[Jap. J. Parasit., Vol. 32, No. 4, 287-292, August, 1983]

Studies on Filariasis II: Exsheathment of the Microfilariae of in subalbatus

Hisashi YAMAMOTO, Nobuo OGURA, Mutsuo KOBAYASHI and Yuichi CHIGUSA

(Received for publication; March 20, 1981)

Key words: filariasis, microfilariae, exsheathment, sheathed larvae, Brugia pahangi, Armigeres subalbatus

Microfilariae of some filarial species such of susceptible mosquitoes (Ewert, 1965). as , Brugia malayi In this paper, the authors present the and B. pahangi are enclosed within the result of the observations on the exsheath egg shell which forms a tubular sheath ment of B. pahangi microfilariae in Armi around the larval body (Devaney and geres subalbatus, one of the susceptible Howells, 1979). It has been reported that mosquitoes to the parasite. The results the sheath is cast in the midgut of the suggest the strong possibility that the ex and then unsheathed microfilariae sheathment of the microfilariae takes place burrow into the midgut wall to enter the in the abdominal haemocoel rather than abdominal haemocoel in susceptible mos in the midgut. quitoes (Aoki, 1971a). In fact, cast-off sheath of W. bancrofti microfilariae is Materials and Methods found among the ingested erythrocytes in the midgut of the vector mosquitoes Parasite (Wilkocks and Manson-Bahr, 1972). A mongolian jird, Meriones unguicula- It has been also presented that the ex tus, infected with B. pahangi was used for sheathment of B. pahangi microfilariae the source of blood meal. The average takes place almost immediately on their number of microfilariae in blood was 50.7 arrival to the midgut of Anopheles quad- per cmm. After brief anaesthesia by ethyl- rimaculatus and those which emerge from ether, the jird was fixed on a fixing appa the ingested blood mass are usually devoid ratus to expose to the mosquitoes. of sheath (Esslinger, 1962). However, the details of in vivo exsheathment in Brugia Mosquitoes spp. have not been fully understood, Armigeres subalbatus, Rendaiji strain, though it is known that the cast-off of the were reared at 26±1 C and 60±5% RH sheath occurs within a few hours after the in the insectarium. Mean infective rate of infective blood meals among some species the mosquitoes on the 9th day after in fective blood meal was 100%. This study was supported in part by a Scientific Research Grant from the Ministry of Education Dissection of mosquitoes and an outline (No. 557101). of in vitro cultures Department of Medical Zoology, Dokkyo Univer sity School of Medicine, Tochigi 321-02, . Ten or 30 minutes after infective blood

( 37 ) meal, the abdomen of the was hollow slide containing Ringer-Tyrode's dissected without tearing the midgut in a solution. After 1 hour, microfilariae wrig drop of distilled water on a slide. The gling on the bottom were sucked by a head, thorax and midgut were taken out capillary tubule and transferred again to and then the remainder was crushed. another hollow slide, in which they were Then, the specimens were covered with a kept for another 1 hour. Then, they were coverslip for examining the presence of injected into abdominal haemocoel of the the sheath under a microscope. female Ar. subalbatus by two ways, using The abdomen of mosquito was dissected a capillary tubule pointed at head. One in Ringer-Tyrode's solution containing group of the mosquitoes whose thoraco- penicillin (200 units/ml) and streptomycine abdominal junction were ligated by a fine (100 jug/ml), within 30 minutes of infective thread and the other one without ligation blood meal. The intact midgut taken out were received an innoculant of approxi carefully from a mosquito was submerged mately 0.8 pi GMA containing 30 to 50 in Grace's tissue culture medium microfilariae individually. The former was (GMA) which had been dropped on a kept at 26 C for 12 hours and then was hollow slide glass previously. Both the subjected to the examination of the sheath, thorax and abdomen of mosquitoes were while the latter was dissected for examin dissected at 2 hours after feeding and the ing the presence of the 3rd stage larvae intact alimentary canal was placed in on the 10th day after injection. GMA in the same way. On the other hand, collected micro The culture vessel, hollow slide glass, filariae were packed in a glass tubule, was kept at 26±1 C under the light for 2 10mm long, 1.0mm in diameter, sealed hours, then the larvae which emerged from both side, as a control and it was kept at the midgut were collected on a slide glass 26 C for 12 hours. Then, all the micro t oobserve the presence or absence of the filariae were examined whether the ex- sheath. GMA was supplemented with 20% sheathment had occurred spontaneously. heat-inactivated foetal bovine serum and the antibiotics were also added at the con Results centrations mentioned above. Dissection of mosquitoes after the infective Injection of microfilariae into mosquitoes blood meal About 10 jul of blood of a jird infected As shown in Table 1, 68 (83.9%) out of with B. pahangi was transferred to a. 81 which were recovered from abdominal

Table 1 Sheathed larvae* of Brugia pahangi in the abdominal haemocoel of Armigeres subalbatus dissected 10 minutes and 30 minutes after an infective blood meal

Time after infective No. (%) of sheathed larvae per larvae recovered blood meal In each abdominal haemocoel Total

22/29(75.8), 6/7(85.7), 19/22(86.3): 10 minutes 68/81(83.9) 17/19(89.4), 4/4(100)

2/8(25.0), 7/50(14.0), 1/7(14.2), 30 minutes 16/96(16.6) 6/31(19.3)

* Sheathed larvae are the larvae which have just come out from the midgut wrapped in the sheath.

( 38 ) 289 haemocoel 10 minutes after infective blood 93.7%. Forty (42.1%) out of 95 were ap meal were found to be in sheath, that is, parently microfilariae, i.e., sheathed, as a apparently microfilariae. Concerning to whole. the terminology of the larvae that just came out of the midgut and that were still Injection of microfilariae into mosquitoes in sheath will be discussed later. It was found that only 4 (2.0%) out of In case of 30 minutes-dissection 16 200 microfilariae were devoid of sheath (16.6%) out of 96 were sheathed. among those collected from the bottom of a hollow slide containing GMA. As shown Results obtained by the in vitro cultures in Table 3, it was also confirmed that the of the infected midgut microfilariae kept in sealed glass tubules The rate of microfilariae per larvae re for 12 hours showed only 1.4% exsheath- covered from each culture vessel ranged ment. from 22.5 to 98.5% in the culture of ex Microfilariae which had been inocu posed midgut taken out from mosquitoes lated into the abdominal haemocoels of within 30 minutes after an infective blood the ligated abdomens showed exsheath- meal. After all, 276 (64.9%) out of 425 ment of 45.3% on 12 hours post-injection. were found sheathed as seen in Table 2. Among 30 mosquitoes injected with 30 In those of alimentary canals of 2 hours to 50 microfilariae into the haemocoel after infection, it ranged from 10.5 to without ligation, 25 mosquitoes were alive

Table 2 Sheathed larvae of Brugia pahangi emerged from the midgut of Armigeres subalbatus to the artificial medium at 2 hours culture

No. (%) of sheathed larvae per larvae Time until setting recovered from the medium of in vitro culture In each culture vessel Total

7/31(22.5), 70/140(50.0), 26/46(56.5), Within 30 minutes 25/38(65.7), 32/46 (69.5), 16/20 (80. 0), 276/425 (64. 9) of blood meal 9/10(90.0), 21/23(91.3), 70/71(98.5)

Two hours after 2/19(10.5), 2/12(16.6), 3/9(33.3), 40/95 (42.1) blood meal 9/20(45.0), 9/19(47.3), 15/16(93.7)

The term of the sheathed larvae is explained in Table 1.

Table 3 Exsheathment of Brugia pahangi microfilariae in the haemocoel of the isolated abdomen by ligation between thoraco-abdominal junction of Armigeres subalbatus

No. (%) of exsheathed larvae per larvae recovered

In each abdominal haemocoel Total

Isolated abdomen 11/46(23.9), 12/36(33.3), 6/17(35.2), 20/54(37.0), 19/40(47.5), 9/15(60.0), 135/298(45.3) 15/25(60.0), 19/29(65.5), 24/36(66.6)

0/58 (0. 0), 0/196(0.0), 2/120(1.6), Glass tubule 7/480(1.4) 5/106(4.7)

Thrity to 50 microfilariae were injected into each isolated abdomen. Results were read 12 hours after injection of microfilariae.

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Table 4 The 3rd stage larvae obtained from Armigeres subalbatus injected with microfilariae of Brugia pahangi which were collected from blood of Meriones unguiculatus

No. of mosquitoes No. of the 3rd stage larvae Average injected obtained from each mosquito

0, 0, 0, 0, 0, 0, 0, 1, 1, 30 (5) * 1, 2, 2, 2, 3, 3, 3, 3, 4, 3.04 5, 5, 5, 7, 7, 10, 12

Each mosquito was injected with 30 to 50 microfilariae. Figure in parentheses* shows the number of mosquitoes died before 10 days after injection. on 10 day post-injection. Number of the filariae which once emerged from the mos 3rd stage larvae per mosquito are shown quitoes' midgut, regardless to sheathed or in Table 4. It was found that 3.04 (rang not, might have already started their de ing 0 to 12) larvae in average could com velopment. Therefore, the authors prefer plete their development in the mosquitoes the term "sheathed larvae" to indicate when microfilariae were injected. specifically those that just came out of the midgut and still wrapped in the sheath,

Discussion tentatively. As the term "sheathed larvae" will remain different opinions, the matter In the abdominal haemocoel of Armi should be considered and a decision be geres subalbatus 10 minutes after an in made in near future. fective blood meal with Brugia pahangi On the dissection of mosquitoes 30 microfilariae, about 80% of the larvae have minutes after infective blood meal, 85% been observed as microfilariae, judging of the larvae, recovered from haemocoel from the presence of the sheath. The term, had already exsheathed (Table 1). This microfilariae, has been commonly used in result suggests that the exsheathment takes the larvae found in the peripheral blood place in the abdominal haemocoel, and and/or tissues of the definitive host. On that sheathed larvae found early in the the other hand, there have been some haemocoel may abort their development confusions on the term which should be and some other larvae which exsheathed used exactly for the larvae of the very in the midgut just prior to penetrate it early stage in the intermediate host. could begin further development. In the For example, microfilariae, microfilari-form in vitro culture of the infected midgut, larvae, sheathed microfilariae and so on about a half of the larvae piecing out have been adopted in many literatures to through the midgut wall were provided describe those found in the midgut and/or with sheath irrespective of the time after abdominal haemocoel just after coming infective blood meal (Table 2). It was also out of the midgut. In the present study, found that the exsheathment of micro it was found that many larvae wrapped in filariae had occurred in 45.3% at the the sheath, apparently looked like micro abdominal haemocoel of the mosquitoes filariae, could come out of the midgut. ligated at the thoraco-abdominal junction The authors' opinion is that the term (Table 3). "microfilariae" should be used only in the Summarizing these results obtained, it stage of the definitive host. It seems may be concluded that the exsheathment reasonable to consider that the micro of microfilariae of B. pahangi ingested by

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At. subalbatus takes place in the abdomi some 45% of them exsheathed during 12 nal haemocoel. A finding that the injected hours post-injection. microfilariae into mosquitoes developed to 4) Average number of 3.04 3rd stage the 3rd stage strongly support this sugges larvae were found from the batch of 25 tion (Table 4). mosquitoes that had been injected with 30 Ewert (1965) reported the presence of to 50 microfilariae into haemocoel. sheathed larvae of B. pahangi in the thorax 5) From the results, it is clarified that and abdominal haemocoel of Anopheles the exsheathment of microfilariae of B. quadrimaculatus, Aedes aegypti and Ae. pahangi ingested by Ar. subalbatus takes albopictus. Therefore, the sheathed larvae place in the abdominal haemocoel of this of B. pahangi may also come out of the species. midgut even in other species, such as Ae. togoi and Culex pipiens. Acknowledgments In vitro exsheathment of microfilariae The authors wish to express their thanks to Miss in Wuchereria bancrojti, B. malayi, B. Junko Shimizu and Miss Masako Tamura in our pahangi and Litomosoides carinii has been laboratory for rearing jirds and mosquitoes. studied by some authors (Weinstein, 1963; Katamine and Aoki, 1970; Aoki, 1979a, References 1979b; Devaney and Howells, 1979). How 1) Aoki, Y. (1971a): Exsheathing phenomenon of ever, the factors responsible for inducing microfilaria in vitro (I). Tropical Med., 13, 134- exsheathment in vivo have not been eluci 140. dated (Devaney and Howells, 1979). 2) Aoki, Y. (1971b): Exsheathing phenomenon of Results obtained from the present ex microfilaria in vitro (II). ibid, 13, 170-179. 3) Devaney, E. and Howells, R. E. (1979): The periments indicate the importance of ab exsheathment of Brugia pahangi microfilariae dominal haemocoel for in vivo exsheath under controlled conditions in vitro. Ann. Trop. ment of B. pahangi microfilariae. Med. Parasit., 73, 227-233. 4) Esslinger, J. H. (1962): Behavior of micro filariae of Brugia pahangi in Anopheles quadri Summary maculatus. Amer. J. Trop. Med. Hyg., 11, 749- 758. 1) Microfilariae of Brugia pahangi taken 5) Ewert, A. (1965): Exsheathment of the micro by Armigeres subalbatus could penetrate filariae of Brugia pahangi in susceptible and the midgut wall without casting off their refractory mosquitoes. Amer. J. Trop. Med. sheath. On the dissection of 10 minutes Hyg., 14, 260-262. 6) Katamine, D. and Aoki, Y. (1970): Studies on after infective blood meal, 68 (83.9%) out exsheathing of microfilariae in vitro. In the of 81 were sheathed. Joint conference on Parasitic Diseases, The U.S. 2) In the short time in vitro cultures Japan Co-Operative Med. Sci. Programme. of the infected midgut which were taken 7) Wilcocks, C. and Manson-Bahr, P. E. C. (1972): out of mosquitoes, about a half of the Manson's Tropical Diseases, pp 1164, Bailliere Tindall, London. larvae were found sheathed in the medium. 8) Weinstein, P. P. (1963): Development in vitro 3) Among the microfilariae injected into of the microfilariae of Wuchereria bancrojti and the abdominal haemocoel which had been Litomosoides carinii as far as the sausage form. ligated at the thoraco-abdominal junction, Trans. Roy. Soc. Trop. Med. Hyg., 57, 236.

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