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Exsheathment of the Microfilariae of Brugia Pahangi in Armigeres Subalbatus

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Exsheathment of the Microfilariae of Brugia Pahangi in Armigeres Subalbatus [Jap. J. Parasit., Vol. 32, No. 4, 287-292, August, 1983] Studies on Filariasis II: Exsheathment of the Microfilariae of Brugia pahangi in Armigeres 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 Wuchereria bancrofti, 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 insects 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, Japan. Ten or 30 minutes after infective blood ( 37 ) meal, the abdomen of the mosquito 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 insect 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. ( 39 ) 290 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.
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