Sandfly diet and Leishmania

Parasitology Today, voL 2, no, 6, 1986

175

Sandfly Diet and Leishmania Y. Schlein The diet of female phlebotomine sandflies is made up of regular sugar meals and the infrequent blood meals which induce oogenesis. Thus the environment for Leishmania development in the gut comprises two different nutrient media and their respective digestive enzymes. Amastigote forms of the parasr~eare ingested with the blood meal which is gradually digested and is followed by sugar meals. During this time the amastigotestransform into various flagellated forms, including the infective forms which move forward into the proboscis to be transmitted to a new host when another blood meal istaken L2. Very little is known about the effect of sandfly diet or gut enzymes on the development of Leishmania. But there is evidence that establishment of infection in the gut and transmission by bite, both essentialfor the perpetuation of the para~ sites, depend on components of the sandfly meal. Blood Meal and Vector Specificity

Each vector of Old World Leishmania tends to transmit only one species of the paras~e. In contrast, New World vectors can support infections of various Leishmania species. The selectivity of Old World vectors was shown by artificial membrane feeding of Phlebotomus papatasi with meals that contained a small number of parasites in 50% rabbit serum 3. The susceptibility to infection with parasite strains not normally transmitted by this sandfly increased considerably when the flies ingested a large number of promastigates or when the meals were made up of 10% serum or saline.This suggeststhat under natural conditions the non-transmitted strains of Leishmania are destroyed by products of serum digestion3. In the sandfly gut, levels of proteases rise after the ingestion of blood4, and these enzymes could be the effectors of Leishmania selection. We compared the effects of the naturally transmitted L. major and of L. donovani (introduced artificially by membrane feeding) on the gut enzymes of P. papotasi; substrate digestion by gut preparations of flies harbouring L. major was a third less, and of similar preparations of L. donovani a third greater, than that of uninfected controls. But promastJgotes of both Leishmania species

enhanced the proteolytic activrty when added in vitro to control gut preparations (Y. Schlein and H. Romano, unpublished), This enhancement of activity is the direct effect of the parasites on the enzymes, so the decrease caused in vivo by L. major seems to result from inhibition of enzyme production. L. donovani, which is not adapted to this vector, seems unable to inhibit the production of gut enzymes, Other evidence for the apparent role of enzymes in vector competence is the death of L. major in P. papatasi fed on turkey blood before or after the infective meal. In this case the measured DNAase level induced bythe avian blood was much higher than that measured following rabbit blood meals5. These observations suggest that Leishmania can tolerate only a certain level of some enzymes in the gut. This level varies according to the source of the blood meal and could thereby render the vector unsuitable for harbouring the Leishmania it transmits in nature. The indiscriminate viability of Leishmania in the New World vectors is a trait of the sandflies.L major (= k tropica) from Israel, which is normally transmitted by P. papatasi in the Old World, can also infect the New World vectors Lutzomyia Iongipalpis and Lu. renei6. On the basis of their behaviour in the vector, mammalian species of Leishmania have been classified in two groups: the peripylaria which multiply in the hindgut, and suprapylaria which develop only anterior to the hindgut The peripylaria includes the L braziliensis complex, and the suprapylaria comprises other neotropical and Old World species7. However, suprapylarians can change their location. This is shown by studies of the

tolerance of South American sandflies(Lu. Iongipalpis and Lu. renei) to various infections - suprapylarian species such as L. major6, L. donovani 8 and L. mexicana 9, invaded the hindgut of these sandflies in about the same propo~on as the peripylarian L. braziliensis Io. It may be that location in the hindgut confers protection and enables survival of the various Leishmania species, because enzymes are secreted and food is digested in the midgut and not in the hindgut I'. The evidence that gut enzymes function in the elimination of non-transmitted Leishmania from the vector is circumstantJal; further investigation is needed to substantiate the findings and to clarify the mechanism. It would be interesting to study the manner in which the non4ransmitted infe~ons die out in the gut. and which enzymes are affected by the vectorspecific Leishmania, The role of enzymes other than proteases and DNAases (such as the glycosidases that have been described in Stomoxys.2) should also be investigated. Another aspect which merits study is the effect of different blood meals on the digestion and on the Leishmania parasites. Potential factors are the trypsin inhibitors present in the sera of animals which affect the enzyme activity of haematophagous Diptera to different degrees4. Sugar Meals Sugar meals are required for the transmission by bite of Leishmania, as was shown by the successful transmission of L donovani by P. argentipes, after feeding the sandflies on raisinsf3. However, the routine inclusion of sugar or raisins in the laboratory diet of sandfliesdoes not result in consistent transmission, apparently due to the absence of other necessarycomponents which are present in the natural diet of vectors. This point is illustrated by the location of L infantum in the gut of laboratory infected P. ariasi; in flies maintained in the laboratory, infections were limited to the midgut- whereas similarly infected flies, that had been released and recaptured, had parasites in the fore-gut and had therefore become potentJaltransmitt e r s 14,

Fig. I. Phlebotomuspapatasi female feeding from the stem of a caper plant (Capparis spin.a).

There is little information on the source of natural sugar meals for sandflies,except for the observation that P. papatasi feeds

~)1986, Elsevier SciencePublishers BM, Amsterdam 0169 4758/8(:~02 00

175

Parasitology Today, voi. 2, no. 6, I986

Sandfly Diet and Lei&monio Y. Schlein The diet of female phlebotomlne sandflles IS made up of regular sugar meals and the Infrequent blood meals which induce oogenesls. Thus the environment for Lershmonro development in the gut comproses two different nutrient media and their respective digestive enzymes. Amastigote forms of the parasite are ingested with the blood meal which is gradually digested and is followed by sugar meals. Duringthis time the amastlgotes transform Into various flagellated forms, Including the Infective forms which move forward Into the proboscis to be transmitted to a new host when another blood meal istaken l,2. Very little is known about the effect of sandfly diet or gut enzymes on the development of Leishmonia But there is evidence that establishment of infection m the gut and transmlsslon by bite, both essential for the perpetuation of the parasites, depend on components of the sandfly meal. Blood Meal and Vector Specificity

Each vector of Old World lelshmania tends to transmit only one species of the parasrte. In contrast. New World vectors can support infections of various Lelshmania species. The selectivity of Old World vectors was shown by artificial of Phlebotomus membrane feeding pupatasl with meals that contained a small number of parasites in 50% rabbit serum3 The susceptlbllity to infection with parasite strains not normally transmitted by this sandfly Increased considerably when the flies Ingested a large number of promastigotes or when the meals were made up of IO% serum or saline. This suggeststhat under natural condrtlonsthe non-transmitted strains of Lelshmanra are destroyed by products of serum digestjoG. In the sandfly gut levels of proteases rise after the ingestion of blood4, and these enzymes could be the effecters of Lenhmon/a selection. We compared the effects of the naturally transmitted I_.major and of 1. donovoni (Introduced artificially by membrane feeding) on the gut enzymes of P. popatasi; substrate digestion by gut preparations of flies harbouring L. major was a third less, and of similar preparations of I_.donovani a third greater, than that of uninfected controls. But promastigotes of both Leishmania species

enhanced the proteolytlc a&Q when added in vitro to control gut preparations (Y. Schlein and H. Romano, unpublished). This enhancement of activity is the direct effect of the parasites on the enzymes, so the decrease caused in vivo by L. major seems to result from inhibition of enzyme productlon. L. donovani, which IS not adapted to this vector, seems unable to Inhibit the production of gut enzymes. Other evidence for the apparent role of enzymes in vector competence is the death of L. major In P. pupatos~ fed on turkey blood before or after the Infedive meal. In this case the measured DNAase level Induced by the avian blood was much higher than that measured following rabbit blood meals5. These observations suggest that Leishmanio can tolerate only a certain level of some enzymes in the gut. This level varies according to the source of the blood meal and could thereby render the vector unsuitable for harbounng the Leishmania it transmits in nature. The IndiscrimInate viability of Leishmania in the New World vectors ISa trait of the sandflies. L major (= I_tropica) from Israel, which IS normally transmitted by P. papatas~ IN-I the Old World, can also Infect the New World vectors Lutzomyia longipa/pis and Lu. renei6. On the basis of thelr behaviour in the vector, mammalian species of Leishmonia have been classified In two groups: the peripylaria which m&ply in the hindgut, and suprapylarla which develop on!y anterior to the hindgut The perlpylarla Includes the L braziliensis complex, and the suprapylaria comprises other neotropical and Old World species’. However, suprapylanans can change their location. This IS shown by studies of the

Fig. I. Phlebotomus papatasi female feeding from the stem of 0 caper plant (Capparis spincsa).

tolerance of South American sandflies (LLJ longipa/pis and Lu. renei) to various Infectrons - suprapylarian species such as L. majoP, L. donovani and L mexiconag, Invaded the hlndgut of these sandflies in about the same proportron as the penpylarian L. brazilrensislo. It may be that location in the hindgut confers protection and enables survival of the various Leishmania species, because enzymes are secreted and food is digested In the midgut and not in the hlndgutll. The evidence that gut enzymes function In the ellminatlon of non-transmitted Leishmanla from the vector is circumstantial; further investigation ISneeded to substantiate the findings and to clarify the mechanism. It would be Interesting to study the manner in which the non-transmitted infectrons die out In the gut+ and which enzymes are affected by the vectorspecific Leishmania. The role of enzymes other than proteases and DNAases (such as the glycosidases that have been described in Stomoxys’2) should also be Investrgated. Another aspect which merits study ISthe effect of different blood meals on the dIgestion and on the Leishmania parasites. Potential factors are the trypsin Inhibitors present in the seta of animals which affect the enzyme a&v&y of haematophagous Dlptera to different degrees? Sugar

Meak

Sugar meals are required for the transmlsslon by bite of Lershmania, as was shown by the successful transmlsslon of L donovanr by P. orgentipes, after feeding the sandflies on raisinsl3. However, the routine inclusion of sugar or raisins in the laboratory diet of sandflies does not result in consistent transmission, apparently due to the absence of other necessary components which are present in the natural diet of vectors. This point is illustrated by the location of I_ infontum in the gut of laboratory Infected P. arias); In flies maintained in the laboratory, infectrons were limited to the midgut-whereas similarly infected flies, that had been released and recaptured, had parasites in the fore-gut and had therefore become potential transmitters14. There ISMe Information on the source of natural sugar meals for sandflies, except for the observation that P. pupatas~ feeds

Parasitology Today, vot 2, no. 6, 1986 may exert a marginal, but nevertheless constant selectrve pressure on the Leishmania, RA~ferel'l~ I Killick-Kendrick, I~ (1979) in Biology of the Kinetaplastida, vol. 2 (Lumsden, W.H.R. and Evans, D.A, eds) pp. 395-449, Academic Press, London 2 Molyneux, D.H. (1977)Adv. Para~tol. 25, 1-82 3 Adler, S. (1938)Harefuafl 14, I-6 4 Gooding, R.H. (1975) in Blood digestion rn flaemataphagous insects (Freyvogel, T.A. ed.) Acta Trap. 32, 96-111 5 Schlein, Y. et al. (1983)Acta Trap. 40, 65-70 6 Coelho, M.V., Falcao,A.FLand Falcoa,A.L. (I 967) Rev. Inst. Meal Trap Sao Paulo 9, 192-196 7 Lainson, FL and Shaw,J.J.(I 979) in Biology of the Kinetaptastida vol. 2 (Lumsden, W.H.R and Evans, DA. eds) pp. I 98, Academic Press London

177 8 Coe/ho, M.V.,Falcao,A.I~ and Falcao,A.L. (I 967) Rev. Inst. Med, Trap. Sao Paulo 9, 361-366 9 Coelho, M.V.,Falcao, AP~andFalcao, A.L.(1967) Rev. Inst. Med, Trap. Sao Paulo 9, 299-303 I 0 Coelho, M.V., Falcao,A.K and Falcao,A.L, (I 967) Rev. Inst. Trap. A4ed, Sao Paulo 9, 177-19 I I I Chapman, R.L. (1982) The Insects. Structure and Function 3rd edn, Hodder and Stoughton, London 12 Deloach, I.R and Spates, G,E (1984) Insect Biochem. 14, 169-173 13 Smith, P~O.A.,Holder, K.C and Ahmed, I.( 1941) Ind. J. Med. Res. 29, 799402 14 Killick-Kendrick, R, and PJoux, IA. (1981) in Parasitological Topics(Canning, E.U. ed.) pp. 136145, Societyof Protozoology SpecialPublicatJons 15 Schlein, Y. and Warburg, A. (1986)J. Mad. Entomol. 23, 11-25 16 Lis, H. and Sharon, N. (I 977) in The antigensvol. IV (Sela, M.ed.) pp. 429-529, Academic Press, New York 17 Jackobson, R.L, et al. (1982) Ann. Trap. Mad.

ParasitoL 76, 45-52 18 Brown, K.S.Jr (1975) Chem. Soc Rev.4, 263-288 19 AIjeboori, T,I. and Evans, DA. (1980)Trans. P~ Soc. Trap. Med. Hyg. 74, 169-I77 20 LeBlancq, S.M., Schnur, LF. and Peters, W. (1986) Trans. R. Soc. Trap. Med. Hyg. 80, 99-I 12 21 LeBlancq, S.M.(1985)ZymodemesofLe~hmania ~n the Old World. Vfl International Congress of Protozoology, p. 149, Abstract 392 22 Adler, S. and Theodor, O. (1929) Ann. Trap. Med. Parasitol. 23, I - 18 23 Adler, S. (1964)Adv. Parasitol. 2, 35-39 24 Schlein, Y. Warburg, A. and Yuval, B. Insect Sci Appl. (in press) 25 Schlein, Y., Polacheck, I. and Yuval, B. (1985) Parasitology 90, 57--66

YosefSchleinIsat the Departmentof Parasitology, Hadassah Medical School, PO Box 1172, Jerusalem, Israel

Microtubular Cytoskeletons of Parasitic Protozoa D.G. Russell and J.-F. Dubremetz Shape, motility and division of eukaryotic cells are all determined to some extent by intracellular microtubules. Parasites, particularly protozoan parasites, offer important models for the study of microtubule organization, which may also provide useful leads for novel chemotherapeutic agents against parasite-specific forms of these organelles. Research on the biology of microtubules has advanced rapidly in the last 2-3 years, from purely descriptive studies to an understanding of some of the genetic and biochemical aspects. In this article, David Russell and Jean-Franc~ois Dubremetz summarize the progress and discuss the potential of tubulin as a drug target for antiprotozoal chemotherapy. The microtubule is a ubiquitous structure, represented in all eukaryotic cells during at least some stage of the life cycle. Microtubules are formed by the polymerization of dimers consisting of non-identical subunits, designated ct and 13tubulin, with molecular weights around 50 kDa. The cellular functions fulfilled by these polymers are highly diverse although normally related to motility or maintenance of cell shape. The mechanism by which a cell exerts control over the assembly of unrelated microtubular organelles is at present one of the more popular questions in cell biology, and parasitic protozoa have two properties that recommend them as useful models. As eukaryotic cells they tend to possess highly differentiated and ordered microtubular organelles, and the different morphological stages of the life cycles of most parasitic protozoa offer opportunities for studying the differentiation and morphogenesis of these structures.

The Flagellates Amongst the flagellates the most extensively studied group are the

trypanosomatids, which all show the same basic cellular pattern of microtubular organelles (Fig. I). Their microtubules may be classified by virtue of location and function into three groups: (I) the flagellar microtubules that constitute the 9 plus 2 axoneme responsible for cell locomotion; (2) the subpellicular microtubular corset that dictates the cell body shape; and (3) the intranuclear spindle (Fig. I c) which is a transient structure present only during nuclear division. Amongst trypanosomatids, the major variation on this basic theme is the point of origin and length of the flagellum L The first observation that the tubulins in trypanosomatids may differ biochemically according to their cellular location was made by Gallo and Anderton 2, who raised a monoclonal antibody that recognized an epitope present on only the flagellar tubulin in Trypanosoma brucei. Shortly afterwards, using the insect pathogen Crithidia fasciculata, we isolated tubulin from the flagellum and pellicular microtubules and from the cytoplasmic pool independently 3. This facilitated biochemical analysis of microtubular proteins from the different subcellular locations. The 13

tubulin subunits appeared identical regardless of source, whereas some heterogeneity was detectable amongst the cLtubulin subunits. Tubulin from the cytoplasmic pool contained a tubulin subunits ~1 and ~2 (separable by isoelectric focusing)3, the axonemal tubulin possessed only the c~3 subunit, while the pellicular microtubule fraction contained all three ct tubulin subunit forms. As the relative gel migration characteristics of the different a tubulin subunits are similar to those reported for the unicellular alga Chlamydomonas 4, we adopted the same nomenclature. An intriguing observation was the absence of ct3 subunit in the cytoplasmic pool fraction. We had expected the cytoplasm to contain microtubular protein capable of polymerizing to form all the microtubular organelles present in these cells. The in vitro translation products from mRNA isolated from Crithidia, in the process of regenerating their flagella following mechanical deflagellation, also lacked the c~3 subunit, It has subsequently been shown that the c~3 subunit is generated by post-translational modification of cytoplasmic c~ tubulin during its polymerization into microtubules s. In connection with this post-translational modification, Stieger and coworkers 6 partially purified tubulin from Trypanosoma bruce/ and demon~ strated that part of the ~ tubutin in these cells was post-translationally modified by tyrosinolation. Subsequent experimentation (R. Schneider et al., unpublished) has demonstrated that the a3 subunit,

~31986,Elsev~rSciencePubflshersB~. Amsterdam0169-475~86/$0200