D. H. Molyneux

Honeydew sugars in wild-caught Phlebotomus ariasi detected by high performance liquid chromatography (HPLC) and gas chromatography (GC)

Abstract. Phlebotomus ariasi Tonnoir sandflies were caught in light traps hung in oak trees and in a house in the Cévennes focus of leishmaniasis in the South of France. The flies were cryopreserved either immediately on removal from the traps, or after starvation for 6–7 days, or after 6–7 days starvation followed by exposure to oak infested with the aphid genera Lachnus or Thelaxes.

Lectins (haemagglutinins) in the haemolymph of Glossina fuscipes fuscipes: Isolation, partial characterization, selected physico-chemical properties and carbohydrate-binding specificities

Abstract Glossina fuscipes fuscipes haemolymph contained agglutinins (lectins), titre range 2 −11 –2 −18 , against red blood cells (RBC) of human ABO(H) blood group with highest values detected against “AB” RBC. The use of protease- and neuraminidase-treated RBC in many cases increased titres whilst treatment with galactosidases or glucosidases caused decreased levels. Haemolymph adsorption with “O” RBC reduced titres against “O” and “AB” but to a lesser extent anti-A or -B activity indicating lectin heterogeneity. The carbohydrate-binding specificities for human RBC were directed towards N -acetylated and deoxy derivatives of glucose and/or galactose. In addition the haemagglutinins were reactive against some oligosaccharides, ribose, deoxymannose, deoxygalactose, xylose and xylan with certain of the RBC types. The agglutinins were glycoprotein in nature, thermo-labile, affected by storage, freezing and thawing treatments and exposure to a high dosage of γ-radiation, possessed limited disulphide and hydrogen bonds, and depended upon slightly acid to neutral conditions for optimum agglutination. The haemag-glutinins did not require the presence of divalent cations (Ca 2+ , Mn 2+ or Cu 2+ ions) for activity although an elevated concentration of Mg 2+ ions resulted in increased endpoint titres. However heavy metal ions (Pb 2+ and Fe 2+ ) in the buffer lowered agglutinin levels. The intact lectin molecule had an isoelectric point of 6.2, a relative molecular weight of 710 kDa and comprised approx. 70 kDa subunits.

The ultrastructure of Leishmania major in the foregut and proboscis of Phlebotomus papatasi

The relationship between Leishmania parasites and their sandfly vectors has been the subject of several studies over the past decade (see Killick-Kendrick 1979, 1986; Molyneux and Killick-Kendrick 1987; Molyneux et al. 1986). Transmission and scanning electron microscopy of infected flies (Killick-Kendrick et al. 1974; Molyneux etal. 1975; Warburg et al. 1986; Walters et al. 1987; Lawyer et al. 1987) have been complemented by biochemical and physiological studies (Sacks et al. 1985; Schlein 1986) and, as a result, we have a much better, yet incomplete, picture of parasite morphogenesis of the development of infectivity to the vertebrate host and of transmission. Confirmation of the existence of metacyclic promastigotes in stationary phase cultures and midgut populations in sandflies (Sacks and Perkins 1984, 1985; Sacks et al. 1985; Franke et al. 1985; Howard et al. 1987) has led to the importance of the investigation of foregut infections, particularly of attached parasites. Phlebotomus papatasi forcefed L. major could later egest promastigotes, although apparently no parasites were attached to the sandfly foregut (Warburg and Schlein 1986). Walters et al. (1987) have recently suggested that foregut infections are ephemeral and of little significance, since in the foreguts of Lutzomyia abonnenci, which they infected with L. mexicana mexicana (of which the former is not the natural vector), they only found a few parasites that appeared to be degenerating. This has prompted us to publish electron micrographs from one of very few natural vector/parasite combinations studied in detail (Table 1), which demonstrate intense foregut infections with few degenerating parasites, and which are, as far as we are aware, the first published

Trypanosoma (Megatrypanum) incertum from Pipistrellus pipistrellus : development and transmission by cimicid bugs

Trypanosoma (Megatrypanum) incertum Pittaluga 1905 was found in 33 out of 206 Pipistrellus pipistrellus caught at various sites in Britain. The trypanosome is described from blood smears. Development took place in laboratory-reared Cimex pipistrelli and Cimex lectularius. Epimastigote forms initially multiplied rapidly in the ventriculus and midgut of Cimex. Metacyclic trypanosomes were found in the rectum of both species of Cimex after 8 days when bugs were maintained at 20 degrees C and as early as 3 days at 30 degrees C. Electron microscopy of infected bugs revealed that there was no attachment to epithelial cells of the ventriculus or midgut, but within the rectum epimastigotes were attached by their flagella to the cuticle of the rectum by hemidesmosomes. Transmission was achieved by feeding experimentally infected bugs to bats kept in the laboratory. These bats were negative as judged by xenodiagnosis using laboratory-reared Cimex. Bats which had been caught in the wild demonstrated low-grade or sub-patent parasitaemias (positive in xenodiagnosis) for up to 400 days after the day of capture. Despite an extensive search of impression smears of tissues immediately after trypanosomes first appeared in the blood of experimentally infected bats no multiplicative stages were found.

Sugar specificities of anti-human ABO(H) blood group erythrocyte agglutinins (lectins) and haemolytic activity in the haemolymph and gut extracts of three Glossina species

Relatively heat-labile, human ABO(H) blood group non-specific lectins or lectin-like agglutinins, titre range 2−9–2−16, were detected in Glossina morsitans morsitans, Glossina palpalis gambiensis and Glossina tachinoides haemolymph. The haemagglutinins exhibited wide specificities for carbohydrate residues on the surface of human erythrocytes, indicative of heterogeneity, which varied according to the tsetse species examined and the type of erythrocyte used. Haemolymph agglutinin reactivities were directed mainly towards sorbose, trehalose, glucose, 2-deoxygalactose and to a lesser extent the deoxy, [1–4]- and/or [1–6]-linked derivatives of glucose. Occasionally fructose, mannose, sucrose, turanose, stachyose and melezitose minimally inhibited agglutination. Midgut haemagglutinins, titres 2−6 or 2−7, were only found in G. m. morsitans exclusively against “AB” erthrocytes whilst hindgut extracts in all threee Glossina species caused agglutination (titres 2−1–2−7) of most erythrocyte types. Heat-labile, possibly protease but not trypsin, haemolytic molecules were present in most gut preparations. Conversely, a non-proteolytic, partially thermostable haemolysin(s) was detected in G. m. morsitans midgut samples. Gut haemagglutinin specificities were less diverse than those of haemolymph and effective agglutination inhibitors were glucose, galactose or mannose and their deoxy, aminated and N-acetylated derivatives. Additionally sorbose, sucrose, turanose, gluconic acid and methyl glucoside inhibited in G. m. morsitans. Both agglutinin and lytic activities were either negated or reduced following freezing and thawing treatments of gut extracts and haemolymph.

Two populations of Phlebotomus ariasi in the Cévennes focus of leishmaniasis in the south of France revealed by analysis of cuticular hydrocarbons

ABSTRACT. Two distinct populations of Phlebotomus ariasi Tonnoir have been identified in the Cevennes focus of leishmaniasis in the south of France using gas‐liquid chromatography (GLC) of cuticular hydrocarbons extracted from individual dried female flies. Results were obtained after analysis of flies collected from CDC light traps from a domestic and a sylvatic site separated by a distance of 900 m. Flies were provided for GLC analysis as six blind samples. Using cluster and discriminant analysis techniques, five of the samples were shown to form two distinct groups, while a sixth was identified as a mixture.

Naturally occurring agglutinins against trypanosomatid flagellates in the haemolymph of insects

In vitro studies of the behaviour of the trypanosomatid flagellates Trypanosoma brucei and Leishmania hertigi in the presence of cell-free haemolymph of locusts, Schistocerca gregaria and cockroaches, Periplaneta americana revealed the presence of parasite agglutinins. The range of normal values of agglutination titres was 2(-4) to 2(-13). Physico-chemical treatment of haemolymph indicated that these agglutinins are protein or glycoprotein in nature and are only partially affected by heat treatment below 65 degrees C, at which temperature incubation of haemolymph for 30 min abrogated all agglutination. Agglutination was not dependent on the presence of Ca2+ or Mg2+. Prior injection of locusts and cockroaches with T. brucei and L. hertigi significantly increased agglutinin titres between Days 4 and 6 in cockroaches (P less than 0.05) and from Days 2 to 4 when L. hertigi was inoculated into locusts. The induced differences in titres observed in locusts infected with T. brucei were not significant. Lysozyme levels were significantly increased after inoculation of T. brucei into cockroaches compared with placebo-inoculated and uninoculated controls. L. hertigi inoculation produced significant increases in lysozyme levels compared with controls between Days 1 and 7 in locusts and 3 to 6 in cockroaches. These studies indicate that, at least in easily manipulated model systems, induced responses to intrahaemocoelic inoculation to trypanosomes and Leishmania can occur. As far as we are aware this is the first report of an induced response of an insect to such important parasites. The possibility that induced responses in natural vector to this parasites occurs requires investigation.

A study of the use of gas chromatography of cuticular hydrocarbons for identifying members of the Anopheles gambiae (Diptera: Culicidae) complex

Cuticular hydrocarbons were analysed by gas chromatography (GC) in 564 specimens of the Anopheles gambiae Giles complex from several West African sites, to determine whether individual A. gambiae sensu stricto and A. arabiensis Patton can be reliably identified, and to investigate the extent to which distinct chromosomal forms of A. gambiae sensu stricto , which are ecologically restricted as well as in some cases sexually isolated, can be distinguished by their cuticular hydrocarbons. Sympatric A. arabiensis and A. gambiae sensu stricto at Banambani, Mali could be distinguished with 90% correct identifications using the concentrations of four hydrocarbons in a linear discriminant function, but at a second site in Moribabougou, Mali, A. arabiensis was indistinguishable from a small sample of Bamako form of A. gambiae sensu stricto . Sympatric chromosomal forms of A. gambiae sensu stricto could be separated and clearest differences were found between the Mopti and Bamako forms. Direct gene flow between these forms has been found to be completely lacking despite partial intergradation of each form with the savanna form. Ethological isolating mechanisms between these forms have not however been demonstrated. Estimates of the rates of misclassification between the savanna form and the Mopti and Bamko forms reflect the degree of integradation observed amongst these forms by analysis of karyotype frequency in the wild. Discrimination was poor when an allopatric sample of the Mopti form was compared with other samples. An overall test shows that the proportion of correct classifications in discriminant analysis tends to be higher between sympatric than between allopatric populations; however, more extensive sampling would be needed for a rigorous test. The involvement of cuticular hydrocarbons in specific mate recognition systems is discussed.

Schizotrypanum in British bats

Two species of Schizotrypanum, T. (S.) dionisii and T. (S.) vespertilionis, were identified from British bats. Laboratory studies on stocks of isolated trypanosomes from 5 species of bat (Pipistrellus pipistrellus, Nyctalus leisleri, N. noctula, Eptesicus serotinus and Myotis brandti) indicated that the predominant species was T. d. dionisii. Collections and dissection of the bat bug Cimex pipistrelli from bat roosts revealed flagellate infection in a total of 12 out of 20 bugs; 7 of these bugs had metacyclic trypanosomes present. C. pipistrelli and the human bed bug, C. lectularius were reared in the laboratory and allowed to feed on wild-caught bats known to be infected with T. d. dionisii. Development occurred in both species of Cimex. Cimex spp. could be used to detect subpatent Schizotrypanum infections by xenodiagnosis. This technique was used to test the parasitological status of bats collected in the wild or reared in captivity. On a single occasion an apparent transmission of T. d. dionisii to an uninfected (by xenodiagnosis) laboratory reared bat was achieved. A stock of Schizotrypanum isolated from a wild-caught C. pipistrelli collected in a N. leisteri roost was identified by DNA buoyant density centrifugation as T. (S.) vespertilionis. A P. pipistrellus known to be infected with T. d. dionisii was found to have cyst-like structures in thoracic skeletal muscle containing amastigotes. The study provided the strongest evidence yet that C. pipistrelli is the vector of Schizotrypanum in British bats.

Variation in cuticular hydrocarbons among strains of the Anopheles gambiae sensu stricto by analysis of cuticular hydrocarbons using gas liquid chromatography of larvae.

Cuticular hydrocarbons of larvae of individual strains of the Anopheles gambiae sensu stricto were investigated using gas liquid chromatography. Biomedical discriminant analysis involving multivariate statistics suggests that there was clear hydrocarbon difference between the Gambian(G3), the Nigerian (16CSS and, its malathion resistant substrain, REFMA) and the Tanzanian (KWA) strains. The high degree of segregation (95%) in hydrocarbons among the four strains investigated indicates that further analysis is needed to enable understanding of hydrocarbon variation in samples of An. gambiae especially from areas where these populations co-exist.