was evaluated. The following experiments were conducted: (1) A. aegypti midgut-derived mff were inoculated into A. aegypti, (2) A trivittutus midgut-derived mff were inoculated into A. trivittutus, and (3) A. aegypti midgut-derived mff were inoculated into A. trivittutus. To further examine effects of chemical
AND
FORTON
exsheathment on microfilarial viability, sheathed and chemically exsheathed mff were examined with transmission electron microscopy. Microfilariae were fixed overnight at 4°C in Karnovsky fixative (3% glutaraldehyde and 4% paraformaldehyde in 0.2 M sodium cacodylate-HCl buffer) and placed in a drop of molten 3% agar solution following the procedure of Scott et al. (1970). The agar cube, containing mff, was postfixed in 1% osmium tetroxide for 2 hr, dehydrated through graded alcohols to propylene oxide, and embedded in Epon expoxy resin. Thin sections were cut with a diamond knife on an LKB Ultratome III 8800, mounted on uncoated 300-mesh copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips EM 410 at 60 kV. Results were analyzed statistically using R x C contingency tables with Chi-square analysis and Student’s t test on a HewlettPackard HP-86 microcomputer. Differences were considered significant at P < 0.05. RESULTS
AND DISCUSSION
The melanization response of A. aegypti against chemically exsheathed B. pahangi mff was significantly reduced on all days examined compared with controls (Fig. 1). Likewise, the percentage of exsheathed mff living was significantly greater through 4 days PI (Table 1). In A. trivittutus, however, the response against sheathed and chemically exsheathed mff was the same, except on Day 1 PI. Approximately 91% (107/118) of chemically exsheathed mff recovered from 20 A. trivittutus at Day 1 PI were melanized, whereas 100% (lOl/lOl) of sheathed mff from 15 mosquitoes were melanized (P < 0.01). On Days 2-5 PI, however, no difference was noted in the percentage of 444 sheathed and 453 exsheathed mff melanized from 56 and 72 A. trivittutus, respectively. Essentially 100% (97- 100%) of recovered mff were melanized in either group following Day 1 PI. A. trivittutus can naturally support development of B. puhangi to a certain degree (Christensen et
MOSQUITO
RESPONSE
TO MICROFILARIAL
277
SHEATH
sheathed m
31
exsheathed
185
31 184
r
31 212
T
24 83
21 27 124
82
::z :gg E:.J$ A.. . A..... ~@~
:::::: ::::::: 2:s ;:g . $&;:g ...A.... A. :::::::::;::::: 5. A. .. ...A.... ...A. A. ::::::::::::::: ::::::::::::::: ::::::::::::::: x:::::::::::: (.,.,.,.,.,.,. y:::::::::::: ::::::::::::::: y:::::::::::: :::::::m: ... .. ... ...n. :::::::::::::::
31 169
20
::: ::::::. ::: ::::::. :.. i:.:. :;:#:$:i y::::::::: i.......... g$g; g$$$$ ggg :<::::s::: $ggg; ,:::::::: y . ... .. ...i ‘gg$ 1
11
i$$$g :::::::.:::;::: :.:.;.&:.:.: :g:ggs $$@;:; :gw ::::::::::::::: gg$g$~ ~~~:~:~:~
3
~
4
:.:.:.: :;:g
.:.:.: #:$
i;$$,.:$g :::.:::::g: g$g$; y::::::::::: :g:::::::: ::::::::::::::: ;.:.:.:.:.:.:.: .:+:.:.:.:.:. :::::::::$j:: . . . .A.... r.. v . . ..A. . . . . ggg . ..a.. ..a. $?:m . . . ..A . ..A. ::y:s$:z ::::::.:.:.:.:. ::::::::::::::: :::~:~:~:~:::~: sw:z
II 5
DAYS (POSTINOCULATION) FIG. 1. Response of Aedes aegypri against inoculated sheathed and chemically exsheathed Brugia pahangi microtilariae (mff). Top number indicates mosquitoes examined; bottom number indicates mff recovered. Range bars indicate standard error of the mean. All comparisons between sheathed and exsheathed groips are significant (P < 0.05).
al., 1984), but this mosquito still has a very effective defense response against inoculated mff, even when they are exsheathed. Although we significantly reduced the response in A. aegypti by chemically exsheathing mff, more than 50% of the mff were in melanotic capsules by Day 5 PI. The black-eyed Liverpool strain of A. aegypti has been genetically selected for filarial worm susceptibility, and is an extremely effective laboratory vector. We have seldom noted a melanization response in this mosquito against B. pahangi obtained by natural feeding; therefore, we believed our chemical exsheathment procedure was not the entire answer as regards the avoidance of the mosquito response by B. pahangi.
It is possible that incubation of mff in enzyme not only results in exsheathment, but also may damage the cuticular surface or alter the physiological state of mff so that viability is reduced. Chemically treated mff thus may not be as competent in avoiding the mosquito response and (or) may be more “visible” to mosquito responses as
compared with naturally exsheathed mff. However, comparison of sheathed (Fig. 2) and chemically exsheathed (Fig. 3) mff revealed no apparent morphological change in the cuticle of artificially exsheathed mff. Exsheathed mff possess a normal cuticle consisting of three electron-dense outer membranes, with the outermost membrane showing breaks of continuity in the region of the annular grooves, as was described for B. pahangi by Laurence and Simpson (1974) and for five species of mff by McLaren (1972). It also should be noted that only exsheathed mff showing normal movement and activity were chosen for inoculation into mosquitoes. Our data indicate that artificially exsheathed mff were morphologically and physiologically normal, and it was the absence of the sheath that significantly reduced immunogenicity of mff to A. aegypti. When mff were allowed to migrate through mosquito midguts in in vitro before being inoculated into mosquitoes, we obtained some interesting results (Table 2). Brugia pahangi mff almost totally avoided
278
SURVIVAL
SUTHERLAND,
OF SHEATHED
CHRISTENSEN,
AND
FORTON
TABLE 1 (S) AND CHEMICALLY EXSHEATHED (ES) Brugia pahangi FOLLOWING INOCULATION INTO Aedes aegypti No. mosquitoes examined
Percentage mff living (*SE)
(MFF)
mff
Days PI
S ES
1 1
30 30
233 162
60.1 81.4
k 4.1 2 4.6
0.001
S ES
2 2
31 31
209 169
45.1 74.5
2 5.6 t 5.2
0.001
S ES
3 3
31 27
212 124
38.1 56.2
k 4.6 k 5.5
0.007
S ES
4 4
31 21
184 82
26.9 61.8
Z!Y3.8 _c 7.8
0.001
S ES
5 5
31 24
185 83
20.5 f 4.2 30.4 z? 5.2
0.072
the response in Liverpool strain A. aegypti if they were previously allowed to penetrate a Liverpool midgut, but penetration through an A. trivittatus midgut before inoculation did not allow them to avoid the response in A. trivittatus. In fact, very few mff were capable of penetrating an A. trivittatus midgut; therefore, only 10 mosquitoes were inoculated. We have subsequently examined the percentage of B. pahangi mff penetrating the midgut of A. trivittatus, and found that only 4.1% (161 389) of the mff ingested penetrated within 2 hr in the 34 midguts examined in vitro. Microfilariae allowed to penetrate A. aegypti midguts before inoculation into A. trivittatus showed a significantly improved ability to avoid the response in this species. Nearly 60% of the mff inoculated were developing as L, larvae (Table 2). In all previous inoculation studies we have conducted with mff and A. trivittatus, 100% of the mff have been in melanotic capsules by Day 5 PI. We believe there are three possible explanations for the increased ability of B. pahangi to avoid the mosquito response following penetration of the midgut of Liverpool strain A. aegypti: (1) During penetration of the midgut wall, mff acquire host materials and therefore are not rec-
No. mff recovered
MICROFILARIAE
P
ognized as foreign by the mosquito; (2) exposure to the midgut environment modifies or stimulates mff in such a way that they are able to actively inhibit the response in mosquitoes; or (3) both of the above reasons might be occurring. Subsequent investigations indicate that the method of exsheathment has little effect on the ability of mff to avoid the response. Brugia pahangi mff usually do not exsheath within the midgut of A. aegypti, but rather the sheath is probably weakened or broken at the anterior end of mff during penetration through the midgut, and is shed after mff have migrated into the hemocoel (Christensen and Sutherland, 1984). The hypothesis that B. pahangi mff become “coated” with mosquito material(s) during migration through the midgut seems to be the best explanation for data reported in this study. Host antigen sharing as a mechanism of evasion of the vertebrate immune response by parasites has been well documented (Damian, 1964, 1979), although a similar mechanism has not previously been suggested for a mosquito-parasite system. In other invertebrate-parasite models, however, this phenomenon has received considerable support, especially in the Schistosoma mansoni-Biomphalaria glabrata system (Stein and Basch, 1979;
MOSQUITO
RESPONSE
TO MICROFILARIAL
SHEATH
FIGS. 2, 3. Brugia pahangi microfilariae. Figure 2, sheathed microfilaria. Electron micrograph showing sheath (s) and cuticle (c). x 99,000. Figure 3, chemically exsheathed microfilaria. Electron micrograph showing cuticle (c). x 110,000. Note breaks in continuity of structure in annular rings in both figures (arrows).
279
280
SUTHERLAND,
RESPONSEOF Aedes aegypti MICROFILARIAE (MFF) Source of mff A. aegypti A. trivittatus A. aegypti
AND Aedes INOCULATED
Inoculated into A. aegypti A. trivittatus A. trivittatus
CHRISTENSEN,
trivittatus
AND FORTON
TABLE 2 AT 5 DAYS POSTINOCULATIONAGAINST Brugia
AFTER MIGRATING THROUGH MIDGUTS IN VITRO
A. aegypti
pahangi
OR A. trivittatus
No. mosquitoes examined
Percentage mff melanized
Percentage mff alive (developing L,s)
20 10 36
6.3 (8/128) 98.1 (51152) 42.7 (73/171)
93.7 (120/128)b 1.9 (l/52) 57.3 (981171)
a No. melanizedino. recovered. b No. developing as L,s/no. recovered.
Yoshino and Bayne, 1983). Lackie (1976) has provided evidence for antigen sharing as a means for Hymenolepis diminuta cysticercoids to avoid recognition as nonself by two genera of beetles, Tribolium and Tenebrio, and a locust, Schistocerca; although these studies require further clarification. Surface similarities of Moniliformis dubius larvae to insect host tissues (Periplaneta americana) is likely the mechanism whereby this parasite avoids the encapsulation response (Lackie, 1975), but Lackie and Lackie (1979) provide convincing evidence that the “mimetic” properties of the surface are inherent in the larvae and not due to incorporation of host material. At this time it is difftcult to speculate as to whether the gut environment in Liverpool strain A. aegypti stimulates the parasite to actively inhibit the normal defense reaction in the vector. Our present data indicate, however, that the avoidance or inhibition of the mosquito response is not inherent in the mff, but rather that this phenomenon is in some way host induced. It also is evident that the A. aegypti Liverpool strain midgut environment is different than that found in A. trivittatus. Why mff that migrate through a Liverpool midgut can avoid and (or) inhibit the response, even in A. trivittatus, but mff penetrating an A. trivittatus midgut cannot, remains to be determined. McGreevy et al. (1974) suggested that the genetic control of filarial worm susceptibility is related to the tissue site of localization and development within the mos-
quito vector. Perhaps the series of multiple alleles controlling Brugia susceptibility in A. aegypti also influence the properties of the midgut wall. ACKNOWLEDGMENTS We thank Becky Lasee and Karen Harris for technical assistance. This study was supported by National Institutes of Health Grant AI 19769.
REFERENCES CHRISTENSEN, B. M., AND SUTHERLAND, D. R. 1984. Brugia pahangi: Exsheathment and midgut penetration in Aedes aegypti. Tr. Amer. Microsc. Sot., 103. in press. CHRISTENSEN, B. M., SUTHERLAND. D. R., AND GLEASON, L. N. 1984. Defense reactions of mosquitoes to filarial worms: Comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and DirojZaria immitis microfilariae. J. Invertebr. Pathol., 44, 267-274. DAMIAN, R. T. 1964. Molecular mimicry: Antigen sharing by parasite and host and its consequences. Amer. Nat., 98, 129-149. DAMIAN, R. T. 1979. Molecular mimicry in biological adaptation. In “Host Parasite Interfaces” (B. B. Nickel, ed.), pp. 103-126. Academic Press, New York. DEVANEY, E., AND HOWELLS, R. E. 1979. The exsheathment of Brugia pahangi microtilariae under controlled conditions in vitro. Ann. Trop. Med. Parasitol.,
73, 227-233.
J. H. 1962. Behavior of microtilariae of Brugia pahangi in Anopheles quadrimaculatus. Amer. J. Trop. Med. Hyg., 11, 749-758. EWERT, A. 1965. Comparative migration of microfilariae and development of Brugiapahangi in various mosquitoes. Amer. J. Trop. Med. Hyg., 14, 254259. HAYES. R. 0. 1953. Determination of a physiological saline for Aedes aegypti CL.). J. &on. Entomol., 46, 624-627. ESSLINGER,
MOSQUITO
RESPONSE
TO MICROFILARIAL
LACKIE, A. M. 1976. Evasion of the haemocytic defense reaction of certain insects by larvae of Hymenolepis diminuta (Cestoda). Parasitology, 73, 97- 107. LACKIE, A. M., AND LACKIE, J. M. 1979. Evasion of the insect immune response by Moniliformis dubius (Acanthocephala): Further observations on the origin of the envelope. Parasitology. 79, 297-301. LACKIE, J. M. 1975. The host-specificity of Moniliformis dubius (Acanthocephala), a parasite of cockroaches. Int. J. Parasitol.. 5, 301-307. LAURENCE, R. R., AND SIMPSON, M. G. 1974. The ultrastructure of the microfilaria of Bragia, Nematoda: Filarioidea. Int. J. Parasitol., 4, 523-536. MCGREEVY, P. B., MCCLELLAND, G. A. H., AND LAVOIPIERRE, M. M. J. 1974. Inheritance of sucepti-
bility to Dirofilaria gypti.
281
SHEATH
Ann.
Trop.
immitis infection Med. Parasitol., 68,
MCLAREN, D. J. 1972. Ultrastructural crofilariae (Nematoda: Filarioidea).
in Aedes ae97-109. studies on miParasitology,
6.5, 317-332.
Scorr, K., TARIN, D., AND SHARP. J. A. 1970. Orientation of spherical specimens for ultra thin sectioning in selected planes by embedding in agar. J. Microsc.,
91, 217-220.
STEIN, P. C., AND BASCH, P. F. 1979. Purification and binding properties of a hemagglutinin from Biomphalaria
glabrata.
J. Invertebr.
Pathol.,
33, 10-18.
YOSHINO, T. P., AND BAYNE, C. J. 1983. Mimicry of snail host antigens by miricidia and primary sporocysts of Schistosoma mansoni. Parasite Immunol.. 5, 317-328.
OF INVERTEBRATE
PATHOLOGY
4,
275-281 (1984)
Defense Reactions of Mosquitoes to Filarial Worms: Role of the Microfilarial Sheath in the Response of Mosquitoes to Inoculated Brugia pahangi Microfilariae DANIEL R. SUTHERLAND,
BRUCE M. CHRISTENSEN,
AND KEITH
E FORTON
Department of Veterinary Science, 1655 Linden Drive, University of Wisconsin, Madison, Wisconsin 53706 Received January 24, 1984; accepted April 23, 1984 The melanization response of Aedes trivittatus and A. aegypti (black-eyed Liverpool strain) against intrathoracically inoculated sheathed and chemically exsheathed Brugia pahangi microfilariae (mff) was assessed daily through 5 days postinoculation (PI). Response of A. aegypti against exsheathed mff was significantly reduced on all days compared with the response against sheathed mff, and a significantly greater percentage of exsheathed mff were alive through 4 days PI than were sheathed mff. The melanization response of A. trivittatus was nearly 100% effective against either sheathed or exsheathed mff by Day 2 PI. When mff were allowed to migrate through A. aegypti midguts in vitro before inoculation into intact A. aegypti, nearly 94% (120/128) of the parasites recovered had avoided the response and were developing. Penetration of A. trivittatus midguts in vitro by mff before inoculation into intact A. trivittatus did not prevent a melanization response. Inoculation of mff into A. trivittatus following A. aegypti midgut penetration, however, resulted in almost 60% (98/171) of the mff avoiding the response and developing as normal L, larvae after 5 days PI. The possibility of mff acquiring host antigens during midgut penetration and therefore avoiding recognition as nonself by mosquitoes, and (or) the possibility of the midgut environment modifying or stimulating mff to inhibit the response of mosquitoes are discussed. 0 1984 Academic
Press, Inc.
Brugia pahangi; Aedes aegypti; Aedes trivittatus; exsheathment, chemical; encapsulation; melanization; response, defense; sheath: microtilaria; papaya extract; mosquitoes; Filarioidea. KEY WORDS:
INTRODUCTION
The ability of mosquitoes (either susceptible or refractory strains) to encapsulate and melanize Dirofiluria immitis microfilariae (mff) after intrathoracic inoculation was easily explained by the abnormal environment in which mff were placed (Christensen et al., 1984). The response observed against Brugia pahangi mff inoculated into the hemocoel of normally susceptible mosquitoes, however, was not expected. This filarial worm normally is exposed to the hemocoel environment during its migration from the midgut to the thoracic musculature, where development occurs. Because B. puhungi mff have been reported to exsheath within the midgut prior to penetration (Esslinger, 1962; Ewert, 1965), it was postulated that perhaps the sheath present on inoculated mff stimulated a response in
mosquitoes we examined (Christensen et al., 1984). The present study therefore was designed to determine the role the microtilarial sheath might play in the defense response of mosquitoes to intrathoracically inoculated B. puhungi mff. MATERIALS
AND METHODS
Two mosquito species (Aedes trivittutus and A. uegypti black-eyed Liverpool strain), normally susceptible to B. puhangi infections, were used in this study. Source of mosquitoes and B. puhangi mff, and methods for rearing and inoculating mosquitoes were as previously described (Christensen et al., 1984). In order to determine if exsheathed mff could avoid the mosquito response, B. puhangi mff were isolated and concentrated from jird blood as described by Christensen
275 0022-201 l/84 $1.50 Copyright .,. r.
6 1984 by Academic Press. Inc. .c .-. A.._. :__ :_ “_.. ‘Ye... -^r^r,,aA
276
SUTHERLAND,
CHRISTENSEN,
et al. (1984). Isolated mff were then chemically exsheathed using methods of Devaney and Howells (1979). Briefly, mff were incubated for 30 min in Hank’s balanced salt solution (HBSS) containing 3 active units/ml protease (Type III crude papaya extract, Sigma) at 37°C and a pH of 7.0. Following incubation, mff were washed twice in Aedes saline (Hayes, 1953) by mixing and centrifuging before inoculation into mosquitoes. Control mosquitoes received mff treated in the same manner except that they were incubated in HBSS without enzyme. Four- to 6-day-old adult female mosquitoes were individually inoculated with 510 exsheathed or sheathed mff, and individuals were dissected daily through 5 days postinoculation (PI). Number of mff recovered, percentage melanized, and percentage living were determined for each mosquito dissected. One replicate of experiments with A. trivittutus and two replicate experiments with A. uegypti were conducted. Results of replicate experiments were not significantly different (x’ analysis); therefore, data were combined. To ascertain if our methods of chemical exsheathment altered mff as regards the mosquito response, we allowed mosquitoes to fully engorge by blood feeding on a microfilaremic jird. Immediately after feeding the midgut was removed (with foregut, hindgut, and Malpighian tubules attached) and placed in Aedes saline in a depression slide. Microfilariae that penetrated through the midgut were aspirated into a micropipette, and 5-10 were inoculated into individual 4- to 6-day-old adult mosquitoes. Inoculated mosquitoes were held for 5 days PI and then were dissected, and the response was evaluated. The following experiments were conducted: (1) A. aegypti midgut-derived mff were inoculated into A. aegypti, (2) A trivittutus midgut-derived mff were inoculated into A. trivittutus, and (3) A. aegypti midgut-derived mff were inoculated into A. trivittutus. To further examine effects of chemical
AND
FORTON
exsheathment on microfilarial viability, sheathed and chemically exsheathed mff were examined with transmission electron microscopy. Microfilariae were fixed overnight at 4°C in Karnovsky fixative (3% glutaraldehyde and 4% paraformaldehyde in 0.2 M sodium cacodylate-HCl buffer) and placed in a drop of molten 3% agar solution following the procedure of Scott et al. (1970). The agar cube, containing mff, was postfixed in 1% osmium tetroxide for 2 hr, dehydrated through graded alcohols to propylene oxide, and embedded in Epon expoxy resin. Thin sections were cut with a diamond knife on an LKB Ultratome III 8800, mounted on uncoated 300-mesh copper grids, stained with uranyl acetate and lead citrate, and examined with a Philips EM 410 at 60 kV. Results were analyzed statistically using R x C contingency tables with Chi-square analysis and Student’s t test on a HewlettPackard HP-86 microcomputer. Differences were considered significant at P < 0.05. RESULTS
AND DISCUSSION
The melanization response of A. aegypti against chemically exsheathed B. pahangi mff was significantly reduced on all days examined compared with controls (Fig. 1). Likewise, the percentage of exsheathed mff living was significantly greater through 4 days PI (Table 1). In A. trivittutus, however, the response against sheathed and chemically exsheathed mff was the same, except on Day 1 PI. Approximately 91% (107/118) of chemically exsheathed mff recovered from 20 A. trivittutus at Day 1 PI were melanized, whereas 100% (lOl/lOl) of sheathed mff from 15 mosquitoes were melanized (P < 0.01). On Days 2-5 PI, however, no difference was noted in the percentage of 444 sheathed and 453 exsheathed mff melanized from 56 and 72 A. trivittutus, respectively. Essentially 100% (97- 100%) of recovered mff were melanized in either group following Day 1 PI. A. trivittutus can naturally support development of B. puhangi to a certain degree (Christensen et
MOSQUITO
RESPONSE
TO MICROFILARIAL
277
SHEATH
sheathed m
31
exsheathed
185
31 184
r
31 212
T
24 83
21 27 124
82
::z :gg E:.J$ A.. . A..... ~@~
:::::: ::::::: 2:s ;:g . $&;:g ...A.... A. :::::::::;::::: 5. A. .. ...A.... ...A. A. ::::::::::::::: ::::::::::::::: ::::::::::::::: x:::::::::::: (.,.,.,.,.,.,. y:::::::::::: ::::::::::::::: y:::::::::::: :::::::m: ... .. ... ...n. :::::::::::::::
31 169
20
::: ::::::. ::: ::::::. :.. i:.:. :;:#:$:i y::::::::: i.......... g$g; g$$$$ ggg :<::::s::: $ggg; ,:::::::: y . ... .. ...i ‘gg$ 1
11
i$$$g :::::::.:::;::: :.:.;.&:.:.: :g:ggs $$@;:; :gw ::::::::::::::: gg$g$~ ~~~:~:~:~
3
~
4
:.:.:.: :;:g
.:.:.: #:$
i;$$,.:$g :::.:::::g: g$g$; y::::::::::: :g:::::::: ::::::::::::::: ;.:.:.:.:.:.:.: .:+:.:.:.:.:. :::::::::$j:: . . . .A.... r.. v . . ..A. . . . . ggg . ..a.. ..a. $?:m . . . ..A . ..A. ::y:s$:z ::::::.:.:.:.:. ::::::::::::::: :::~:~:~:~:::~: sw:z
II 5
DAYS (POSTINOCULATION) FIG. 1. Response of Aedes aegypri against inoculated sheathed and chemically exsheathed Brugia pahangi microtilariae (mff). Top number indicates mosquitoes examined; bottom number indicates mff recovered. Range bars indicate standard error of the mean. All comparisons between sheathed and exsheathed groips are significant (P < 0.05).
al., 1984), but this mosquito still has a very effective defense response against inoculated mff, even when they are exsheathed. Although we significantly reduced the response in A. aegypti by chemically exsheathing mff, more than 50% of the mff were in melanotic capsules by Day 5 PI. The black-eyed Liverpool strain of A. aegypti has been genetically selected for filarial worm susceptibility, and is an extremely effective laboratory vector. We have seldom noted a melanization response in this mosquito against B. pahangi obtained by natural feeding; therefore, we believed our chemical exsheathment procedure was not the entire answer as regards the avoidance of the mosquito response by B. pahangi.
It is possible that incubation of mff in enzyme not only results in exsheathment, but also may damage the cuticular surface or alter the physiological state of mff so that viability is reduced. Chemically treated mff thus may not be as competent in avoiding the mosquito response and (or) may be more “visible” to mosquito responses as
compared with naturally exsheathed mff. However, comparison of sheathed (Fig. 2) and chemically exsheathed (Fig. 3) mff revealed no apparent morphological change in the cuticle of artificially exsheathed mff. Exsheathed mff possess a normal cuticle consisting of three electron-dense outer membranes, with the outermost membrane showing breaks of continuity in the region of the annular grooves, as was described for B. pahangi by Laurence and Simpson (1974) and for five species of mff by McLaren (1972). It also should be noted that only exsheathed mff showing normal movement and activity were chosen for inoculation into mosquitoes. Our data indicate that artificially exsheathed mff were morphologically and physiologically normal, and it was the absence of the sheath that significantly reduced immunogenicity of mff to A. aegypti. When mff were allowed to migrate through mosquito midguts in in vitro before being inoculated into mosquitoes, we obtained some interesting results (Table 2). Brugia pahangi mff almost totally avoided
278
SURVIVAL
SUTHERLAND,
OF SHEATHED
CHRISTENSEN,
AND
FORTON
TABLE 1 (S) AND CHEMICALLY EXSHEATHED (ES) Brugia pahangi FOLLOWING INOCULATION INTO Aedes aegypti No. mosquitoes examined
Percentage mff living (*SE)
(MFF)
mff
Days PI
S ES
1 1
30 30
233 162
60.1 81.4
k 4.1 2 4.6
0.001
S ES
2 2
31 31
209 169
45.1 74.5
2 5.6 t 5.2
0.001
S ES
3 3
31 27
212 124
38.1 56.2
k 4.6 k 5.5
0.007
S ES
4 4
31 21
184 82
26.9 61.8
Z!Y3.8 _c 7.8
0.001
S ES
5 5
31 24
185 83
20.5 f 4.2 30.4 z? 5.2
0.072
the response in Liverpool strain A. aegypti if they were previously allowed to penetrate a Liverpool midgut, but penetration through an A. trivittatus midgut before inoculation did not allow them to avoid the response in A. trivittatus. In fact, very few mff were capable of penetrating an A. trivittatus midgut; therefore, only 10 mosquitoes were inoculated. We have subsequently examined the percentage of B. pahangi mff penetrating the midgut of A. trivittatus, and found that only 4.1% (161 389) of the mff ingested penetrated within 2 hr in the 34 midguts examined in vitro. Microfilariae allowed to penetrate A. aegypti midguts before inoculation into A. trivittatus showed a significantly improved ability to avoid the response in this species. Nearly 60% of the mff inoculated were developing as L, larvae (Table 2). In all previous inoculation studies we have conducted with mff and A. trivittatus, 100% of the mff have been in melanotic capsules by Day 5 PI. We believe there are three possible explanations for the increased ability of B. pahangi to avoid the mosquito response following penetration of the midgut of Liverpool strain A. aegypti: (1) During penetration of the midgut wall, mff acquire host materials and therefore are not rec-
No. mff recovered
MICROFILARIAE
P
ognized as foreign by the mosquito; (2) exposure to the midgut environment modifies or stimulates mff in such a way that they are able to actively inhibit the response in mosquitoes; or (3) both of the above reasons might be occurring. Subsequent investigations indicate that the method of exsheathment has little effect on the ability of mff to avoid the response. Brugia pahangi mff usually do not exsheath within the midgut of A. aegypti, but rather the sheath is probably weakened or broken at the anterior end of mff during penetration through the midgut, and is shed after mff have migrated into the hemocoel (Christensen and Sutherland, 1984). The hypothesis that B. pahangi mff become “coated” with mosquito material(s) during migration through the midgut seems to be the best explanation for data reported in this study. Host antigen sharing as a mechanism of evasion of the vertebrate immune response by parasites has been well documented (Damian, 1964, 1979), although a similar mechanism has not previously been suggested for a mosquito-parasite system. In other invertebrate-parasite models, however, this phenomenon has received considerable support, especially in the Schistosoma mansoni-Biomphalaria glabrata system (Stein and Basch, 1979;
MOSQUITO
RESPONSE
TO MICROFILARIAL
SHEATH
FIGS. 2, 3. Brugia pahangi microfilariae. Figure 2, sheathed microfilaria. Electron micrograph showing sheath (s) and cuticle (c). x 99,000. Figure 3, chemically exsheathed microfilaria. Electron micrograph showing cuticle (c). x 110,000. Note breaks in continuity of structure in annular rings in both figures (arrows).
279
280
SUTHERLAND,
RESPONSEOF Aedes aegypti MICROFILARIAE (MFF) Source of mff A. aegypti A. trivittatus A. aegypti
AND Aedes INOCULATED
Inoculated into A. aegypti A. trivittatus A. trivittatus
CHRISTENSEN,
trivittatus
AND FORTON
TABLE 2 AT 5 DAYS POSTINOCULATIONAGAINST Brugia
AFTER MIGRATING THROUGH MIDGUTS IN VITRO
A. aegypti
pahangi
OR A. trivittatus
No. mosquitoes examined
Percentage mff melanized
Percentage mff alive (developing L,s)
20 10 36
6.3 (8/128) 98.1 (51152) 42.7 (73/171)
93.7 (120/128)b 1.9 (l/52) 57.3 (981171)
a No. melanizedino. recovered. b No. developing as L,s/no. recovered.
Yoshino and Bayne, 1983). Lackie (1976) has provided evidence for antigen sharing as a means for Hymenolepis diminuta cysticercoids to avoid recognition as nonself by two genera of beetles, Tribolium and Tenebrio, and a locust, Schistocerca; although these studies require further clarification. Surface similarities of Moniliformis dubius larvae to insect host tissues (Periplaneta americana) is likely the mechanism whereby this parasite avoids the encapsulation response (Lackie, 1975), but Lackie and Lackie (1979) provide convincing evidence that the “mimetic” properties of the surface are inherent in the larvae and not due to incorporation of host material. At this time it is difftcult to speculate as to whether the gut environment in Liverpool strain A. aegypti stimulates the parasite to actively inhibit the normal defense reaction in the vector. Our present data indicate, however, that the avoidance or inhibition of the mosquito response is not inherent in the mff, but rather that this phenomenon is in some way host induced. It also is evident that the A. aegypti Liverpool strain midgut environment is different than that found in A. trivittatus. Why mff that migrate through a Liverpool midgut can avoid and (or) inhibit the response, even in A. trivittatus, but mff penetrating an A. trivittatus midgut cannot, remains to be determined. McGreevy et al. (1974) suggested that the genetic control of filarial worm susceptibility is related to the tissue site of localization and development within the mos-
quito vector. Perhaps the series of multiple alleles controlling Brugia susceptibility in A. aegypti also influence the properties of the midgut wall. ACKNOWLEDGMENTS We thank Becky Lasee and Karen Harris for technical assistance. This study was supported by National Institutes of Health Grant AI 19769.
REFERENCES CHRISTENSEN, B. M., AND SUTHERLAND, D. R. 1984. Brugia pahangi: Exsheathment and midgut penetration in Aedes aegypti. Tr. Amer. Microsc. Sot., 103. in press. CHRISTENSEN, B. M., SUTHERLAND. D. R., AND GLEASON, L. N. 1984. Defense reactions of mosquitoes to filarial worms: Comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and DirojZaria immitis microfilariae. J. Invertebr. Pathol., 44, 267-274. DAMIAN, R. T. 1964. Molecular mimicry: Antigen sharing by parasite and host and its consequences. Amer. Nat., 98, 129-149. DAMIAN, R. T. 1979. Molecular mimicry in biological adaptation. In “Host Parasite Interfaces” (B. B. Nickel, ed.), pp. 103-126. Academic Press, New York. DEVANEY, E., AND HOWELLS, R. E. 1979. The exsheathment of Brugia pahangi microtilariae under controlled conditions in vitro. Ann. Trop. Med. Parasitol.,
73, 227-233.
J. H. 1962. Behavior of microtilariae of Brugia pahangi in Anopheles quadrimaculatus. Amer. J. Trop. Med. Hyg., 11, 749-758. EWERT, A. 1965. Comparative migration of microfilariae and development of Brugiapahangi in various mosquitoes. Amer. J. Trop. Med. Hyg., 14, 254259. HAYES. R. 0. 1953. Determination of a physiological saline for Aedes aegypti CL.). J. &on. Entomol., 46, 624-627. ESSLINGER,
MOSQUITO
RESPONSE
TO MICROFILARIAL
LACKIE, A. M. 1976. Evasion of the haemocytic defense reaction of certain insects by larvae of Hymenolepis diminuta (Cestoda). Parasitology, 73, 97- 107. LACKIE, A. M., AND LACKIE, J. M. 1979. Evasion of the insect immune response by Moniliformis dubius (Acanthocephala): Further observations on the origin of the envelope. Parasitology. 79, 297-301. LACKIE, J. M. 1975. The host-specificity of Moniliformis dubius (Acanthocephala), a parasite of cockroaches. Int. J. Parasitol.. 5, 301-307. LAURENCE, R. R., AND SIMPSON, M. G. 1974. The ultrastructure of the microfilaria of Bragia, Nematoda: Filarioidea. Int. J. Parasitol., 4, 523-536. MCGREEVY, P. B., MCCLELLAND, G. A. H., AND LAVOIPIERRE, M. M. J. 1974. Inheritance of sucepti-
bility to Dirofilaria gypti.
281
SHEATH
Ann.
Trop.
immitis infection Med. Parasitol., 68,
MCLAREN, D. J. 1972. Ultrastructural crofilariae (Nematoda: Filarioidea).
in Aedes ae97-109. studies on miParasitology,
6.5, 317-332.
Scorr, K., TARIN, D., AND SHARP. J. A. 1970. Orientation of spherical specimens for ultra thin sectioning in selected planes by embedding in agar. J. Microsc.,
91, 217-220.
STEIN, P. C., AND BASCH, P. F. 1979. Purification and binding properties of a hemagglutinin from Biomphalaria
glabrata.
J. Invertebr.
Pathol.,
33, 10-18.
YOSHINO, T. P., AND BAYNE, C. J. 1983. Mimicry of snail host antigens by miricidia and primary sporocysts of Schistosoma mansoni. Parasite Immunol.. 5, 317-328.