Leishmania. Taxonomie et phylogenèse. Applications éco-épidémioiogiques. (Coll. int. CNRS/INSERM. 1984). IMEEE, Montpellier, 1986, 311-324.

The -S3ináí\y interface

D.H. Molyneux*, L. Ryan**, R. Lainson** and J.J. Shaw**

SUMMARY — The factors affecting the behaviour of Leishmania in sandflies are described against the background of our knowledge of the basic hfe cycles of suprapylarian and peripylarian species. A diagramma- tic representation is used to tabulate a variety of known and possible interactions between parasite and sandfly based on our knowledge of other trypanosomatid-insect associations. The physical interactions between Leishmania and sandfly are described and the áreas of productive further study highlighted.

The natural Leishmania-ssLnáríy combination, L. amazonensis in Lutzomyia flaviscutellata, is discussed in the light of recent results and compared with earlier findings on host parasite associations. The finding of promastigote infections in the oesophageal diverticulum is described in this system; the significance of such a finding is unknown. This observation is discussed in the light of present ideas on the relationships between Leishmania promastigotes and carbohydrates, carbohydrates on inhibition of agglutination, insect lectins and Leishmania transmission. It is emphasised that the interactions will be complex and species specific, whilst modulated by carbohydrates derived from a variety of plant sources. Generalisations about such interactions in the field may be difficult to predict.

KEY-WORDS — Le;s/imanía-sandfly interactions — Peritrophic membrane — Hagellum-cuticle attachment — Leishmania] infection in crop.

INTRODUCTION

Over the past decade there has been increased interest in ali aspects of Leishmania biology. The topic discussed in this paper has benefitted particularly as a result of the colonisation in the laboratory of neotropical, silvatic sandflies as well as the availability of well characterised stocks of parasites. However, although sevoral interesting problems have been resolved and new concepts have emerged there are important áreas which remain to be thoroughly investigated. One advantage of studying Leishmania parasites in the sandfly is that comparisons can be made with other trypanosomatid-insect systems. Findings in, for example, studies on - Glossina or T. cruzi-Rhodnius can be extrapolated into Leishmania-sar\àí\y studies with some degree of confidence. In this paper the present knowledge of the Leishmania-sanà{\y interface will be summarised, new findings introduced and further áreas for research identified; in particular those which, in view of recent findings in trypanosomatid-insect systems, may be relevant. Recent reviews on the behaviour of Leishmania in the sandfly [17, 18, 36] and of other trypanosomatids in insects [31, 32] have been published. The review of Lainson and Shaw [25] introduced the concept of peripylarian and suprapylarian Leishmania based on the development of parasites in sandflies, a system of categorisation similar to that introduced by Hoare [11] in his division of mammalian trypanosomes into Salivaria and Stercoraria sections. The conclusions of Lainson and Shaw [25] were based on extensive studies of laboratory and natural infections in sandflies of a large number of well characterised and documented stocks [26, 27]. Lainson et ai [26] concluded that hindgut development of the peripylarian L. braziliensis complex is primitive and has disap- peared from suprapylarian life-cycles. An alternative explanation could be that peripylarian and suprapylarian Leishmania may have separate origins. This is supported by large differences in kDNA between L. braziliensis and L. mexicana, compatible with a distant common origin [3]. The comparative aspects of the relationships of trypanosomatids in insects have been reviewed by

* Department of Biological Sciences, University of Salford, Salford, United Kingdom. ** Wellcome Parasitology Unit. Instituto Evandro Chagas. Belém. Pará. Brazil. 312

Molyneux [31, 33]. From these studies certain generahsations can be made about the nature of such associations and ahow us to extrapolate ideas about the Leishmania-sandíly interface where appropriate. This paper presents some approaches to these problems, many of which are pertinent to Leishmania taxonomy and phylogeny. The remarkable restriction of the genus Leishmania to certain species of Phlebotominae as efficient vectors is the basis of disease dissemination. A thorough understanding of the relationship is thus not only relevant to the phylogeny of the genus Leishmania itself, but also to epidemiology.

MATERIALS AND METHODS

The materiais and methods are described in the original texts which are quoted in the appropriate sections. Details of the range of techniques employed have thus been omitted for the sake of brevity. The specimens of Lutzomia flaviscutellata were from the 62nd generation of the closed colony from the Wellcome Parasitology Unit, Belém. They were infected with stock MHOM/BR/83/M 7631 of L. amazonensis of the same laboratory, by feeding them on the skin lesions on an infected hamster. Flies were dissected at 4 days, and the guts fixed immediately in glutaraldehyde and then processed by methods previously described [19]. This section is divided into appropriate subject áreas; reference is made primarily to new ideas which may be determinants in the Leishmania-s&náí[y association or interface. Newer aspects of the host parasite relationships are also emphasised. Figure 1 with its accompanying legend is an attempt to summarise the known life cycle in the sandfly and define the factors which may influence establishment and transmission of Leishmania.

INTERPRETATION OF FIGURE

The Figure 1 is divided into two halves. The legends and figures on the right-hand side indicate the stage of Leishmania found in different áreas of the sandfly gut and their morphological forms. The recognised sequence is: I. Initial infection by amastigotes and a multiplication in the amastigote stage. II. Transformation and elongation to promastigotes within the peritrophic membrane of the abdominal midgut (am); following break-up of the peritrophic membrane (pm). III. Migration of nectomonad promastigotes to the thoracic midgut (tm). IV. Some insertion of the flagella between microvilli of the midgut epithelial cells occurs in the midgut (sce text). In peripylarian parasites (IIIA) there is colonisation of the pylorus (py) and ileum (i) by paramastigotes attached to the cuticle; this begins around Day 3 of infection. The process of anterior migration in such parasites is not well documented; however, in suprapylarian parasites, establishment of haptomonads (attached promastigotes) on the stomodeal valve (sv) occurs following the break-up of the bloodmeal (V). VI. Anterior migration of parasites and attachment to oesophagus (o) and pharynx (p) then occurs, with the development of paramastigotes in these sites. Division has not been seen anterior to the midgut, the major site of division being in the abdominal midgut. VII. Occasional infection of various parasite forms have been found in the crop (c). The possible significance of these infections is discussed in the text. VIII. Infective motile promastigotes are found in the proboscis in small numbers; these represent the terminal stage of the cycle in the sandfly. The left-hand side of the table is an attempt to categorise those factors which might operate at the parasite-sandfly interface and may act as determinants of specificity through their ability to determine establishment, migration and transmission. This attempt is necessarily speculative and draws on our knowledge of, and suggestions from, other insect/trypanosomatid systems. Numbers between brackets refer to appropriate references in bibliography. It is however necessary to indicate áreas where research should be concentrated to assess the molecular and genetic basis of Leishmania-sandíly interactions. The factors listed below have either been demonstrated or are suggested to be necessary for the essential changes of the life cycle to be completed. The order listed is in approximate sequence. An expanded discussion of some points is in the text. 313

Changes in feeding behaviour mediated through interference with sensilla in proboscis and cibarium [21 Infective small promastigotes (VIII)

Effects of parasites on flow reception by receptors [15]

Attachment mediated Transformation to proboscis promastigotes (VIII) via lectins in cuticle. Carbo-hydrates block attachment and induce ^o"^ Paramastigotes attached in pharynx and anterior migration [4,7] oesophagus (VI) Parasites in crop (VII) Lectin induced transformation [45] Plant derived carbo- Intensa attached infections of hydrates in crop. Role in oesophagus , stomodeal valve and parasite migration via thoracic midgut (V) chemotaxis [4,47] Role of excretory factor in sandfly 143] Flagellar-microvillus-midgut cell insertion (IV) Genetics of sandfly [17, 28, 29] Nature of subsequent bloodmeal [39] Initial multiplication in abdominal midgut of amastigotes (I) Role of haemolymph responses [13, 14]

Sterile midgut prerequisite for multiplication [I] Transformation to promastigotes (II) Role of symbionts in susceptibility

Breakup of peritrophic Peritrophic membrane physiology membrane prerequisite for [9] anterior migration (III)

Specific receptors on peripylarian Colonisation of pylorus and ileum of Leishmania flagellum for binding to O) peripylarian leishmanias, pyloric-ileum cuticle ~ by paramastigotes (IMA)

Figure 1 — Life cycle of Leishmania in the sandfly. Factors vifhich may involve development, establishment and transmission in various parts of the gut. Legend: numbers on left of gut refer to references appropriate to suggested factors. Roman numerais on right refer to stages and sequential development of sites in the sandfly. am. abdominal midgut; c. crop or oesophageal diverticulum; ci. cibarium; i. ileum; mt. malpighian tubules; o. oesophagus; p. pharynx; pm. peritrophic membrane; pr. proboscis; py. pylorus; sv. stomodeal valve; tm. thoracic midgut. 314

a — A sterile midgut free from bactéria or yeasts has been shown to be cssential for vigorous Leishmania multiplication [1]. b — The peritrophic membrane must break-up at the end of the digestion of the bloodmeal to allow escape of suprapylarian parasites anteriorly or peripylarian parasites posteriorly [9], c — Specific receptors for peripylarian-pylorus-ileum cuticle interaction must exist; simi- larly the stomodeal valve and foregut will provide an establishment site for attachment of compatible Leishmania-sandíly combinations. d — Stimulus to transformation must be provided by the sandfly micro-environment. Lectin induced transformation in vitro has been observed [45]; the presence of lectins in insects is recognised [35. 37]. e — The nature of the bloodmeal subsequent to the infective meai could influence the physiologj' of the digestive processes and effect behaviour of parasites within the fly [39]. f — The role of carbohydrates in the chemotaxis of promastigotes has been investigated in vitro; it is known that sandflies take in sugars in nature and there is evidence suggesting that laboratory transmission may depend on provision of sucrose (earlier the provision of raisins) [44]. This combination of circumstances suggests complex interactions between parasite, carbohydrates (including aminosugars) and the likely lectins in sandflies. These effects are suggested to operate interactively via the capacity of carbohydrates to block lectin mediated attachment [7, 41], the capacity of parasites to migrate chemotactically in the presence of sugars [4], the different receptor sites of different Leishmania species and morphological stages, and the suggested capacity of lectins to influence transformation within the fly as it does in vitro in other kineto- plastid flagellates. g — A variety of sensilla have been described in the cibarium and proboscis of sandflies. It was suggested that parasite receptor interaction could influence feeding behaviour [21] on the basis of experimental observations on transmission. Direct interaction between parasites and cibarial and labral sensilla have yet to be demonstrated as they have in trypanosome-infected Glossina [32] but theoretical studies on fluid mechanics indicated that parasite colonisation could affect reception by mechanoreceptive sensilla in cibarium and proboscis [15]. h — Establishment of Leishmania in sandflies may be determined directly by genetically regulated phenomena. Much work remains to be done on this subject but studies on the Glossina and Rhodnius/Trypanosoma systems [28, 29] indicate that similar mechanisms could exist in Leishmania-sanàíly relationships to add to strong epidemiological evidence of í\y-Leishmania specificities [18, page 430-8]. Other mechanisms which could determine susceptibility are the role of symbionts and haemolymph responses of sandflies. Does excreted factor (EF) so much studied in vitro [43] play any role in control of infection in the sandfly?

THE PHYSICAL INTERFACE

The interface between Leishmania parasites and sandflies can be categorised into several distinct physical and chemical associations; further information can be obtained from the reviews quoted in the introduction. Lúmen of abdominal and thoracic midgut and enzyme-parasite interactions. There is extremely limited information available on this topic. However, in view of the necessity for rapid multiplication if a successful life-cycle is to be completed, it is apparent that those Leishmania which fail to multiply and establish are in an unfavourable environment. There is therefore a need for extensive studies on sandfly gut physiology in both uninfected and infected flies. A recent study [39] has suggested that sandfly digestive enzymes could play a role in determining the ability of Leishmania to establish. Schlein et ai [39] found that flies fed on turkey blood subsequent to an infective mammalian bloodmeal had a much lower infection rate than control flies maintained on a mammalian diet. It was suggested that this difference in infection rate was related to the different leveis of DNA-ase secretion in response to the higher DNA content of turkey erythrocytes. It is abundantly clear that even the basic information on sandfly gut physiology is not available. Such studies should be initiated on easily reared species to enable comparisons to be made at a later stage between vector and non-vector species and the different sandfly groups which can transmit Leishmania. 315

Association with peritrophic membrane. This topic has been reviewed by Killick-Ken- dricli [18]; and more recently [38] a study of the gut morphology of Lutzomyia longipalpis has been published. Rudin and Hecker [38] state that they confirm the observations of Gemetchu [10] working with Phlebotomus longipes, but no peritrophic membrane micrographs were provided. Earlier reviews [1, 29] emphasised the importance of the physiology of the peritrophic membrane for development of Leishmania in the sandfly based on the work of Feng [9] who suggested that in sandflies in which the peritrophic membrane remained intact, enclosing the parasites to be subsequently voided with the faeces, Leishmania did not establish. When the peritrophic mem• brane broke up the promastigotes were freed to move anteriorly. There is no doubt that a more detailed study of the peritrophic membrane of sandflies is required. This is particularly so when the importance of lectins as determinants of specificity are under consideration as immunocytochemical labelling has indicated that Con A receptor molecules are present on Calliphora peritrophic membrane [37]. A more intense study, in parallel with gut physiology studies, is also needed to define the role of the peritrophic membrane in both intrinsically susceptible vector and in non-vector species. Midgut cell-microvilli associations. Our initial studies showed [19] that the thoracic and abdominal midgut microvilli were long structures intruding extensively into the lúmen of the midgut. There is clearly variation in microvillar length not only in the different parts of the midgut, but at different times in the digestive cycle. However, in view of the intensity of parasite infections in the thoracic midgut and the relatively short length of the microvilli in Lu. flaviscutellata infected with L. amazonensis, the possibility may exist that such intense infections themselves affect microvillar micromorphology (Fig. 2)A similar suggestion was first made by Desser [6] who observed loss of microvilli in the gastric caeca of the leech Batracobdella picta infected with Trypanosoma rotatorium. Similarly we have recently observed, in Blastocrithidia gerridis infec• tions of Gerris [45], close association of flagella with midgut cells where microvilli are absent. These observations on related systems indicate that a more detailed look at natural, vector/ parasite combinations is required. It is clear that microvillar-glagellar interactions occur and it is necessary to resolve what effects intense parasite colonisation of the thoracic midgut has on microvilli and underlying cells, particularly in view of the frequent finding of flagellates and parasites within the midgut cells themselves [22, 34]. A feature of the heavy midgut infections of L. amazonensis in Lu. flaviscutellata is the large number of flagella penetrating thoracic midgut cells of the sandfly (Fig. 3). This phenomenon has also been observed, with the electron microscope, in experimental studies on L. amazonensis in Lu. longipalpis by Molyneux et al. [34], by Killick-Kendrick et al. [22] with L. braziliensis promastigotes in the gut wall of naturally infected Psychodopygus wellcomei; and more recently by Kaddu and Mutinga [16] in a naturally infected P. pedifer, presumed to be infected with L. aethiopica. As in the relationship between trypanosomes and midgut cells of Glossina [8] the significance of this cell invasion is at present unclear. It is not uncommon and is possibly a regular occurrence not confined to laboratory initiated infections. Flagellum-cuticle attachment in: a. peripylarian leishmanias in pylorus and ileum, and foregut; b. attachment of suprapylarian leishmanias to foregut. The descriptions of these associations were made by Killick-Kendrick and co-workers in the mid-1970s and were revie• wed [18, 33]. However in initial studies L. amazonensis in Lu. longipalpis was the experimental system employed and, although this is not a natural parasite/vector combination, Lu. longipalpis did transmit the infection [21]. Similarly in a study of L. braziliensis in the pylorus and ileum of the same species of fly, the pattern of development and establishment was similar to that observed in the natural vector, Psychodopygus « wellcomei » [18]. Further studies on natural systems, e.g. L. infantum in P. ariasi, L. braziliensis in Ps. well• comei [18], L. amazonensis in Lu. flaviscutellata {this paper) (Figs. 2 and 4) generally confirm these earlier observations using Lu. longipalpis as a laboratory model, a fact emphasised by Lainson et al. [25]. The study of L. amazonensis in Lu. flaviscutellata has provided confirmation of mechanisms of attachment to midgut, stomodeal valve and oesophagus, but has also revealed the presence of parasites in the oesophageal diverticulum (or crop) (Figs. 2 to 8)'". Attachment of suprapylarian parasites to cuticle of the foregut and of peripylarian Leish• mania to the pylorus and ileum (and presumably later to foregut) is a prerequisite for establishing

1. Figures 2 to 8 are electron micrographs of Lu. flaviscutellata experimentally infected with L. amazonensis, 4 days after infected meai. sv

1-2 1 - 'i

Figure 2 — Electron micrograph of the junction of the thoracic midgut (tm) and stomodeal valve (sv) of Lutzomyia flaviscutellata experimentally infected with L. amazonensis, 4 days after infected meai. Note microvillar lining (mv) of midgut epithelial cells (me) is gradually lost at the anterior part of the midgut close to the point where the cuticular lining of the stomodeal valve (sv) commences. Small arrows under the cuticular intima of the valve indicate site of attached flagella of parasites, x 8,740.

dense infections which colonise the designated áreas. Ali attachment is mediated through fla- gellum-cuticle hemidesmosomes formed immediately the flagella of the parasites contact the cuticle of the stomodeal valve where it invaginates into the thoracic midgut (Figs. 1 and 4). Attached flagella have expanded intraflagellar regions particularly at the distai point of contact with the sandfly gut cuticle. A similar pattern of development has been observed in ali Leish- mania-sanàíly combinations. Experimental, immunocytochemical studies are required to investi- gate the nature of this association which, it is suggested, could be lectin mediated [37], Figure 3 — Section through thoracic midgut showing penetration of flagellum (f) between microvilh (mv) of epithelial cell (me) and into the cytoplasm of the cell itself. x 11,600.

Figure 4 — Higher magnification of thoracic midgut (tm)/stomodeal valve (sv) junction showing parasite attachment along cuticular lining of the valve. Note the expanded distai end of the attached flagella (large arrow); small arrows indicate sites of attached flagella of parasites which are not in the section. X 8,940. Figure 6 — Higher magnification of part of Fig. 5 showing attached promastigotes. Flagella (f) are attached to the thin cuticular lining of the crop by hemidesmosomes (arrowed). Note double axoneme within a flagellar membrane (small arrows). x 18,500. 319

Figure 7 — Section of crop of Lutzomyia flaviscutellata showing parasites and indicating the highly folded appearance and the very thin wall of the crop. Double axoneme within flagellar membrane arrowed. x 11,600.

Figure 8 — Multiple axoneme within a flagellar membrane which is also attached by hemidesmosome to the thin wall of the crop. x 27,750. 320

Infection of L. amazonensis in oesophageal diverticulum (crop) of Lu. flaviscutellata. Although infections of the oesophageal diverticulum have been seen with «L. trópica» (= L. major) in P. papatasi [2] and with L. amazonensis in 5 out of 8 naturally infected Lu. flaviscutellata ([24] and Ward, pers. comm.), few authors have considered the significance of such infections. Recent studies of material examined with the transmission electron microscope (TEM) of L. amazonensis in its natural vector sandfly Lu. flaviscutellata have again shown extensiva infections of the crop, in 4 day old laboratory infected sandflies, with 3 out of 4 infected sandflies examined with the TEM showing such infections. These sandflies were fed sucrose, post-infective meai. The oesophageal diverticulum is a thin-walled evagination of the gut which joins the gut close to the junction of the stomodeal valve and the oesophagus. Sugars, when imbibed either artificially or naturally, pass to the oesophageal diverticulum. When full of fluid the crop is an extended translucent sac. The lining of the diverticulum is made up of a thin cuticular layer under which lie « two very fine muscle layers, one longitudinal and internai, the other circular and exter• nai » [2]. When not completely filled with fluid the crop collapses to a highly folded sac (Fig. 7). The rapidity of the dissection employed and fixation, the dimensions of valve opening into the diverticulum and the fact that infection of the guts was determined by the appearance of the stomodeal valve without recourse to compound microscope examination under a coverslip suggest that the infections observed were not artefacts. The presence of attached forms, seen by ordinary light microscopy, surely renders ali other arguments superfluous. This finding is supported by TEM findings of attached parasites, and the unusual appearance of some of them (Figs. 6 to 8). Infection of the crop was sometimes heavy, but not intense (Fig. 5). Promastigote types observed were the nectomonad type and the shorter haptomonad. No parasites were observed as paramastigotes in the sections of the crop examined. Parasites were attached by their flagella to the cuticular intima by hemidesmosomes indicating the association was not a transitory one (Figs. 6 to 8). In addition, flagella with double and possiblv multiple axonemes were seen in the crop (Figs. 7 and 8). This is a rare phenomenon and one which has previously been recorded only for T. lewisi in the rectum of the flea, Nosopsyllus fasciatus [30]. Other aspects of parasite cytology and ultrastructure were normal. These and previous observations suggest that crop infections of Leishmania in sandflies may be of common occurrence. Their significance, if any, is not yet known, but the presence of the flagellates in this site may be related to the chemotaxis of promastigotes towards carbohydrates [4]. Bray [4] has presented a considerable body of evidence to show that sugars can act as chemotaxins and can influence the movement of promastigotes in vitro. The role of sugar has been the subject of much debate in relation to transmission of Leishmania since the addition of raisins to the diet of bloodfed sandflies successfully resulted in the first laboratory transmission [44]. In addition to earlier observations of sandflies feeding on plants, Killick-Kendrick [18] emphasised the importance of this facet of the feeding of sandflies. The exact nature of the sugar-Leishmania- sandfly interaction, however, is likely to be a complex which again may vary depending on the system under study. Early studies on the agglutinating abilities of plant lectins (such as Concanavalin A) with promastigotes of L. donovani [7] gave valuable Information on the nature of the cell surface on the basis of carbohydrate inhibition studies. The studies of trypanosomatid surface cytochemistry using lectins and inhibitors have been extended to T. cruzi and Leishmania as a method of parasite Identification (see for example [40, 41, 42]). Pereira et al. [35] have shown that there are lectin-like molecules in Rhodnius prolixus midgut, hindgut and haemolymph which vary in their capacity to agglutinate T. cruzi epimastigotes and erythrocytes of various animal species. Ibrahim et al. [12] have similarly demonstrated haemagglutinins and trypanosome agglutinins in Glossina. These agglutination reactions are inhibited by a variety of different sugars. Our own studies have shown that locusts (Schistocerca gregária) and cockroaches (Periplaneta americana) have high leveis of trypanosomatid agglutinins in haemolymph [13,14]. The range of normal agglutinating values was 2"'' to 2^"; various treatments of haemolymph revealed that the agglutinins are protein or glycoprotein in nature; of great interest however, was the observation of induced increases in agglutinin titres and leveis of lysozyme [13,14]. This was the first recorded induction of a response in an insect to a trypanosomatid challenge. These observations are pertinent to the Leishmania-sandí[y interaction. There is little doubt that lectin or lectin-like molecules occur in insects [37, 48]. It has been shown [35] that such molecules isolated from different parts of the gut have different agglutinating activity for T. cruzi epimastigotes and in Glossina for T. brucei procyclic forms [12]. Schottelius [40, 41] has shown 321

Carbohydrate from plant sources

Oesophageal Lectins on surfaces diverticulum of sandfly gut and peritrophic membrane

t Receptors on parasite Carbohydrate for lectins released into midgut

Detachment of parasites by carbohydrate inhibition of attachment

Promastigote migration in presença of carbohydrates

Transmission by unattached small proboscis promastigotes through migration under carbohydrate gradients.

Figure 9 — Suggested Leishmania-c&rhohydrate interactions in relation to attachment and transmission.

that strains of T. cruzi and Leishmania promastigotes have different agglutinating activity when exposed to a bank of plant lectins. Inhibition of lectin mediated-agglutination by carbohydrates is a major method of cell surface characterisation experiments. Carbohydrates are believed to be an integral part of the diet of female sandflies [47] and promastigotes behave chemotactically towards certain sugars [4]. Promastigotes have been found in the crop ([24] and this paper). Studies on the flagellate Herpetomonas samuelpessoai [5] have revealed that configuration transformation can be induced by Concanavalin A in vitro. The implications of ali these possible interactions remain to be elucidated in the Leis/imania-sandfly system, but the possibility must exist that such factors interact to modulate the distribution and migration of parasites in the various sites in the vector. The possible interactions between these interactive and modulating factors are shown in Figure 9.

ACKNO WLEDGEMENTS

We are grateful to the Wellcome Trusf and the UNDP/World BankAVHO Special Programme for Research and Training in Tropical Diseases for generous financial support. 322

L'interface Leis/irna/Jia-Phlébotome

RÉSUMÉ — Les facteurs intervenant dans le comportement des Leishmania sont décrits sur la base de nos connaissances du cycle élémentaire des espèces de Suprapylaria et de Peripylaria. Les diverses interactions connues et supposées entre les parasites et les Phlébotomes sont synthétisées dans un schéma regroupant nos connaissances sur les autres associations entre les Trypanosomatidae et les Insectes. Les interactions physiques entre les Leishmania et les Phlébotomes sont envisagées. Les avenues de recherche futures sont dégagées. La relation naturelle entre Leishmania amazonensis et son vecteur Lutzomyia flaviscutellata est discutée à la lumière de resultais récents, et comparée aux informations déjà obtenues sur les associations hôte-parasite. La présence de promastigotes dans le diverticule oesophagien est rapportée dans cette asso- ciation, mais sa signification reste inconnue. Cette observation est discutée à la lumière des idées actuelles surtes relations entre les promastigotes et les hydrates de carbone, entre les hydrates de carbone et Vinhibition de Vagglutination, entre les lectines dlnsectes et la transmission des Leishmania. Laccent est mis sur la complexité et la spécificité de ces interactions, modulées en fait par les glucides provenant de sources végétales variées. La généralisation possible de telles interactions dans les conditions naturelles doit être envisagée avec prudence.

MOT-CLÊS — Interactions heishmanm-Phlébotomes — Membrane péritrophique — Fixation flagelle-cuticule — Infestation leishmanienne du jabot.

BIBLIOGRAPHY

[I] Adler S. — Leishmania. Adv. Parasitai., 1964, 2, 35-96. [2] Adler S., Theodor O. — The mouth parts, alimentary tract, and salivary apparatus of the female in Phlebotomus papatasii. Ann. trop. Med. Parasitai.. 1926, 20, 109-143. [3] Barker D.R., Butcher J. — The use of DNA probes in the Identification of leishmanias: discrimination between isolates of the Leishmania mexicana and L. braziliensis complexes. Trans. R. Soe. trop. Med. Hyg., 1983, 77, 285-297. [4] Bray R.S. — Leis/ima«ia.-chemotaxic responses of promastigotes and macrophages in vitra. J. Pratozaal., 1983, 30, 322-329. [5] De Souza E.T., Thomas E.M., Esteves M.J.G., Angluster J., de Souza W. — Concanavalin A-induced cell differentiation in the protozoan Herpetomonas samuelpessoai. J. ProtozooL, 1980, 66, 985-988. [6] Desser S.S. — The ultrastructure of the epimastigote stages of Trypanosoma rotatorium in the leech Batracobdella picta. Can. J. ZooL, 1976, 54, 1712-1723. [7] Dwj'er D.M. — Lectin binding saccharides on a parasitic protozoan. Science, 1974, 184, 471-473. [8] Evans D.A., EUis D.S. — Recent observations on the behaviour of certain trypanosomes within their insect hosts. Adv. Parasitai., 1983, 22, 1-41. [9] Feng L.C. — The role of the peritrophic membrane in Leishmania and trypanosome infections of sandflies. Peking Nat. Hist. Buli, 1951, 19, 327-334. [10] Gemetchu T. — The morphology and fine structure of the midgut and peritrophic membrane of the adult 68femal, 111-124e Phlebotomus. longipes Parrot and Martin (Diptera, Psychodidae). Ann. trop. Med. Parasitai, 1974, [II] Hoare CA. — Morphological and taxonomic studies on mammalian trypanosomes. X. Revision of the systematics. J. Pratozaal, 1964, 11, 200. [12] Ibrahim E.A.R., Ingram, G.A., Molyneux, D.H. — Haemagglutinins and parasite agglutinins in hae- molymph and gut of Glossina. Z. Tropenmed. Parasitai, 1984, 35, 151-156. [13] Ingram G.A., East, J., Molyneux D.H. — Agglutinins of Trypanosoma, Leishmania and Crithidia in insect haemolymph. Devi comp. Immunal, 1983, 7, 649-652. [14] Ingram G.A., East J., Molyneux D.H. - Naturally occurringagglutinins against trypanosomatid flagella- tes in the haemolymph of insects. Parasitalagy, 1984, 89, 435-451. [15] Jefferies D., Livesey J.L., Molyneux D.H. — Fluid mechanics of blood meai uptake by Leishmania - infected sandflies. Acta trap., 1986, 43, 43-53. [16] Kaddu J.B., Mutinga M.J. — Leishmania in Kenyan phletobomine sandflies. 1. Leishmania aethiapica in the midgut of naturally infected Phlebotomus pedifer. Insect Sei. appl, 1981, 2, 245-247. [17] Killick-Kendrick R. — Recent advances and outstanding problems in the biology of phlebotomine sandflies. Acta trop., 1978, 35, 297-313. [18] Killick-Kendrick R. — Biology of Leishmania in phlebotomine sandflies. In: Biology of the Kineto- plastida. Vol. 2. Lumsden W.H.R., Evans D.A., eds. Academic Press. London/New York, 1979, 396-460. 323

[19] Killick-Kendrick R., Molyneux D.H., Ashford R.W. — Leishmania in phlebotomid sandflies. I. Modifi- cations of the flagellum associated with attachment to the mid-gut and oesophageal valve of the sandfly. Proc. R. Soe., London, ser. B, 1974, 187, 409-419. [20] KilUck-Kendrick R., Molyneux D.H., Leaney A.J., Rioux J.A. — Aspects of the life-cycle of Leishmania in the sandfly. Proc. 2nd Europ. Multicoll. ParasitoL, Trogir, 1975, 89-95. [21] Killick-Kendrick R., Leaney A.J., Ready P.D., Molyneux D.H. — Leishmania in phlebotomid sandflies. IV. The transmission of Leishmania mexicana amazonensis to hamsters by the bite of experimentally infected Lutzomyia longipalpis. Proc. R. Soe, London, ser. B, 1977, 196, 105-115. [22] Killick-Kendrick R., Lainson R., Leaney A.J., Ward R.D., Shaw J.J. — Promastigotes of Leishmania b. braziliensis in the gut wall of the natural vector, Psychodopygus wellcomei. Trans. R. Soe. trop. Med. Hyg., 1977, 71, 381. [23] Killick-Kendrick R., Molyneux D.H., Hommel M., Leaney A., Robertson E.S. — Leishmaniain phleboto• mid sandflies. V. The nature and significance of infections of the pylorus and ileum of the sandfly by leishmaniae of the braziliensis complex. Proc. R. Soe., London, ser. B, 1977, 198, 191-199. [24] Lainson R., Shaw J.J. — in Brazil. I. Observations on enzootic rodent leishmaniasis. Incrimination of Lutzomyia flaviscutellata (Mangabeira) as the vector in the lower Amazon Basin. Trans. R. Soe. trop. Med. Hyg., 1968, 62, 385-395. [25] Lainson R., Shaw J.J. — The role of animais in the epidemiology of South American leishmaniasis. In: Biology of the Vol. 2. Lumsden W.H.R., Evans D.A., eds. Academic Press, New York/ London/San Francisco, 1979, 1-116. [26] Lainson R., Ward R.D., Shaw J.J. — Leishmania in phlebotomid sandflies. VI. Importance of hindgut development in distinguishing between parasites of the Leishmania mexicana and L. braziliensis complexes. Proc. R. Soe, London, ser. B., 1977, 199, 309-320. [27] Lainson R., Ready P.D., Shaw J.J. — Leishmania in phlebotomid sandflies. VIL On the taxonomic status of Leishmania peruviana, causative agent of Peruvian « uta » as indicated by its development in the sandfly, Lutzomyia longipalpis. Proc. R. Soe, London, ser. B, 1979, 206, 307-318. [28] Maudlin I. — Inheritance of susceptibility to infection in Rhodnius prolixus. Nature, London, 1976, 262, 214-215. [29] Maudlin I. — Inheritance of susceptibility to Trypanosoma congolense infection in Glossina morsitans. Ann. trop. Med. ParasitoL, 1982, 76, 225-227. [30] Molyneux D.H. — The fine structure of the epimastigote forms of Trypanosoma lewisi in the rectum of the flea, Nosopsyllus fasciatus. Parasitology, 1969, 59, 55-56. [31] Molyneux D.H. — Vector parasite relationships in the Trypanosomatidae. 7n.-Advances in Parasitology. Dawes B., ed. Academic Press, London/New York/San Francisco, 1977, 1-82. [32] Molyneux D.H. — Host-trypanosome interactions in Glossina. Insect Sei. appL, 1980, 1, 39-46. [33] Molyneux D.H. — Host-parasite relationships of Trypanosomatidae in vectors. In: Current topics in vector research. Vol. 1. Harris K.F., ed. Praeger Scientific Press, 1983, 117-148. [34] Molyneux D.H., Killick-Kendrick R., Ashford R.W. — Leishmania in phlebotomid sandflies. III. The ultrastructure of Leishmania mexicana amazonensis in the midgut and pharynx of Lutzomyia longi• palpis. Proc. R. Soe., London, ser. B., 1975, 199, 341-357. [35] Pereira M.E.A., Andrade A.F.B., Ribeiro J.M.C. — Lectins of distinct specificity in Rhodnius prolixus interact selectively with Trypanosoma cruzi. Science, 1981, 211, 597-600. [36] Peters W., Killick-Kendrick R. — The leishmaniases in biology and medicine. Academic Press, 1986 (in press). [37] Peters W., Kolb H., Kolb-Bachofen V. — Evidence for a sugar receptor (lectin) in the peritrophic membrane of the blowflv larva, Calliphora erythrocephala Mg. (Diptera). J. Insect PhysioL, 1983. 29, 275-280. [38] Rudin W., Hecker H. — Functional morphology of the midgut of a sandfly as compared to other hematophagous Nematocera. Tiss. Celi, 1982, 14, 751-758. [39] Schlein Y., Warburg A., Schnur L.F., Shlomai J. — Vector compatibility of Phlebotomus papatasi dependent on differentially induced ingestion. Acta trop., 1983, 40, 65-70. [40] Schottelius J. — The Identification by lectins of two strain groups of Trypanosoma cruzi. Z. Parasi- tenKde, 1982, 68, 147-154. [41] Schottelius J. — Lectin binding strain specific carbohydrates on the cell surfaces of Leishmania strains from the Old World. Z. ParasitenKde, 1982, 66, 237-247. [42] Schottelius J., Uhlenbruck G. — Comparative studies of Trypanosoma cruzi and T. cruzi - like stocks from different South American countries using lectins. Z. ParasitenKde, 1983, 69, 727-736. [43] Schnur L.F. — The immunological Identification and characterization of leishmanial stocks and strains with special reference to excreted factor serotyping. In: Biochemical characterization of Leishmania. Chance M.L., Walton B.C., eds. UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Geneva, 1982, 25-47. [44] Short H.E., Smith R.O.A., Swaminath C.S., Krishnan C.V. — Transmission of Indian kala-azar by the bite of Phlebotomus argentipes. Indian J. med. Res., 1931, 18, 1373-1375. 324

[45] Tieszen K., Hewood P., Molyneux D.H. — Ultrastructure and host-parasite association of Blastocri- thidia gerridis in the ventriculus of Gerris odontogaster {RemÍTptera, Gerridae). Can. J. ZooL, 1983, 61, 1900-1909. [46] Yeaton R.W. — Invertebrate lectins. I. Occurrence. Devi comp. Immunol., 1981, 5, 391-402. [47] Young C.J., Turner D.P., Killick-Kendrick R., Rioux J.A., Leaney A.J. — Fructose in wild caught Phlebotomus ariasi and the possible relevance of sugars taken by sandflies to the transmission of leishmaniasis. Trans. R. Soe. trop. Med. Hyg., 1980, 74, 363-366.

Since this manuscript was submitted the following relevant papers have been published and should be consulted.

Beach R., Kiilu G., Hendricks L., Oster C., Leeuwenburg J. — in Kenya: transmission of to man by the bite of a naturally infected Phlebotomus duboscqi. Trans. R. Soe. Med. Hyg., 1984, 78, 747-751. Beach R., Kiilu G., Leeuwenburg J. — Modification of sandfly biting behaviour by Leishmania leads to increased parasite transmission. Am. J. trop. Med. Hyg., 1985, 34, 278-282. Sacks D.L., Perkins P.V. — Identification of an infective stage of Leishmania promastigotes. Science, 1984, 223, 1417-1419. Sacks D.L., Perkins P.V. — Development of infective stage Leishmania promastigotes within phlebo- tomine sandflies. Am. J. trop. Med. Hyg., 1985, 34, 456-459. Schlein Y., Warburg A. — Phytophagy and the feeding cycle of Phlebotomus papatasi (Diptera, Psychodidae) under experimental conditions. J. med. EntomoL, 1986, 23, 11-15. Warburg A., Hamada G.S., Schlein Y., Shire D. — Scanning electron microscopy of Leishmania major in Phlebotomus papatasi. Z. ParasitenKde, 1986, 72, 423-431. Yuval B., Schlein Y. — Leishmaniasis in the Jordan Valley. III. Nocturnal activity of Phlebotomus papatasi (Diptera, Psychodidae) in relation to nutrition and ovarian development. J. med. EntomoL, 1986, 23, 411-415.