Inst. OswaldoCruz, Rio de Janeiro,Vol. 82 (3) :425-430,jul./set., 1987

THE TRANSMISSION OF SUPRAPYLARIAN LEISHMANIA BY THE BITE OF EXPERIMENTALLY INFECTED SAND FLIES (DIPTERA: PSYCHODIDAE)

RYAN, R. LAINSON, J.J. SHAW & K. R. WALLBANKS*

The Wellcome Parasitology Unit, lnstituto Evandro Chagas,CaixaPostal3, 66001 Belem, PA, Brasil *Departament of Biological Sciences,University of Salford, Salford, M54WT, England

Lutzomyia furcata transmitted Leishmaniachagasi to a hamster10 days after being experi- mentallyfed on an infected spleen.An individualfemale Psychodopygus carrerai carrerai that had fed on a hamsterlesion causedby Leishmaniamexicana amazonensis transmitted this parasite6 days later to another hamster.Transmission electron microscopy of this fly's headrevealed a small number of degeneratepromastigotes in the foregut, but only a few wereattached.

Key word: sandflies Leishmania -transmission -Brazil

During the past 10 years, a number of al., 1973 for description). Flies were left in the workers have recorded the experimental trans- forest overnight(16.05.1985) in a holding cage mission of suprapylarian leishmanias (Lainson that contained a hamsterinfected with L. m. & Shaw, 1979) by the bite of sand fly species amazonensis (IFLA/BR/67/PH8 reference not thought to play any role in the epizootology stock). The hamster was anaesthetizedand of these parasites (Killick-Kendrick, 1979, restrained,with only the lesionsof the rear feet 1986). exposed(see Ryan et al., 1987a& b for ptoce- dures). The 85 femalesthat fed were taken to We report here the experimental transmis- our laboratory in Belem and of those that sion of Leishmania (L.) chagasi Cunha & Cha- survived oviposition, nine subsequently at- gas, 1937, by the bite of Lutzomyia furcata tempted to refeed on individual hamstersof- (Mangabeira, 1941), and L. (L.) mexicanaama- fered to them, some 6 days after their infec- zonensis by the bite of a sand fly of the genus tive meal. Immediately after the insects had Psychodopygus, P. ca"erai ca"erai (Barretto, probed and/or fed, the head of e~~h fly was 1946). placed in 2.5% glutaraldehydein Q:.07Mcac- odylate buffer for 1 hour and stored in buffer L. (L.) chagasi has previously been experi- for 5 weeksuntil it could be processedfurther. mentally transmitted (Lain son et al., 1977, The head was post fixed in 1% osmium tetro- 1984, 1985; Gonyalves et al., 1985; Stephenson xide for 1 hour, dehydratedin an ethnol series, & Ward, 1987) only by its natural vector, L. and treated in an ethanol/Spurr'slow viscosity longipalpis (Lutz & Neiva, 1912) and, as far as resinmixture before finally being embeddedin we are aware, there has been no laboratory the sameresin. Ultrathin sectionswere cut on a transmission reported of a suprapylarian Leish- LKB Ultratome III, mounted on uncoatedcop- mania by a Psychodopygus species. per grids, stained for 25 minutes in uranyl acetate and 10 minutes in lead citrate, and examined with a Corinth electron microscope MATERIALS AND METHODS at 60 kV. Transmissionof L. chagasiby L. furcata - Transmission of L. m. amazonensis by P. c. The parasite used was stock HOM/BR/75/L. ca"erai -We collected 149 female sand flies in dPP75 (M.2682) isolated from man in Bahia, a Shannon trap (Shannon, 1934) in the high Brazil (Lainson et al., 1977). The sand flies forest of Serra dos Carajas (N2 area, seeWard et were from our colony of L. furcata originally rearedby Dr. R.D. Wardand, at the time, in its 54th generation. The sand flies (6 days old) This work was carried out under the auspices of the were fed on a suspensionof heavily infected Wellcome Trust, London, the Instituto Evandro Chagas hamster spleentriturated in defibrinated, heat of the Funda~ao Servi~s de Saude PUblica of Brazil inactivated rabbit blood (Ward, 1977). The and the UNDP/World Bank/WHO Special Programme infected flies were.refed on hamsters10 days for Researchand Training in Tropical Diseases. later, after oviposition,and were offered a satu- Received February 3, 1987. rated sucrose solution throughout their main- Accepted April 14, 1987. tainance.

Mem.L. 426 L.RYANETAL.

RESULTS sick 8 months later and was killed -the liver Transmission of L. m. amazonensis -Of the and spleen were heavily infected with amasti- 9 flies which had probed or refed, two were gotes. uninfected, 2 were not examined and the remaining 5 had well developed infections of DISCUSSION the'cardia and proventriculus (Richards & Ri- This the first transmissionof L. chagasiby chards, 1971). Four of these infected flies were a sandfly specieswhich is not the natural vector P. wellcomei and the other was P. c. ca"erai. (Table) and the second record of L. furcata The latter fly had probed the rear right foot of transmitting Leishmaniaby bite. As mentioned a hamster for over 5 minutes and had succeeded elsewhere(Ryan et al., 1986a),L. furcata has, in obtaining a partial blood meal. The hamster to our knowledge,never beenfound naturally used for the P. c. ca"erai feed developed a small infected with flagellates,nor do we consider lesion at the site of the probe which was posi- this sand fly of importance in the transmission tive for amastigotes. Electron microscopic of L. (L.) chagasito man as it is not highly examination of the head showed only a light anthropophilic (Ryan et al., 1986b). infection of the pharynx and cibarium, and P. c.carrerai has recently been found infected most of these flagellates were unattached and by a Leishmaniaspecies (Caillard et al., 1986) degenerate(fig). No transmissions were obtained which is not of the mexicanacomplex. We have by the bite of P. welicomei. not detectedflagellates in 54~ wild caughtP c. Transmission of L. chagasi-Three of the L. carrerai(Ryan et al., 1987a& ) but this species furcata females that refed on the hamster were remainsa suspectas a vector of diseaseto man dissected, and all were heavily infected in the becauseof its markedanthropophilic behaviour cardia and proventriculus. The animal appeared (Wardet al., 1973).

Longitudinal section of the head of a female Psychodopygusca"erai ca"erai, infected with Leishmania mexicana amazonensis and fixed immediately post feed (and trans- mission), X6K, transmIssion electron micrograph. This shows the cuticular linings of the anterior pharynix (P) and remains of Leishmania with flagellar axonemes (L). TRANSMISSION OF LEISHMANIA 427

TABLE

Experimental transmissions of Leishmania speciesby the bite of sand flies

Leishmania Sand fly References

L. b. braziliensis P. wellcomei* Ryan et aI., (1987a) L. braziliensis P. s. maripaensis Ryan et al., (1987b) complex P. s.squamiventris** L. chagasi L. longipalpis Lainson et al. (1977, 1984, 1985); Gon~alves et aI. (1985), Stephenson & Ward (1987). L. furcata This paper L. donovani P. argentipes Shortt et al. (1931); Napier et aI. (1933); Smith et al. (1936,1940,1941) Swaminath et al. (1942). P. chinensis Feng & Chung (1941); Ho et al. (1943) Chung et aI. (1951); Yuan et al. (1943) P. longiductus Dergacheva & Strelkova (1985) P. smimovi Dergacheva & Strelkova (1985) L. garnhami L. townsendi Scorza et aI. (1984) L. infantum P. ariasi Rioux et a1. (1979) P. pemiciosus Pozio et al. (1985) L. major P.papatasi Adler & Ber (1941); Kryukova (1941); Killick~Kendrick et aI. (1985a & b); Warburg & Schlein (1986)*** P. dubosqi Beachetal.(1984) .' L. m. amazonensis L. flaviscutellata Ward et a1.(1977); Ryan et aI. (19863:)' Lainson et al. (1987) L. furcata Ryan et aI. (1986a) L. longipalpis Killick-Kendrick et a1. (1977) P. c. carrerai This paper L. m. mexicana L. anthophora Endris & Young, in K.-Kendrick (1986) L. cruciata Williams (1966) L. diabolica Lawyer & Young, in K.-Kend.itk (1986) L. longipalpis Coelho & Falcao (1962); Coelho et aI. (1967a&b) L. renei Coelho & Falcao (1962); Coelho et aI. (1967a&b) L. shannoni Lawyer & Young, in K.-Kendrick (1986) P. spp.+ Strangeways-Dixon & Lainson (1962,1966)

*This fly was possibly P. complexus, which occurs in the study area at proportions of 7 P. wellcomei: 1 P. complexus. **This fly was possibly P. chagasi, which occurs in the study area at proportions of 30 P. s. squamiventris: 1 P. chagasi. ***Transmission here refers to the ejection of Leishmania during a forced probe of the fly. + The identity of this species is discussedby Ready & Lainson (1982) and Williams (1983). This table is an unpdate of information given by Killick-Kendrick (1979, 1986).

The transmissionof L. m. amazonensisby sites at the proventriculus,resulting in so many this fly, which had a few degeneratingparasites of the flagellatesfound there being degenerate. in the pharynx and cibarium, possibly lends In our observationsthe degenerationmay pos- supportto the "blocked fly" theory of transmis- sibly be attributed to the delay in processing sion (Shortt & Swaminath, 1928), recently the fly. The difficulty experiencedby this fly discussedby Jefferieset al. (1986) and Warburg in obtaining a blood meal in the presentexpe- & ScWein(1986). Walters et al. (1987) suggest riment supports the suggestionof Killick-Ken- that the forms in the pharynx and cibariumare drick & Molyneux (1981) that infection with ephemeral,continuously generatedfrom para- Leishmaniain some way reducesthe ability of 428 L.RYANETAL. the sand fly to feed: a theory that has found COELHO, M. de V.; FALCAO, A.R. & FALCAO, A. support from other investigators(Beach et al., L., 1967a. Desenv01vimentode especies de genero Leishmania em especiesbrasi1eiras de flebotomos 1986). de genero Lutzomyia Franca, 1924. 111.Cicio vital de L. mexicana em L. longipalpis e L. renei. Rev. RESUMO Inst. Med. Trap. SIlO Paulo, 9 :229-303. A transmissaode Leishmaniasuprapilaria pe- COELHO, M. de V.; FALCAO, A.R. & FALCAO, A. Ia picada do t1ebotomineo infectado experi- L., 1967b. Desenvolvimento de especiesde genero Lei- mentalmente -0 protozoario Leishmania(L.) shmania em especies brasilieras de flebotomos de genero Lytzomyia Franca, 1924. IV. Infectividade chagasi foi transmitido experimentalmentea de leptomonas evoluindo no flebotomo e experien- urn hamsterpela picada do flebotomineo Lut- cias de transmissao de leishmanioses. Rev. Inst. zomyia furcata. Os insetos foram infectados Med. Trop. SIlO Paulo, 9 :367-373. atrav6sde uma membrana(pele de pinto), uti- DERGACHEVA, T.I. & STRELKOVA, M.V., 1985. lizando-sefonnas amastigotasprovenientes do Epidemiological role of sand flies Phlebotomus bayo de um hamsterinfectado. 0 ba~o foi tri- smirnovi Perfiliev, 1941 and P. longiductus Parrot, turado em ganguede coelho. A L. (L.) amazo- 1928 in visceral leishmaniasis foci in the Kazakh nensis foi transmitida a um hamsterpela pica- SSR. Trans. Roy. Soc. Trap. Med. Hyg., 79 :34-36. da do flebotomineo Psychodopygusc. carrerai. FENG, L.C. & CHUNG, H.L., 1941. Experiments on previamentealimentado em lesaode pele de um the transmission of kala-azar from dogs to hamsters outro hamsterinfectado com 0 parasita.0 exa- by Chinese sandflies. Chin. Med. J, 60 :489-496. me desse flebotomineo, atrav6s de microsc6- GON('ALVES, M. de N.A.; RYAN, L.;LAINSON,R. pia eletrfJnico,revelou um n6rnero pequenode & SHAW, J.J., 1985. The retained capacity of flageladosdegenerados, livres no lumen do in- Lutzomyia longipalpis (Lutz & Neiva) to transmit Leishmania chagasi (Cunha & Chagas)after 8 years testino anterior. Palavras-chave:flebotomlneos -Leishmania - (64 generations) in a closed laboratory colony. Mem. Inst. Oswaldo Cruz, 80 :337-338. transmissao -Brasil HO, E.A.; CHU, H.J. & YUAN, I.C., 1943. Transmis- sion of leishmaniasis to the Chinese hamster (Cri- cetulus griseus) by the bite of Chinese sandflies ACKNOWLEDGEMENTS (Phlebotomus chinensis). Chin. Med. J., 62 :207- We are grateful to Augusto F.N. Filho, lor- 209. lando da R. Barata,Sued de N.F. Silvas,J. Ita- JEFFERIES, D.; LIVESY, J. L. & MOLYNEUX, D. H., mar de Almeida, Manoel C.M. de Souza, J.B. 1986. Fluid mechanics of bloodmeal uptake by Leishmania-infected sandflies. Acta Trop., 43 :43- Palhetada Luz, A. Julio O. Monteiro, D. Ga1liza 53. Primo, J. Paulo N. Cruz and D. Lavin for inval- KILLICK-KENDRICK, R., 1979. 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