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tease. If "'as activated in Research Note prophenoloxidase the circulating haemolymph of the mosqui- toes when the parasites invaded them, a I･n vibro adhesion of enzyme(s) related total melanizatien of the haemolymph would to melanin formation in the pupal occur. However, this has never seen in mosquito haemolymph to the surfaee the haemolymph of Armigeres subalbatus, of Brugia paha･ngi mierofilaria though a strong encapsulation "'as observed around microfilariae (Mf) of Bru.aia mata)'i Mutsuo KoBAy.xsHi,* Keilco YAMttDA* (Yamamoto et al. 1985). and Hisashi YAMAMoTo" From an ultrastructural observation of the

melanization in Culex Department of AIedical Zoolog),. Dokkyo processes pipi`ni.s larvae to the nematode, AieoaPlectana University School of AIedicine, carPocaPsae, and Tochigi 321-02. JaPan Poinar Leutenegger (1971) proposed that melanin is forined from April 2, 19g7) {Received: eomponents in the noncellular portion of the haemolymph coagulated on the surface Key words: mclanin formation, Tnosquito of the nematode. Other ultrastructural

haernolymph, enzyme adhesion, observations ef encapsulated )'If in tl]e microfilaria, Brugia Pahangi, mosquitoes strongly suggest that capsule Armigeres subalbatus. materials contain melanin (Chen and Lau- rence, 1985; Forton et al., 1985),. Vey and G6tz (1975) also observed that in vitro humoral encapsulation occurred on ent,onio. Numereus reports have been publislied on fungi, a certain foreign bod}r and the melanization and!or encapsulation of the genous nematodes in the ]arval Chironomus haemo- filarial larvae in mosquitoes (Burton, 1963J Iymph. Kobayashi et al. descrihecl Oothman et al., 1974; Christensen, 1981; (1986a) the adhesion of a light brown substanee to Yamamoto et ag,, 1985; Kobayashi et al., the B. mata),i Mf in the abdomirml haemo- 19.86a,b). However, the manner in which coele of Ar. subalbatus within 30 rriin melanin is formed on the larval surface is i]e$t- infection. To date, however, the chemical not understood. There are two hypotheses: natures of such capsules adhered to the sur- 1) the haemoc>,tes initiate the melanin for- face of the invading during the mation (Forton et al., 1985), and 2) hu- parasite initial processes of humoral encapsulation rnoral encapsulation takes place around the have net been anal}'zed. The present study larvae followed by cellular participation was carried out to learn the participation (Chen and Laurence, 1985; Koba}'ashi ct aJ., of enzymes in the haernolymph in the mela- 1986a). Humoral encapsulation is known nin forrnation on the surface of A'If, only in certain dipteran insects such as Brztgia )ff were collected from Culicidae and Chironomidae and the reac- pahangi the infected jirds by injecting warmed tion is correlated with the low count of Hanks' balanced salt solution /['HBSS) into haemocytes in the haemo]}'mph. In Insecta the cavity and then sucking it out. er Crustacea melanin is known to be svnthe- peritoneal ' The flLii'd containing )v{f was incubated for sized from aromatic amino acids such as 30min in plastic Petri dishes to eliminate tyrosine by phenoloxidase which is produced leucoci'tes from the host. Then )ff were i by an activation of prephenoloxidase. It collected by centrifugation after washing is probable that the activation of prophenol- t"'ice with HBSS. Packed },lf svere resus- exidase in the mosquitoes folleivs the path- '"'ere pended in disti]led "'ater and killed way proposed by Ashida and S6derh511 in a hot water bath (950C) for 5min. An (1984), that is, the activation is initiated aliquot of the A{f suspension "'as smeared by B-1,3-glucans with the aid of serine pro- on albuminized glass slides and the rest was -300C kept in a freezer at for later use. *- ,S'ik me ts, ph mi{i:, tu ts za : re th za ev<#eg ut -.L.,h JF 'es Haemo]ymph of 2-day old female of lp ## (T321-O: Sfi 1< JEJ, Uts Xl IE Ai wr ik ,lx pt pupae strain) was collected by 88e) Ar. subalbatus (406

143

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the centrifugation method according to ,: / ' Ogura et al. with some modifica- (1985) :- tt tions, that is, an addition of a small amount ' ' ; of phenylthiourea (PTU) to centrifugation tubes and spinning at 1,500g for 5min in

a second centrifugation to eliminate cells or

cellular components. Five to 6pl of the supernatant was immediately put on the ' glass slides previously smeared with Mf as described above. These had been kept in a wet chamber for 2 hr at room temperature t. ttt t before the second incubation for pigment Fig. 1 In vitro melanization of heat-killed deposition on the Mf. Haemelymph on the rnicrofilaria of Brugia pahangi incubated slides was found to have no formation glass with the supernatant of thermocoagulated of blackish pigment during this preincuba- haernolymph of pupal mosquito, Armigeres tion. Thereafter, the glass slides were washed subalbatus. twice with HBSS and covered with 5-6 Ml supernatant of thermocoagulated haemo- Table1 Adhesion of enzyme(s) related to lymph which had been prepared by heating melanin formatien in the haernolymph col- at 95eC for 5sec. They were incubated lected from pupal mosquitoes, Armigerej again for 1hr in a wet chamber at room subalbatus to the surface of heat-killed micro- temperature, and melanin formatien occur- filariae of Brugia pahangi in vitro. rin.ff on the Mf was observed under a micro-

scope. Fully andlor partia]ly melanized Mi Rate of Condition of No. of Mf , melanization were counted as positive (Fig. 1). "Then reactlon observed <%) Mf were incubated with both PTU-treated

and thermocoagulated haemolymph, 63.2% PTU-Haemo." and TC-Haemo.** 155 of Mf was melanized during the second 63.2 PTU-Haemo, and incubation (Table 1). In a contrel experi- HBSS*** 130 7.7 ment of l,If incubated with P[l"U-treated PTU-Haemo., haemolymph and HBSS, a small number of Heat-treatment**** 101130) showed a reac- Mf (7.7%; positive and TC-Haemo. 70149 o.oo.o tion though deposited on partially pigment TC-Haemo, only Mf was very thin as compared with that on * Mf incubated with thermocoagulated haemo- Preincubation of heat-killed pt{f on glass slides with PTU-treated haemolymph of lymph. When Mf on the glass slides were for 2 hr. incubated with PTU-treated haemolymph pupae '* Incubation with supernatant of thermo- and then heated at 950C for 20 sec no pig- coagulated(TC-) haemolymph of pupae ment formation on the Mf was observed for 1 hr. the during the second incubation with **' Incubation with HBSS for 1 hr. thermocoagulated haemo]ymph. Mf incu- *'** After preincubation glass slides were with haemolymph were bated PTU-treated heated at 95eC for 20 sec, not pigmented during the secend incubation with therrnocoagulated haemolymph previ-

ouslv added with a small amount of PTU. to detect attached substances around Mf in Theise data suggested that the heat unstable the preincubated specimens. Mf on the glass

enzyme(s) related to melanin formation ad- slides were incubated with PTU-treated hered to the surface of Mf during the pre- haemolymph for 2hr, washed twice with incubation and that a melanization reaction HBSS, and stained with O,05% CBB solution themse]ves were on the Mf surface occurred with the addition for 10min. Although Mf of substrate(s) or heat stable factor(s) dur- stained light blue, the majority showed a color on surface ing the second incubation. heavy blue their (Fig,2). Coomasie brilliant blue (CBB) was used This indicates that some proteinous sub-

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melanin formation. In the experi- ee 1/' present ･g･ "'ti l''ew rnents. in vitro adhesion of enzyme(s) to )If using L-DOPA as a substrate showed that these ]v･lf were melanized the same as those

incubated u.ith thermocoagulated haemo- l>'mph (unpublished data). this report the authors have shown the " In adhesion of an enzyme(s) 1)ossibl>' related

et' to melanin formation on the surface of )If .ti "'ithout apparent participation of the ceils Fig. 2 Adhesion of substances proteinous in the haemolymph of mosquitoes. stainecl "'ith Coomasie Brilliant BILte to the surface of hcat-kMcd microfilaria c,f Brugia AcKNovvLEt]GEMENTs pahangi. Arrows show heavy bltte deposLtion on the technical of surfacc of niicrofilaria, The able assistance Miss Michlko Okazaki is acknowledged.

stances in the haemolymph attach to the REFERENCES surface of Mf, and therefore strongly sug- 'I'he gests that these substances may be the en- Ashida, pt{. and K. S6derhli11 (1984): pro- zyme(s) associated with rnelanin formation phenolexidase activating system in crayfish. Comp. Biochem. Physiol. 77B: 21-26, or proteinous substances containing pro- BuTton, G. J. (1963): Encapsulation of IVuchere- phenoloxidase or active phenoloxidase. In ria bancrofti in seven species of mosquitues in the present experiments the authors did not British Guiana. Am. J. Trop. iVIed. Hyg.. 12: determine whether the enzyme(s) adherin.or 870B76. to the surface of Mf during the preincuba- Chen, C. C. and B. R. Laurellce (1985): An tion was or actiye prophenoloxidase phenol- ultrastructural study in the encapsulation of 'rn oxidase. t'Xnother experimental model is microfilariae of Brugia pahangi the haemo- needed to resolve this. In the definitelv coel of AnoPheles guadrimaculatus. Int. J. isolated haemolympfi of Chironomus Iars･,ae Parasitol., 15: 421-428.

Cheii, C. C. B. R. Laurence foreign organisms were initially completely and <1987a):In enclosed by a soft sticky material, then t,itro study on humoral encapsulation of micro- filariae: Establishment of technique and descrip- hardened, and finally brown pigmentation tion of reactien, Int. J. Parasitol. 17: 781-787. appeared (C6tz. 1986). The substances de- Chen, C. C. and B. R. Laurence (1987b): In tected in the present study might be analo- vitro study on humoral encapsulation of micro- gous to the materials observed in Chirono- filariae: Effects on diethyldithiocarbainate and mus. Haemagglutinin actisrity of the fluid dopachrome on the reactien. Int. J. Parasitol., collected from the of Armigeres sztb- pupae 17: 789-791. albatzts xvas high and the frequency of pig- Christensen, B. )'[. (198r): Observatien on the inoculated inent del)c)s.itien of the B. pahan.ai immune response of Aedes trivittatus against Mf was also high in the pupal stage {Ogura, Dirofilaria immitis. Tran,s. R. Soc, 7irop. J4ed. 1986). There is some possibility that Hyg., 75: 439-443. haemagglutinin mediates the attachment of Forton, K. F., B. M. Christensen and R. Suther- land Ultrastructure of the melanization enz>,me(s) to the surface of }If, although (1985>: response of Aedes trivittatus against inocu!ated the initial process of enzyme adhesion is still Dirofilaria immitis microfilariae. 1. Parasitol., obscure. Chen and Laurence (1987a. b] 71: 331-341. reported that transparent materials xs'ere G6tz, P, (1986): "･Iechanisms of encapsulatiun deposited on the surface of B, Mf Pahangi in dipteran hosts. In: Immune Mechanisms in in vitro using haemoly,mph of AnoPhetes Invertebrate Vectors (ed., I.ackie, A. M,),, pp. the of -17, quadrinzaculatttsin presenee phenol- 1 Oxford Science Publlshcr, New York. rl'suruoka, oxidase inhibitor. Hewever, in their paper Kobayashi, M., N. Ogura, H. Y. Chi- it was not clear whether the transparent gusa and S. Mishima (1986a): Studies on rnaterials contained an enzyme(s) related to filariasis VII: Histological observatlon on the

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ユ46 Jpn． J． San三t． ZooL − encapsu 】ated Brugia malayi larvae in the ab in vitro ・ ParasitotogユP， 70： 77＿86． dominal haemQcoele of the mosquitoes ， Armi − Yamamoto ， H ．， M ． Kobayashi ， N 。 Ogura ， H ． geres subalbatus ． ノPn ．ノ． Sanit． Zool ，， 37 ： Tsuruoka and Y ． Chigusa （1985）： Studies on − 59 65． 丘】ariasis VI ： The encapsulation of Brugia Kobayashi N and malayi and β the mosquito ， M ，， ，Ogura H ．Yamamoto ．pahangi larvaein ， − 「 （1986b）： Studies on 丘lariasis VIII ： Histo ∠drmigeres subalbatus ．ノ汐n ．丿． Sanit． Zoo 乙，36 ： logical QbservatiQn Qn thc abortive develoPエnent レ 6 ， of Brugia malayi larvae in the thoracic muscles of the mosquitoes ，・4rmigeres subalbatus ．∫pn ． 摘 要

． Zoo 〜．37 ： 127− 132． 17Sanit ， In bitr ・ の ニ ン 1986 ； Haemagglutinat ヨng activ 三ty に お け る 蚊 体 液 中 メ ラ 形成 Ogura ， N ． （ ） and 皿 elanin deposition on microfilariae of に 関与 す る酵素 の ミ ク ロ フ ィ ラ リ ア Brugia P α hangi and B ． malavi in the mosquito ， 体 表 へ の 付 着 Armigere∫ 5ubalbatu ∫． ノPn． J， Parasitol．，35 ： 542＿549． オオ ク ロ ヤ ブ カ の 雌蛹 よ り遠心 法 に よ っ て 集 め られ

Ogura N ． M ． Kobayashi and H ． た 体 液 と ，熱処理 で 殺 し た Brugia の ミ ク ロ ， ， YamamQto Pahangi フ M 正 （1985 ）： Haemagglutinating activity in Huid ィ ラ リ ア （ ） と を ス ラ イ ドグ ラ ス 上 で 2 時 間反 応 from the mosquitQ Armiger 彡5 sub − せ た ス ス ハ ン ク ス HBSS collected ， さ ．反応 後 ラ イ ドグ ラ を 液 （ ） albatus by centrifugation method ． ヱ）okkyo ノ． で 2 回 洗 浄 し ，加 熱 凝 固 さ せ た体液 上 清 と 1時 間反 応

ルfed Sci 12 ： −221 せ の 63 2 98 155 の Mf に ． ．， 217 ． さ た と こ ろ ， 全 体 ．％ （ ／ ＞ メ ラ

Oothman P． M ． G ． Simpsoロ and B ． R ． Laurence ニ ン の が こ っ た ． 2 の HBSS で ， ， 沈 着 起 回 目 反 応 を 行

（1974 ）： Abnormal development of a filarial っ た 場 合 は 全 体 の 7．フ％ （10／130）の Mf に 淡褐 色 の 被 worm Bru8ia Buckley Nelson and が せ の の ， patei （ ， 嚢 観 察 され た が ，加熱 凝 固 さ た体 液 上 清 み と

EleiSC］L i am ・ squito AnoPheles 励 ran ・ ＝ ェ ニ レ ）， ロ host， 反応 ， 第 の 反応系 に フ ル チ オ ウ ァ を添加 し た

‘ atro barvus von thOl ： の ス ス hiae ， ThieL ’、rJelmin． ．，48 揚合 ， ま た ，最初 の 2 時 間 反応 後 ラ イ ドグ ラ ．を − ． 161 165 95℃ ，20秒間加熱 した後， 加熱凝固体液上清 と反応 さ Poinar G 0 and R Leutenegger 1971 ： U 】tra・ ， ． ． ． （ ） せ た 場 合 に は 色 素 の 沈 着 が ま ・っ た く起 こ ら な か っ た ． structural investigation of tlle rne 王anizat 玉on 以 上 の 結果 よ り最初 の 反応 で メ ラ ニ ン 形 成 に 関与す る 量 in response process in Culex pipiens （Cu1 cidae ） 酵素 ま た は 酵 素 を含 む 蛋 白質 の Mf 体 表 へ の 付着 が起 to nematode ． ．Ultrastruct． Res ．35 ： 149 − 二 a ノ ， こ り，第 の 反 応 で は 加熱 凝 固体 液 の 上 清 中 に 含 ま れ

ニ 158、 て い る 基質 に よ っ て メ ラ ン の 沈着 が 起 こ っ た も の と Vey A ． and P． G δtz 1975 ： Humoral encapsu − ， （ ） 考 え ら れ る ． ］ation in Diptera （lnsecta）： Cornparative studies

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