Acta Tropica, 47(1990)145-149 Elsevier
145
ACTROP 00060
Separation of Psychodopygus carrerai carrerai and P. yucumensis (Diptera: Psychodidae) by gas chromatography of cuticular hydrocarbons A. Phillips 1, F. Le Pont 2'3, P. Desjeux 2'4, G. Broomfield I and D.H. M o l y n e u x ~ 1Department of Biological Sciences, University of Salford, Salford, U.K., 21BBA, c/o Embajada de Francia, La Paz, Bolivia, 30.R.S.T.O.M. Entomologie Mkdicale, Bondy, France, and aParasitic Diseases Programme, Worm Health Organisation, Geneva, Switzerland. (Received 8 May 1989; accepted 5 September 1989) Specimens of two species of sandfly, Psychodopygus carrerai carrerai and P. yucumensis, vectors of Leishmania braziliensis braziliensis in the subandean lowlands of Bolivia, were subjected to cuticular hydrocarbon analysis to ascertain if the technique could separate the females. A high degree of separation (87%) was obtained based on significant differences in the cuticular hydrocarbon composition of these two species. Statistical treatment of the data shows that these species, initially identified by the colour of the mesonotum, can also be distinguished by their hydrocarbons. Key words: Psychodopygus carrerai carrerai; P. yucumensis; Sandflies; Leishmaniasis; Bolivia; Cuticular hydrocarbons
Introduction
Sandflies o f the genus Psychodopygus are proven vectors o f Leishmania (Viannia) braziliensis braziliensis in South America. P. carrerai carrerai, P. yucumensis and P. llanosmartini have been shown to be vectors o f L. b. braziliensis in Bolivia, using techniques such as isoenzyme typing and m o n o c l o n a l a n t i b o d y characterisation (Le P o n t and Desjeux, 1986; Le P o n t et al., 1986, 1988). In the A m a z o n region o f Brazil, P. wellcomei is recognised as the vector and is often sympatric with other members o f the P. squamiventris series e . g . P , complexus and P. squamiventris s.str. (Lainson et al., 1973; Shaw and Lainson, 1987). The difficulty o f distinguishing females o f certain related taxa o f Psychodopygus species, particularly those involved in the transmission o f leishmaniasis, currently hinders the interpretation o f epidemiological data. This identification problem applies specifically to members o f the P. squamiventris series which have isomorphic females ( R e a d y et al., 1982). Isoenzyme analysis o f P. wellcomei and P. complexus females did n o t reveal any diagnostic enzyme systems which could be used in Correspondenceaddress."Dr. A. Phillips, Department of Biological Sciences, University of Salford, Salford M5 4WT, U.K. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
146 identification (Ready and da Silva, 1984), however, Ryan et al. (1986) used cuticular hydrocarbon analysis to separate the females of these species with a 97% success rate. Phillips et al. (1988) have also described the presence of three hydrocarbons which are characteristic of P. squamiventris s.str, females and which were not found in either P. wellcomei or P. complexus. P. c. carrerai and P. yucumensis are largely sympatric in Bolivia (Caillard et al., 1986; Le Pont et al., 1988) where separation of the females can be achieved by morphological or isoenzyme analysis. The recent description of P. yucumensis (Le Pont et al., 1986) was based on the morphological dichotomy of specimens caught on human bait in the Alto Beni region. Females from this region were separated into two groups according to the colour of the mesonotum. Experienced personnel were able to distinguish P. c. carrerai, which typically has a pale rnesonotum, from the new species P. yucumensis which has a light brown mesonotum. Isoenzyme analysis has also revealed two diagnostic enzyme systems; peptidase and xanthine dehydrogenase which indicate a degree of reproductive isolation between these species (Le Pont et al., 1986; Caillard et al., 1986). Detailed epidemiological study of leishmaniasis transmission by P. c. carrerai and P. yucumensis has not therefore been hampered by severe vector identification problems (Desjeux et al., 1986, 1987; Le Pont and Desjeux, 1986; Le Pont et al., 1988). As part of our continuing studies on sandfly cuticular hydrocarbons (Phillips et al., 1988) and in association with our work on the genus Psychodopygus, the cuticular hydrocarbons of P. c. carrerai and P. yucumensis were examined and the results are detailed below. Hydrocarbon specificity has been of considerable use in the identification of other members of this genus (Ryan et al., 1986; Phillips et al., 1988).
Materials and Methods
Specimens for hydrocarbon analysis were collected by human landing catches in the Alto Beni region of Bolivia - - a tropical lowland area where cutaneous leishmaniasis caused by L. b. braziliensis is endemic (Desjeux et al., 1986 and 1987). Three species of Psychodopygus were present in this area; P. llanosmartini was identified by morphological differences and removed from the collections while P. c. carrerai and P. yucumensis were separated on the basis of mesonotum pigmentation. The cuticular wax layer containing the hydrocarbons was removed, by immersing the insect in solvent, and subsequently analysed by on-column capillary gas chromatography (GC). Full details of the method are given in Phillips et al. (1988). The aim of statistical analyses was to determine whether the specimens could be grouped according to their cuticular hydrocarbon profiles. Initially, outliers in the data set were visualised, by plotting the principal components and removed, where a valid explanation of their difference was available. Outliers may be individuals of other species accidentally included in the sample or flies which have been contaminated in some way (e.g. by fungi). The data were grouped according to species (as determined by mesonotum colour) and discriminant function analysis was performed using BMDP (Dixon, 1988). Error rates were calculated by leaving out an individual each time and using the derived function to subsequently classify the missing individual. This procedure circumvents the problem of using the same data to calculate and test the discriminant function, and is particularly useful for small samples with
147 large n u m b e r s o f variables. The resulting jack-knifed classification table represents an accurate picture o f the success o f allocating u n k n o w n specimens to the groups, using the h y d r o c a r b o n data (for full discussion o f the statistical procedure refer to Phillips et al., 1988).
Results
Fifty three sandflies were analysed; 27 females o f P . c. c a r r e r a i and 26 females o f N o outliers were determined in the samples. Examples o f the h y d r o c a r b o n profiles obtained are shown in Fig. 1 and these have been chosen to highlight the five peaks which a c c o u n t for the separation. The classification table (Table 1) shows a high degree o f separation o f the two species and predicts that 87% P. yucumensis.
Psychodopygus carrerai carrerai ~
Psychodopygus yucumensis ~
Fig. 1. Chromatograms obtained from the extracted cuticular wax of individual female Psychodopygus carrerai carrerai and P. yucumensis. The x-axis represents increasing time, temperature and molecular weight of compound, while the area under each peak represents the concentration. Those hydrocarbon peaks which best separate the two species are marked (*). TABLE 1 The classification success based on differences in the cuticular hydrocarbons Actual group
Predicted group P. c. carrerai
Psychodopygus c. carrerai Psychodopygus yucumensis
Total
P. yucumensis
24
4
3
22 26
27
Average correct classification rate = 87%
148
of unknown flies would be correctly identified using the discriminant function derived from this data.
Discussion P. yucumensis and P. c. carrerai are sympatric only in the lowest altitudes of P. c. carrerai distribution, that is the subandean corridor and the foothills of the first range
of the Andean cordillera, alongside the Beni plain up to an altitude of 500 m. However, they are both important vector species with P . c . carrerai the most abundant while P. yueumensis is the most agressively anthropophilic. The studies described here together with the electrophoretic and morphological information already available provide an accurate description of these species. The results also suggest that differentiation of cuticular hydrocarbons is associated with reproductive isolation and may be an intrinsic part of the isolating mechanism (see Phillips et al., 1988). Examination of the cuticular hydrocarbons of sandfly species therefore enables the separation of cryptic and isomorphic species, as well as the determination of distinct populations within species (Ryan et al., 1986; Kamhawi et al., 1987; Phillips et al., in press). The widespread occurrence of hydrocarbon specificity in closely related species supports the hypothesis that the hydrocarbons have a role in pre-mating recognition and consequently the processes of incipient speciation (Phillips et al., 1988).
Acknowledgements This work received financial assistance from the UK Medical Research Council, the UNDP/World Bank/WHO special Programme for Research and Training in Tropical Diseases, the French Technical Co-operation Programme, the French Ministry of Industry and Investigation and the EEC Health and Development Programme. We are indebted to Paul Milligan for statistical advice.
References Caillard, T., Tibayrenc, M., Le Pont, F., Dujardin, J.P., Desjeux, P. and Ayala, F.J. (1986) Diagnosis by isoenzyme methods of two cryptic species, Psychodopygus earrerai and P. yucumensis (Diptera: Psychodidae). J. Med. Entomol. 23(5), 489-492. Desjeux, P., Le Pont, F., Mollinedo, S. and Tibayrenc, M. (1986) Les Leishmania de Bolivie. I. Leishmania braziliensis Vianna 1911 dans les D6partements de la Paz et du Beni. Premiers isolements de souches d'origine humaine et phylogen6se applications eco-6pidemiologiques (Coll. int. CNRS/INSERM 1984). IMEE, Montpellier, 401-410. Desjeux, P., Mollinedo, S., Le Pont, F., Parades, A. and Ugarte, G. (1987) Cutaneous leishmaniasis in Bolivia. A study of 185 human cases from Alto Beni (La Paz Department). Isolation and characterisation of 26 strains of Leishmania braziliensis braziliensis. Trans. R. Soc. Trop. Med. Hyg. 81,742-746. Dixon, W.J. (1988) BMDP Statistical Software Manual (1988 Software release), Vol. 1, University of California Press, Berkeley, CA. Kamhawi, S., Molyneux, D.H., Killick-Kendrick, R., Milligan, P.J.M., Phillips, A., Wilkes, A.J. and Killick-Kendrick, M. (1987) Two populations of Phlebotomus ariasi in the Cevennes focus of
149 leishmaniasis in the south of France revealed by analysis of cuticular hydrocarbons. Med. Vet. Entomol. 1, 97-102. Lainson, R., Shaw, J.J., Ward, R.D. and Fraiha, H. (1973) Leishmaniasis in Brazil. IX. Considerations on the Leishmania braziliensis complex: importance of sandflies of the genus Psychodopygus (Mangabeira) in the transmission of L. braziliensis in North Brazil. Trans. R. Soc. Trop. Med. Hyg. 67, 184-196. Le Pont, F. and Desjeux, P. (1986) Leishmaniasis in Bolivia. II. The involvement of Psychodopygus yucumensis and P. llanosmartini in the selvatic transmission cycle of Leishmania braziliensis braziliensis in a lowland subandean region. Mem. Inst. Oswaldo Cruz, Rio de Janeiro 81, 31-318. Le Pont, F., Caillard, T., Tibayrenc, M. and Desjeux, P. (1986) Bolivian phlebotomines. II. Psychodopygus yucumensis N.SP. A new man-biting phlebotomine sandfly from subandean region (Diptera: Psychodidae). Mem. Inst. Oswaldo Cruz., Rio de Janeiro 81, 79-85. Le Pont, F., Breni6re, F.S., Mouchet, J. and Desjeux, P. (1988) Leishmaniose en Bolivie. III. Psychodopygus carrerai carrerai (Barretto, 1946) nouveau vecteur de Leishmania braziliensis braziliensis en milieu sylvatique de r6gion subandine basse. C.R. Acad. Sci. Paris 307, 279-282. Phillips, A., Milligan, P.J.M., Broomfield, G. and Molyneux, D.H. (1988) Identification of medically important Diptera by analysis of cuticular hydrocarbons. In: M.W. Service (Ed.), Biosystematics of Haematophagous Insects, Systematics Association Special Volume 37, Chapter 4, Clarendon Press, Oxford, pp. 39-59. Phillips, A., Milligan, P.J.M., Maroli, M., Lane, R., Kamhawi, S. and Molyneux, D.H. Intraspecific variation in the cuticular hydrocarbons of Phlebotomus perfiliewi Parrot (Diptera: Psychodidae) from Italy. Med. Vet. Ent. in press. Ready, P.D., Fraiha, H., Lane, R.P., Arias, J.R. and Pajot, F.X. (1982). On distinguishing the female of Psychodopygus wellcomei, a vector of mucocutaneous leishmaniasis, from other squamiventris series females. I. Characterisation of Ps. squamiventris squamiventris and Ps. s. rnaripaensis stat. nov. (Diptera: Psychodidae). Ann. Trop. Med. Parasitol. 76, 201-214. Ready, P.D. and da Silva, R.M.R. (1984) An alloenzymic comparison of Psychodopygus wellcomei - - an incriminated vector of Leishmania braziliensis in Para State, Brazil - - and the sympatric morphospecies Ps. complexus (Diptera: Psychodidae). Cahier ORSTOM S6r. Ent. M6d. Parasitol. 22, 3-8. Ryan, L., Phillips, A., Milligan, P.J.M., Lainson, R., Molyneux, D.H. and Shaw, J.J. (1986) Separation of female Psychodopygus wellcomei and P. complexus (Diptera: Psychodidae) by cuticular hydrocarbon analysis. Acta Trop. 43, 85-89. Shaw, J.J. and Lainson, R. (1987) Evolution, classification and geographical distribution. In: W. Peters and R. Killick-Kendrick (Eds.), The Leishmaniases in Biology and Medicine, Volume 1, Chapter 7, Academic Press, London, pp. 291-363.
145
ACTROP 00060
Separation of Psychodopygus carrerai carrerai and P. yucumensis (Diptera: Psychodidae) by gas chromatography of cuticular hydrocarbons A. Phillips 1, F. Le Pont 2'3, P. Desjeux 2'4, G. Broomfield I and D.H. M o l y n e u x ~ 1Department of Biological Sciences, University of Salford, Salford, U.K., 21BBA, c/o Embajada de Francia, La Paz, Bolivia, 30.R.S.T.O.M. Entomologie Mkdicale, Bondy, France, and aParasitic Diseases Programme, Worm Health Organisation, Geneva, Switzerland. (Received 8 May 1989; accepted 5 September 1989) Specimens of two species of sandfly, Psychodopygus carrerai carrerai and P. yucumensis, vectors of Leishmania braziliensis braziliensis in the subandean lowlands of Bolivia, were subjected to cuticular hydrocarbon analysis to ascertain if the technique could separate the females. A high degree of separation (87%) was obtained based on significant differences in the cuticular hydrocarbon composition of these two species. Statistical treatment of the data shows that these species, initially identified by the colour of the mesonotum, can also be distinguished by their hydrocarbons. Key words: Psychodopygus carrerai carrerai; P. yucumensis; Sandflies; Leishmaniasis; Bolivia; Cuticular hydrocarbons
Introduction
Sandflies o f the genus Psychodopygus are proven vectors o f Leishmania (Viannia) braziliensis braziliensis in South America. P. carrerai carrerai, P. yucumensis and P. llanosmartini have been shown to be vectors o f L. b. braziliensis in Bolivia, using techniques such as isoenzyme typing and m o n o c l o n a l a n t i b o d y characterisation (Le P o n t and Desjeux, 1986; Le P o n t et al., 1986, 1988). In the A m a z o n region o f Brazil, P. wellcomei is recognised as the vector and is often sympatric with other members o f the P. squamiventris series e . g . P , complexus and P. squamiventris s.str. (Lainson et al., 1973; Shaw and Lainson, 1987). The difficulty o f distinguishing females o f certain related taxa o f Psychodopygus species, particularly those involved in the transmission o f leishmaniasis, currently hinders the interpretation o f epidemiological data. This identification problem applies specifically to members o f the P. squamiventris series which have isomorphic females ( R e a d y et al., 1982). Isoenzyme analysis o f P. wellcomei and P. complexus females did n o t reveal any diagnostic enzyme systems which could be used in Correspondenceaddress."Dr. A. Phillips, Department of Biological Sciences, University of Salford, Salford M5 4WT, U.K. 0001-706X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
146 identification (Ready and da Silva, 1984), however, Ryan et al. (1986) used cuticular hydrocarbon analysis to separate the females of these species with a 97% success rate. Phillips et al. (1988) have also described the presence of three hydrocarbons which are characteristic of P. squamiventris s.str, females and which were not found in either P. wellcomei or P. complexus. P. c. carrerai and P. yucumensis are largely sympatric in Bolivia (Caillard et al., 1986; Le Pont et al., 1988) where separation of the females can be achieved by morphological or isoenzyme analysis. The recent description of P. yucumensis (Le Pont et al., 1986) was based on the morphological dichotomy of specimens caught on human bait in the Alto Beni region. Females from this region were separated into two groups according to the colour of the mesonotum. Experienced personnel were able to distinguish P. c. carrerai, which typically has a pale rnesonotum, from the new species P. yucumensis which has a light brown mesonotum. Isoenzyme analysis has also revealed two diagnostic enzyme systems; peptidase and xanthine dehydrogenase which indicate a degree of reproductive isolation between these species (Le Pont et al., 1986; Caillard et al., 1986). Detailed epidemiological study of leishmaniasis transmission by P. c. carrerai and P. yucumensis has not therefore been hampered by severe vector identification problems (Desjeux et al., 1986, 1987; Le Pont and Desjeux, 1986; Le Pont et al., 1988). As part of our continuing studies on sandfly cuticular hydrocarbons (Phillips et al., 1988) and in association with our work on the genus Psychodopygus, the cuticular hydrocarbons of P. c. carrerai and P. yucumensis were examined and the results are detailed below. Hydrocarbon specificity has been of considerable use in the identification of other members of this genus (Ryan et al., 1986; Phillips et al., 1988).
Materials and Methods
Specimens for hydrocarbon analysis were collected by human landing catches in the Alto Beni region of Bolivia - - a tropical lowland area where cutaneous leishmaniasis caused by L. b. braziliensis is endemic (Desjeux et al., 1986 and 1987). Three species of Psychodopygus were present in this area; P. llanosmartini was identified by morphological differences and removed from the collections while P. c. carrerai and P. yucumensis were separated on the basis of mesonotum pigmentation. The cuticular wax layer containing the hydrocarbons was removed, by immersing the insect in solvent, and subsequently analysed by on-column capillary gas chromatography (GC). Full details of the method are given in Phillips et al. (1988). The aim of statistical analyses was to determine whether the specimens could be grouped according to their cuticular hydrocarbon profiles. Initially, outliers in the data set were visualised, by plotting the principal components and removed, where a valid explanation of their difference was available. Outliers may be individuals of other species accidentally included in the sample or flies which have been contaminated in some way (e.g. by fungi). The data were grouped according to species (as determined by mesonotum colour) and discriminant function analysis was performed using BMDP (Dixon, 1988). Error rates were calculated by leaving out an individual each time and using the derived function to subsequently classify the missing individual. This procedure circumvents the problem of using the same data to calculate and test the discriminant function, and is particularly useful for small samples with
147 large n u m b e r s o f variables. The resulting jack-knifed classification table represents an accurate picture o f the success o f allocating u n k n o w n specimens to the groups, using the h y d r o c a r b o n data (for full discussion o f the statistical procedure refer to Phillips et al., 1988).
Results
Fifty three sandflies were analysed; 27 females o f P . c. c a r r e r a i and 26 females o f N o outliers were determined in the samples. Examples o f the h y d r o c a r b o n profiles obtained are shown in Fig. 1 and these have been chosen to highlight the five peaks which a c c o u n t for the separation. The classification table (Table 1) shows a high degree o f separation o f the two species and predicts that 87% P. yucumensis.
Psychodopygus carrerai carrerai ~
Psychodopygus yucumensis ~
Fig. 1. Chromatograms obtained from the extracted cuticular wax of individual female Psychodopygus carrerai carrerai and P. yucumensis. The x-axis represents increasing time, temperature and molecular weight of compound, while the area under each peak represents the concentration. Those hydrocarbon peaks which best separate the two species are marked (*). TABLE 1 The classification success based on differences in the cuticular hydrocarbons Actual group
Predicted group P. c. carrerai
Psychodopygus c. carrerai Psychodopygus yucumensis
Total
P. yucumensis
24
4
3
22 26
27
Average correct classification rate = 87%
148
of unknown flies would be correctly identified using the discriminant function derived from this data.
Discussion P. yucumensis and P. c. carrerai are sympatric only in the lowest altitudes of P. c. carrerai distribution, that is the subandean corridor and the foothills of the first range
of the Andean cordillera, alongside the Beni plain up to an altitude of 500 m. However, they are both important vector species with P . c . carrerai the most abundant while P. yueumensis is the most agressively anthropophilic. The studies described here together with the electrophoretic and morphological information already available provide an accurate description of these species. The results also suggest that differentiation of cuticular hydrocarbons is associated with reproductive isolation and may be an intrinsic part of the isolating mechanism (see Phillips et al., 1988). Examination of the cuticular hydrocarbons of sandfly species therefore enables the separation of cryptic and isomorphic species, as well as the determination of distinct populations within species (Ryan et al., 1986; Kamhawi et al., 1987; Phillips et al., in press). The widespread occurrence of hydrocarbon specificity in closely related species supports the hypothesis that the hydrocarbons have a role in pre-mating recognition and consequently the processes of incipient speciation (Phillips et al., 1988).
Acknowledgements This work received financial assistance from the UK Medical Research Council, the UNDP/World Bank/WHO special Programme for Research and Training in Tropical Diseases, the French Technical Co-operation Programme, the French Ministry of Industry and Investigation and the EEC Health and Development Programme. We are indebted to Paul Milligan for statistical advice.
References Caillard, T., Tibayrenc, M., Le Pont, F., Dujardin, J.P., Desjeux, P. and Ayala, F.J. (1986) Diagnosis by isoenzyme methods of two cryptic species, Psychodopygus earrerai and P. yucumensis (Diptera: Psychodidae). J. Med. Entomol. 23(5), 489-492. Desjeux, P., Le Pont, F., Mollinedo, S. and Tibayrenc, M. (1986) Les Leishmania de Bolivie. I. Leishmania braziliensis Vianna 1911 dans les D6partements de la Paz et du Beni. Premiers isolements de souches d'origine humaine et phylogen6se applications eco-6pidemiologiques (Coll. int. CNRS/INSERM 1984). IMEE, Montpellier, 401-410. Desjeux, P., Mollinedo, S., Le Pont, F., Parades, A. and Ugarte, G. (1987) Cutaneous leishmaniasis in Bolivia. A study of 185 human cases from Alto Beni (La Paz Department). Isolation and characterisation of 26 strains of Leishmania braziliensis braziliensis. Trans. R. Soc. Trop. Med. Hyg. 81,742-746. Dixon, W.J. (1988) BMDP Statistical Software Manual (1988 Software release), Vol. 1, University of California Press, Berkeley, CA. Kamhawi, S., Molyneux, D.H., Killick-Kendrick, R., Milligan, P.J.M., Phillips, A., Wilkes, A.J. and Killick-Kendrick, M. (1987) Two populations of Phlebotomus ariasi in the Cevennes focus of
149 leishmaniasis in the south of France revealed by analysis of cuticular hydrocarbons. Med. Vet. Entomol. 1, 97-102. Lainson, R., Shaw, J.J., Ward, R.D. and Fraiha, H. (1973) Leishmaniasis in Brazil. IX. Considerations on the Leishmania braziliensis complex: importance of sandflies of the genus Psychodopygus (Mangabeira) in the transmission of L. braziliensis in North Brazil. Trans. R. Soc. Trop. Med. Hyg. 67, 184-196. Le Pont, F. and Desjeux, P. (1986) Leishmaniasis in Bolivia. II. The involvement of Psychodopygus yucumensis and P. llanosmartini in the selvatic transmission cycle of Leishmania braziliensis braziliensis in a lowland subandean region. Mem. Inst. Oswaldo Cruz, Rio de Janeiro 81, 31-318. Le Pont, F., Caillard, T., Tibayrenc, M. and Desjeux, P. (1986) Bolivian phlebotomines. II. Psychodopygus yucumensis N.SP. A new man-biting phlebotomine sandfly from subandean region (Diptera: Psychodidae). Mem. Inst. Oswaldo Cruz., Rio de Janeiro 81, 79-85. Le Pont, F., Breni6re, F.S., Mouchet, J. and Desjeux, P. (1988) Leishmaniose en Bolivie. III. Psychodopygus carrerai carrerai (Barretto, 1946) nouveau vecteur de Leishmania braziliensis braziliensis en milieu sylvatique de r6gion subandine basse. C.R. Acad. Sci. Paris 307, 279-282. Phillips, A., Milligan, P.J.M., Broomfield, G. and Molyneux, D.H. (1988) Identification of medically important Diptera by analysis of cuticular hydrocarbons. In: M.W. Service (Ed.), Biosystematics of Haematophagous Insects, Systematics Association Special Volume 37, Chapter 4, Clarendon Press, Oxford, pp. 39-59. Phillips, A., Milligan, P.J.M., Maroli, M., Lane, R., Kamhawi, S. and Molyneux, D.H. Intraspecific variation in the cuticular hydrocarbons of Phlebotomus perfiliewi Parrot (Diptera: Psychodidae) from Italy. Med. Vet. Ent. in press. Ready, P.D., Fraiha, H., Lane, R.P., Arias, J.R. and Pajot, F.X. (1982). On distinguishing the female of Psychodopygus wellcomei, a vector of mucocutaneous leishmaniasis, from other squamiventris series females. I. Characterisation of Ps. squamiventris squamiventris and Ps. s. rnaripaensis stat. nov. (Diptera: Psychodidae). Ann. Trop. Med. Parasitol. 76, 201-214. Ready, P.D. and da Silva, R.M.R. (1984) An alloenzymic comparison of Psychodopygus wellcomei - - an incriminated vector of Leishmania braziliensis in Para State, Brazil - - and the sympatric morphospecies Ps. complexus (Diptera: Psychodidae). Cahier ORSTOM S6r. Ent. M6d. Parasitol. 22, 3-8. Ryan, L., Phillips, A., Milligan, P.J.M., Lainson, R., Molyneux, D.H. and Shaw, J.J. (1986) Separation of female Psychodopygus wellcomei and P. complexus (Diptera: Psychodidae) by cuticular hydrocarbon analysis. Acta Trop. 43, 85-89. Shaw, J.J. and Lainson, R. (1987) Evolution, classification and geographical distribution. In: W. Peters and R. Killick-Kendrick (Eds.), The Leishmaniases in Biology and Medicine, Volume 1, Chapter 7, Academic Press, London, pp. 291-363.
