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Genetic variability in phlebotomine sandflies: possible implications for Leishmaniasis epidemiology
Focus
Genetic Variability in Phlebotomine Sandflies: Possible Implications for Leishmaniasis Epidemiology G.C. Lanzaro and A. Warburg The vast majonty of genetIc stud/es of phlebotomlne sandflres have had as their goal the resolution of taxonom,c problems. In this paper, Gregory Lonzaro and A/on Warburg summarize the iiterature descnbing levels ofgenetic polymorphism, both w~thrn and between sandfly species, and speculate on the signrficance of vanabrbty in the vector to the complex epdem,o/ogy ofleishmaniavs.
In an earlier review, Ward commented on the shortage of InformatIon on the genetics of phlebotomine sandflies; although some progress has been made, this observation remains true today. Genetic Diversity Epidemiological studies of lelshmaniasis naturally begin with efforts to Identify the vector, This work IS often obfuscated by the fact that closely related species can only be dIstinguIshed morphologically in one sex. A number of molecular-level approaches have been taken to reveal markers of taxonomlc value. Gas chromatographic analysts of cuticular hydrocarbons has been used to distinguish morphologically Indistinguishable females of Phiebotomus we/comei from P. complexus’. Analysis of cuticular hydrocarbons can reveal distinctions even below the species level’ Although these examples illustrate the potential of cutlcular hydrocarbons as taxonomic characten, their utility IS limited because the genetlc basis for observed variability is not clear. Probes have been developed from genomic DNA fragments and used as sandfly species diagnostics4 6. These approaches are useful in the area of taxonomy, but provide no information on genetic relationships among species or on the genetic structure of populations. More recently, a polymetase chain reaction-based technique, known as random amplified polymorphic DNA (RAPD-PCR), has been employed to distinguish morphologically identical females of Lutzomyla spinrcrasso from L. youngi7. This technique uses a single primer in the PCR to amplify DNA fragments at random. Polymorphisms are expressed as the presence or absence of a fragment of a particular size Pormtology
Toduy.
vo’
/ I CL 4 IW‘I
This technique does produce useful genetic information and data generated by this approach have been used by population geneticists. However, there are disadvantages: the majonty of RAPDPCR polymorphisms are Inherited as dominant markers, which prevents tests of random mating within populations because individual genotypes cannot be determined. The molecular technique most widely used to date is analysis of polymorphisms In enzyme encoding genes by electrophoretlc analysis of isozymes. This approach yields information useful both for taxonomy and for detailed analysis of the genetics of populations. Phlebotomous pedrfer and P. elgoonerws are closely related species that can be distinguished only In the males; the former is a vector of Leishmanla aeth/opica in Kenya, the latter is not a vector, and the two occur together over a large portion of their range. A survey of allozyme variablllty revealed four IOCI to be dlagnostlc for the two specie+. The species status of two color forms of Lutzomyla (Psychodopygus) carreral (subsequently i. can-era1 S.S. and L. yucumensis) was confirmed by the discovery of diagnostic allozymes9~~o. Phlebotomlne sandfly species evolve with very little change in gross morphology. In such a group, one would expect species to exist that are Identical morphologically. These so-called sibling or cryptic species are common In other medically Important Diptera and have proven to be of great Importance In cases where there are differences In vectorial capacity among them. A study of two morphological variants of L. long,palprs taken from a single site in Bolivia showed they were not distinguishable by isozyme analysis”. Earlier work on this species indicated that mating barriers, mediated by male pheromones, may exist among different forms”. Recently, lsozymes were used, in conjunction with crossmating expenments, to demonstrate that L. longipalpls is a complex of at least three sibling specles’3. Sibling species In the subfamily Phlebotomlnae are probably common and, as work continues on the population genetics of this group, many more sibling species complexes WIII undoubtedly be uncovered.
Describing the degree of genetic variability in a species is best done by measunng heterozygosity (H), which gives the estimated proportion of genes expected to be heterozygous in an average individual. Ready and Smith14 repot-ted that Isoenzyme variation is limited in sandfIles, but this claim is not supported in the literature. Table I summarizes both observed and expected (based on computing heterozygote frequencies from allele frequency data under the Hardy-Weinberg equilibrium) heterozygoslties from I4 sandfly species in three genera. The overall heterozygosity averaged over species is 0.078. which IS actually slightly higher than the value (0.074) reported for 23 species of Insects in a review by Nevo20. In some cases, there are differences between direct and estimated measures of l-f, as in P. perrvciosus from Tuscany and P. pop&as/ from Egypt (Table I). In these studies, the authon do not suggest significant sub-structuring of these populations and we conclude that these departures are likely the result of sampling en-or-s or errors in gel interpretation. The data in Table I should be viewed with some caution, as many of the values are based on samples of ten, or fewer, loci. Levels of divergence among populations or between taxa are best described by genetic distance statistics, which measure allelic differences at multiple loci. Such values can be used to assemble phylogenies, and may provide clues to establish the taxonomic status of genetlcally distinct populations. Table 2 summarizes genetic distance values reported for a variety of sandfly species at a number of taxonomic levels. The reported levels of genetic divergence agree well with the taxonomic arrangement of the species studied, with distances among local populations of a single species having the smallest values and comparisons of species in different subgenera having the highest. The informal taxonomic rank ‘series’, which represents assemblages of related species within a genus, below the rank of subgenus, also appears to be supported by the data summarized in Table 2. Data from other organisms have shown that the relationship between taxonomic 151
Focus Table
I. Observed and expected heterozygosity for enzyme encoding gene loci in Phlebotomine sandflies Mean sample size
Species Lutzomyio carrerai L yucumensis L bngipolpis L /ongipalpis Brazil Colombia Costa Rica L verrucarum species group
per locus I05 107 138
Phlebotomus perniciosus Sardinia Tuscany P. perfirewi P. papatasi P. langeroni P. ariase L’Aumede Banyuls P. perniciosus Banyuls Port-Vendres
Sergentomyia magna S. africana S. hamoni Yaka-Yaka Mayombe 5. schwetzi
No. of loci studied II II IO
Source of material (Country) Field (Bolivia) Field (Bolivia) Field (Bolivia)
0.0”69 0.126 0.037
0.120 0.145 0.036
0.054 0.072 0.064
Ii
Ref. IO IO II 13
H =P
=
24 22 21
27 27 27
Colony Colony Colony
0.057 0.07 I 0.058
220
21
Field (Colombia)
0.070-O.
44 23 33 51 50
II II II 21 21
Field (Italy) Field (Italy) Field (Italy) Colony (Egypt) Colony (Egypt)
0.033 0.018 0.050 0.062 0.054
0.04 I 0.042 0.055 0.080 0.063
34 21
9 9
Field (France) Field (France)
0.074 0.075
0.062 0.074
55 39
9 9
Field (France) Field (France)
0.067 0.085
0.057 0.08 I
II I 47
6 6
Field (Congo) Field (Congo)
0. I77 0.1 I7
60 84 43
6 6 5
Field (Congo) Field (Congo) Field (Congo)
0.1 IO 0.142 0.035
I76
0.082-o.
I7 I
I5 I6
I6 I7 I7 I8
I8
I9 I9 I9
I9
aHo,,,,observed heterozygosity;H,,,. expected heterozygosity (see text) status and the magnitude of distance values can sometimes be misleading, but they may help to guide workers to pursue further efforts where values are not consistent with existing taxonomlc rankings. Cytogenetics
The karyotypes of I2 species have been described: eght species In the genus Lutzomyia24~25 and four species In the genus Ph/ebotomus1426,2T.Only one species, P. pernroosus. has a karyotype with sexual dimorphism, with males being the heterogametic sex (XV) (with females being XX). Extensive polymorphism in chromosome morphology has been observed, but the karyotype for several species In the genus Lutzomyra
appears to be ldentlcalmq. Old World species have greater variabllrty In chromosome number, 2N = 6 IO. All the iutzomyio species thus far studied have a chromosome number of 2N = 4, except i. traprdor (2N = 3). The general lack of heterogamy suggests that sex determnation IS controlled by Indivldual IOU. Met-y et al I9 have shown slgnificant differences In the distribution of genotypic frequencies at several enzyme encoding gene IOCI between the sexes in several species in the genus Sergentomyio and suggest that these genes may be linked to a sex-detennintng locus In this genus. The polytene chromosomes of larval saIlvary glands have been described for only one species, L. /ong~palp/s’s. Despite the fragile nature of sandfly polytenes,
Table 2. Values for genetic distance at various taxonomic levels in phlebotomine sandflies”
Taxa compared Populations within species Sibling species
No. of comparisons 3 3
Genetic
distance
(‘V WEI 0.004 (~0.004)
Refs 16,17
0.236 (kO.107)
I3
Species within series
21
0. I94 (+O. 127)
I5
Between series
23
0.928 (kO.276)
15
Species within subgenera
4
0.6 I2 (kO.27 I)
IO, 16.2 I ,22
Between subgenera
6
I.250 (kO.40 I)
17.21
’ Data from Ref. 23 I52
the authors conclude that they are suitable for study. Further work on sandfly polytenes is thus warranted and could prove very useful, not only for cytotaxonomy but for the production of genetic maps by utilizing polytenes in in situ hybridizatlon&dies. Genetics
of Susceptibility
to Parasite Infection There is only a single study, reported in tandem paperG30, dealing with the genetic basis of susceptibility/refractoriness to parasite infection in phlebotomine sandflies. In this study, two strains of P. popatasi originating from a common laboratory colony were selected for high and low infection rates (IR) with Leishmania major (strain WR-309). After I7 generations of selection, a susceptible strain (IR = 87.1%) and refractory strain (IR = 5.2%) were developed. That there is a genetic component to susceptibility and refractoriness is shown by the fact that strains did respond to selection. However, the fact that pure lines could not be obtained after I7 generatlons suggests that these traits are not under single locus control. In reciprocal and backcross experiments, it was shown that neither susceptibility nor refractoriness is dominant over the other. Parastology
Today, vol.
I I, no. 4, I995
Focus
-
Sandfly Variability and Variation in Clinical Manifestations of Leishmaniasis Lutzomyia longipalprs IS the vector of Leishmanra chagasl, the causative agent of human visceral leishmaniasis throughout much of South and Central America. Our interest in the genetics of sandflies was aroused when we noticed that bites of I_ longipalpis females from Costa Rica do not leave long-lasting red etythemas (Fig. I), which are characteristic of L. longipalpis from Brazil and Colombia3’. This was particularly interesting because, in Costa Rica, infections with L. chagasi invariably result in a non-ulcerative form of cutaneous leishmaniasis32, while in South America infections are almost exclusively associated with viscera leishmaniasisaa. Whereas parasite isolates from cutaneous cases proved indistinguishable from isolates made from viscera infections, we showed that the two forms of the disease are transmitted by different sandfly species13. We subsequently showed that saliva of Costa Rican L longipalpis has very little vasodilatory activity but strongly enhances cutaneous leishmaniasis in mice. Flies from Brazil and Colombia displayed much higher vasodilatory activity but reduced capacity to enhance cutaneous proliferationa4. Although it remains speculative, we propose that at least some of the variability in the clinical presentations of L chagasi infections in Latin America may be due to the different composition of the saliva of their vectors. If confirmed, this will be the first demonstratron of the importance of genetic variability in vector species to the epidemiology of leishmaniasis and the first example, for any vector-borne disease, of a vector modulating the pathology of the par-a site it transmits. Variability in clinical manifestations is not unique to L chagas/ infections. In North Africa and Southern Europe, L. infanturn (a parasite arguably identical with L chagasr] causes visceral infections in some areas, but primarily cutaneous disease in other geographical region&36. Cutaneous lesions due to L. donovanl, the causative agent of kala-azar in Africa, have also been reportedaT. In the Mediterranean region, a number of Phlebotomus (Larroussit~s) spp are known to transmit L. infanturn and in East Africa P. (Synphlebotomus) spp are the possible vectors of L. donovania*. Data on the saliva of local vectors and their distribution in relation to clinical manifestations and parasite strains may be crucial for understanding the epidemiology of the diseases they transmit. Parasitology Jodoy, vol I I, no 4, 1995
Fig. I. Appearance of erythemas produced by the bites of three sibling species of the sandfly lutzomyio longipalpis (photographed 12 h after feeding): Brazil (Minas Gerais) (a), Colombia (Tolima) (b), Costa Rica (Guanacaste) (c,d). (Reproduced, with permission, from Ref. 34.)
Different clinical manifestations of seemingly identical parasites in separate geographic regions are also known within the L. braziliensis complex. L. b. panomensls and L. b. braziliensls normally produce a cutaneous sore at the site of the sandfly bite and a certain percentage of untreated cases go on to develop secondary chronic infections in the mucosal membranes of the nose and pharynx (=mucocutaneous leishmaniasis). Of those Infected with L. braziliensis, a large percentage develop mucocutaneous manifestations in Brazil, Bolivia and Colombia (2558 I%). In Central America (Costa Rica, Nicaragua, Panama), the percentages are significantly lower (2-5%; compiled from references cited by Grimaldi, et a/.33). There are I5 proven or suspected vectors of L. b. braziliensis and nine suspected vectors of L. b. par-amens/s In Latin America3*. While some of these differences are certainly attributable to host factors and parasite strains, it is not inconceivable that genetlc differences among vectors
also contribute to this complex epidemiological scenario. Research on genetic variability in natural populations of Phlebotomine sandflies has been largely focused on the resolution of taxonomic problems. Distinguishing vector from non-vector species is important, but these efforts should be extended beyond the description of taxonomrc character;. Studies of sandfly genetics, in topics such as vectorparasite interactions, reproductive biology and population genetics are sorely lacking. Exciting research in several areas, including variability in sandfly sex pheromones12J9 and the relationship between sandfly saliva and host immunological response to Leishmanio parasites40m42 suggest promising topics for future research in genetics. Finally, detailed studies of the distribution of polymorphisms at the nucleotide level will provide descriptions of the genetic structure of sandfly populations with a degree of resolution not previously possible. Extending our knowledge of sandfly genetics will 153
Focus improve
of the role
our understanding
of vector
species
in the
epidemiology
of the diseases they transmit provide
information
velopment
of new
and should
critical to the strategies
aimed
deat
their control.
Acknowledgements Thus workwassupported by grant ID-9 IO43 from the UNDPIWotid Bank/WHO/Special Programme for Research and Training in Tropical Diseases (TDR) and from the MacArthur Foundation program on the Molecular Biology of Parasite Vectors. We thank Robert B. Tesh and Richard D. Ward and three anonymous reviewerj for their comments on an earlier version of this paper. References
I 2 3 4 5 6 7 8 9 IO
Ward, R.D. (I 986) Montpellrer Nlbtoekkuer (Coil. Int. CNRS/INSER 1984) IMEEE, 325-329 Ryan, L. et al. (I 986) Acta Tropica 43, 85 -89 Kamhawi, 5. et al. (I 987) Med. Vet Entomoi. I, 97-102 Ready, PD. Smith, D.F. and Killick-Kendnck R ( 1988) Med. Vet Entomoi 2. 109--I I6 Ready, P.D. et al. (I 99 I) Mem inst Oswaido Cruz 86,4 I-49 Adamson. RE. et (71 (I 99 I ) Parassltologra 33 (Suppl. I). 45-53 Adamson, R.E. et al. (1993) Med. Vet Entomol. 7. 203 --207 Rogo, L.M.. Khamala. C.P.M. and Mutinga, M.J. ( 1988) Blochem. Syst Ecol. I 6, 655 - 659 Le Pant. F. et al. (1985) CR Acad. Scr Pans 300.479-482 Calllard. T et 01. (1986) /_ Med. Entomol.
23,489 492 I I Bonnefoy, S. et al. (I 986) Cahlers ORSTOM Entomol. Med. Parasltol. 24, 2 I3 -2 I7 I2 Ward, R.D. et al. (I 988) In Biosystematlcs of Haematophagus Insects (SewIce. M.W.. ed.). pp 257-269. Systematlcs Association Special Vol. 37, Clarendon Press 13 Lanzaro, G.C. et al. (I 993) Am. ] Trap. Med. Hyg. 48, 839-847 14 Ready, P.D. and Smith, D.F. (I 989) In Lershma&s: The Current Status anb New Strategies for Control (Hart, D.T.. ed.). pp 965-972. Plenum Press I5 Krwtzer, R.D. et a\. (I 990) 1. Med. Entomol 27. l-8 I6 Perrottl, E. et al. (199 I) Parassitologia 33 (Suppl. I ), 463--469 I7 Kassem, H.A. et al. (I 990) 1. Med. Entomoi. 27,592-60 I RD., Pasteur, N. and RIOUX, J.A. I8 Ward, (I 98 I) Ann Trap. Med. Parasrtol. 75. 235-245 I9 Mery. A. et al. (1982) Bfochem Syst Ecol. IO, 83 -90 20 Nevo, E. ( 1978) Theor. Popul. Bioi. I 3. I 2 I- I77 2 I Petersen. J.L. ( 1982) in New Approaches to the Identificat!on of Parasrtes and Their Vectors (Newton, B.N. and Michal, F., eds), pp 349-362. TDR Series No. 5 22 Zhang, L-M. and Leng. Y-J. (1991) Parawtologia 33 (Suppl. I). 54 I-550 23 Nei. M. ( 1972) Am. Nat 106.283 -292 24 Kreutzer, R.D. et al. (I 987) J. Med. Entomol.
24, 609-612 25 Kreutzer, R.D. et al. (I 988) 1. Am. Mosq. Control Assoc. 4, 453-455 26 Bhat, U.K.M. and Modi, G.B. (1976) Curr Ser. 45,265 -266 27 Trolano, G. ( 1982) Parassitologra 24, 23 l-236 28 White. G.B. and Killick-Kendrick. R (1975) J. Entomal. 50, I 87- I96 29 Wu. W-K. and Tesh. R.B. (1990) Am. J. Trop.
Med. Hyg. 42, 320-328 30 Wu. W-K and Tesh, R.B. (1990) Am. _/. Trap. Med. Hyg. 42,329-334 3 I Rlbelro. J.M.C. et al. (1989) Science 243, 212-214 32 Zeledon, R. et al. (I 989) Trans. R. Sot. Trop. Med. Hyg. 83, 786 R.B. and 33 Gtimaldi, G., Tesh. McMahon-Pratt, D. (I 989) Am. /. Trap. Med. Hyg. 41,687-725 34 Warbure. A. et al. II 994) Trans. R. Phil. Sot. 345,267-267 ’ ’ 35 Ben-Ismall, R. et al. (I 992) Trans. R Sot. Trap. Med. Hyg, 86,508&5 IO 36 Gramiccla, M. et ai. ( 199 I) Trans. R. Sot. Trap. Med. Hyg. 85,370-37 I 37 Abdalla, R.E. (I 982) Ann. Trap. Med. Parasitol. 76,299 -307 38 Klllick-Kendnck R. (I 990) Med. Vet Entomol. 4. l-24 39 Ward, R.D. et al. ( 1987) in Leishmaniasis: The Current Status and New Strategies for Control (Hart, D.T.. ed.), pp 235-243, Plenum Press 40 Titus, R.G. and Ribeiro, J.M.C. (1988) Soence 239, l306- I308 41 Theodos, C.M., Ribelro. J.M.C. and Titus, R.G. ( I99 I ) Infect Immun. 59, I592- I598 42 Theodos. CM and Titus, R.G. (I 993) Parasite Immunol. 15.48 I-487
Gregory Lonraro Malaria National
Research,
is at
Notional
the Loborutory of Institutes
ofHealth,
and infectious Dlseoses, BuJdrng 4, Room 126, Bethesda,
MD
Institute
20892-0425,
of
Allergy
USA.
Alon
Wartxq
IS
of Parasitology, Hebrew Unwersity-HodassahMedical School,Ein Kerem, /en&em 9 1010, Isroel. Tel: +I 301 496 1700, Fax: + I 301 402 0079, e-mail: [email protected] at
the
Deportment
Fit for Fertilization: Mating in Malaria Parasites L.C. Ranford-Cartwright The human malona paroslte Plasmodium falciparum has on obligate sexual phase in its life cycle. Male and femoie gametes must
mate
in the
mosquito
mIdgut
for
transmission to occur. When mosqu/toes ingest a mixture of two parasrte clones, the inheritance of nuclear genes suggests that mating between gametes IS random. Both cross-fertilization (between unIIke male ond female gametes) and selfing OCCUT. However, ,t has been suggested that the Inheritance of mrtochondrial markers lndicotes non-random mating. An altemotlve hypothesis, which IS presented here by Lisa Ronford-Cartwright, 1s that mating IS random, but differences ip the relative fitnesses of the gametocytes can expbn the inheritance patterns observed. Malaria parasites are haplold, except for a brief diploid stage after rJgote for154
mation in the mosquito gut and prior to the formation of the oocyst stage. An oocyst contains the haploid meiotic products (sporozoites) from a single fertilization event. Recently it has become possible to analyse this stage, allowing the inheritance of both nuclear and extra-chromosomal genes to be studied’ 4. Crossing Experiments and Inheritance of Nuclear Markers Genetic crosses have been made in the laboratory by allowing mosquitoes to feed on a mixture of gametocytes from two different cloned Plasmodium falcrparum Ilnes5 (Fig. I). Analyses of such crossing experiments, involving the clones 3D7 and HB3, showed that zygotes both heterozygous and homo-
zygous for two unlinked nuclear genes (MSPI and MSP2) were producedi,3. Homozygous oocysts are the products of matings between gametes from the same parasite clone (self-fertilization), and heterozygous oocysts the result of matings between gametes from different clones (cross-fertilization). The proportions of the three types of zygotes (homozygous for the 3D7 allele: homozygous for the HB3 allele; heterozygous containing both 3D7 and HB3 alleles) were found to be in accordance with expectations if matings between all gametes are random3. Random mating has also been suggested to occur between gametes of another human malaria parasite, Plasmodium v&ax, from studies on mosquitoes fed on humans naturally Infected with this parasite2.
Genetic Variability in Phlebotomine Sandflies: Possible Implications for Leishmaniasis Epidemiology G.C. Lanzaro and A. Warburg The vast majonty of genetIc stud/es of phlebotomlne sandflres have had as their goal the resolution of taxonom,c problems. In this paper, Gregory Lonzaro and A/on Warburg summarize the iiterature descnbing levels ofgenetic polymorphism, both w~thrn and between sandfly species, and speculate on the signrficance of vanabrbty in the vector to the complex epdem,o/ogy ofleishmaniavs.
In an earlier review, Ward commented on the shortage of InformatIon on the genetics of phlebotomine sandflies; although some progress has been made, this observation remains true today. Genetic Diversity Epidemiological studies of lelshmaniasis naturally begin with efforts to Identify the vector, This work IS often obfuscated by the fact that closely related species can only be dIstinguIshed morphologically in one sex. A number of molecular-level approaches have been taken to reveal markers of taxonomlc value. Gas chromatographic analysts of cuticular hydrocarbons has been used to distinguish morphologically Indistinguishable females of Phiebotomus we/comei from P. complexus’. Analysis of cuticular hydrocarbons can reveal distinctions even below the species level’ Although these examples illustrate the potential of cutlcular hydrocarbons as taxonomic characten, their utility IS limited because the genetlc basis for observed variability is not clear. Probes have been developed from genomic DNA fragments and used as sandfly species diagnostics4 6. These approaches are useful in the area of taxonomy, but provide no information on genetic relationships among species or on the genetic structure of populations. More recently, a polymetase chain reaction-based technique, known as random amplified polymorphic DNA (RAPD-PCR), has been employed to distinguish morphologically identical females of Lutzomyla spinrcrasso from L. youngi7. This technique uses a single primer in the PCR to amplify DNA fragments at random. Polymorphisms are expressed as the presence or absence of a fragment of a particular size Pormtology
Toduy.
vo’
/ I CL 4 IW‘I
This technique does produce useful genetic information and data generated by this approach have been used by population geneticists. However, there are disadvantages: the majonty of RAPDPCR polymorphisms are Inherited as dominant markers, which prevents tests of random mating within populations because individual genotypes cannot be determined. The molecular technique most widely used to date is analysis of polymorphisms In enzyme encoding genes by electrophoretlc analysis of isozymes. This approach yields information useful both for taxonomy and for detailed analysis of the genetics of populations. Phlebotomous pedrfer and P. elgoonerws are closely related species that can be distinguished only In the males; the former is a vector of Leishmanla aeth/opica in Kenya, the latter is not a vector, and the two occur together over a large portion of their range. A survey of allozyme variablllty revealed four IOCI to be dlagnostlc for the two specie+. The species status of two color forms of Lutzomyla (Psychodopygus) carreral (subsequently i. can-era1 S.S. and L. yucumensis) was confirmed by the discovery of diagnostic allozymes9~~o. Phlebotomlne sandfly species evolve with very little change in gross morphology. In such a group, one would expect species to exist that are Identical morphologically. These so-called sibling or cryptic species are common In other medically Important Diptera and have proven to be of great Importance In cases where there are differences In vectorial capacity among them. A study of two morphological variants of L. long,palprs taken from a single site in Bolivia showed they were not distinguishable by isozyme analysis”. Earlier work on this species indicated that mating barriers, mediated by male pheromones, may exist among different forms”. Recently, lsozymes were used, in conjunction with crossmating expenments, to demonstrate that L. longipalpls is a complex of at least three sibling specles’3. Sibling species In the subfamily Phlebotomlnae are probably common and, as work continues on the population genetics of this group, many more sibling species complexes WIII undoubtedly be uncovered.
Describing the degree of genetic variability in a species is best done by measunng heterozygosity (H), which gives the estimated proportion of genes expected to be heterozygous in an average individual. Ready and Smith14 repot-ted that Isoenzyme variation is limited in sandfIles, but this claim is not supported in the literature. Table I summarizes both observed and expected (based on computing heterozygote frequencies from allele frequency data under the Hardy-Weinberg equilibrium) heterozygoslties from I4 sandfly species in three genera. The overall heterozygosity averaged over species is 0.078. which IS actually slightly higher than the value (0.074) reported for 23 species of Insects in a review by Nevo20. In some cases, there are differences between direct and estimated measures of l-f, as in P. perrvciosus from Tuscany and P. pop&as/ from Egypt (Table I). In these studies, the authon do not suggest significant sub-structuring of these populations and we conclude that these departures are likely the result of sampling en-or-s or errors in gel interpretation. The data in Table I should be viewed with some caution, as many of the values are based on samples of ten, or fewer, loci. Levels of divergence among populations or between taxa are best described by genetic distance statistics, which measure allelic differences at multiple loci. Such values can be used to assemble phylogenies, and may provide clues to establish the taxonomic status of genetlcally distinct populations. Table 2 summarizes genetic distance values reported for a variety of sandfly species at a number of taxonomic levels. The reported levels of genetic divergence agree well with the taxonomic arrangement of the species studied, with distances among local populations of a single species having the smallest values and comparisons of species in different subgenera having the highest. The informal taxonomic rank ‘series’, which represents assemblages of related species within a genus, below the rank of subgenus, also appears to be supported by the data summarized in Table 2. Data from other organisms have shown that the relationship between taxonomic 151
Focus Table
I. Observed and expected heterozygosity for enzyme encoding gene loci in Phlebotomine sandflies Mean sample size
Species Lutzomyio carrerai L yucumensis L bngipolpis L /ongipalpis Brazil Colombia Costa Rica L verrucarum species group
per locus I05 107 138
Phlebotomus perniciosus Sardinia Tuscany P. perfirewi P. papatasi P. langeroni P. ariase L’Aumede Banyuls P. perniciosus Banyuls Port-Vendres
Sergentomyia magna S. africana S. hamoni Yaka-Yaka Mayombe 5. schwetzi
No. of loci studied II II IO
Source of material (Country) Field (Bolivia) Field (Bolivia) Field (Bolivia)
0.0”69 0.126 0.037
0.120 0.145 0.036
0.054 0.072 0.064
Ii
Ref. IO IO II 13
H =P
=
24 22 21
27 27 27
Colony Colony Colony
0.057 0.07 I 0.058
220
21
Field (Colombia)
0.070-O.
44 23 33 51 50
II II II 21 21
Field (Italy) Field (Italy) Field (Italy) Colony (Egypt) Colony (Egypt)
0.033 0.018 0.050 0.062 0.054
0.04 I 0.042 0.055 0.080 0.063
34 21
9 9
Field (France) Field (France)
0.074 0.075
0.062 0.074
55 39
9 9
Field (France) Field (France)
0.067 0.085
0.057 0.08 I
II I 47
6 6
Field (Congo) Field (Congo)
0. I77 0.1 I7
60 84 43
6 6 5
Field (Congo) Field (Congo) Field (Congo)
0.1 IO 0.142 0.035
I76
0.082-o.
I7 I
I5 I6
I6 I7 I7 I8
I8
I9 I9 I9
I9
aHo,,,,observed heterozygosity;H,,,. expected heterozygosity (see text) status and the magnitude of distance values can sometimes be misleading, but they may help to guide workers to pursue further efforts where values are not consistent with existing taxonomlc rankings. Cytogenetics
The karyotypes of I2 species have been described: eght species In the genus Lutzomyia24~25 and four species In the genus Ph/ebotomus1426,2T.Only one species, P. pernroosus. has a karyotype with sexual dimorphism, with males being the heterogametic sex (XV) (with females being XX). Extensive polymorphism in chromosome morphology has been observed, but the karyotype for several species In the genus Lutzomyra
appears to be ldentlcalmq. Old World species have greater variabllrty In chromosome number, 2N = 6 IO. All the iutzomyio species thus far studied have a chromosome number of 2N = 4, except i. traprdor (2N = 3). The general lack of heterogamy suggests that sex determnation IS controlled by Indivldual IOU. Met-y et al I9 have shown slgnificant differences In the distribution of genotypic frequencies at several enzyme encoding gene IOCI between the sexes in several species in the genus Sergentomyio and suggest that these genes may be linked to a sex-detennintng locus In this genus. The polytene chromosomes of larval saIlvary glands have been described for only one species, L. /ong~palp/s’s. Despite the fragile nature of sandfly polytenes,
Table 2. Values for genetic distance at various taxonomic levels in phlebotomine sandflies”
Taxa compared Populations within species Sibling species
No. of comparisons 3 3
Genetic
distance
(‘V WEI 0.004 (~0.004)
Refs 16,17
0.236 (kO.107)
I3
Species within series
21
0. I94 (+O. 127)
I5
Between series
23
0.928 (kO.276)
15
Species within subgenera
4
0.6 I2 (kO.27 I)
IO, 16.2 I ,22
Between subgenera
6
I.250 (kO.40 I)
17.21
’ Data from Ref. 23 I52
the authors conclude that they are suitable for study. Further work on sandfly polytenes is thus warranted and could prove very useful, not only for cytotaxonomy but for the production of genetic maps by utilizing polytenes in in situ hybridizatlon&dies. Genetics
of Susceptibility
to Parasite Infection There is only a single study, reported in tandem paperG30, dealing with the genetic basis of susceptibility/refractoriness to parasite infection in phlebotomine sandflies. In this study, two strains of P. popatasi originating from a common laboratory colony were selected for high and low infection rates (IR) with Leishmania major (strain WR-309). After I7 generations of selection, a susceptible strain (IR = 87.1%) and refractory strain (IR = 5.2%) were developed. That there is a genetic component to susceptibility and refractoriness is shown by the fact that strains did respond to selection. However, the fact that pure lines could not be obtained after I7 generatlons suggests that these traits are not under single locus control. In reciprocal and backcross experiments, it was shown that neither susceptibility nor refractoriness is dominant over the other. Parastology
Today, vol.
I I, no. 4, I995
Focus
-
Sandfly Variability and Variation in Clinical Manifestations of Leishmaniasis Lutzomyia longipalprs IS the vector of Leishmanra chagasl, the causative agent of human visceral leishmaniasis throughout much of South and Central America. Our interest in the genetics of sandflies was aroused when we noticed that bites of I_ longipalpis females from Costa Rica do not leave long-lasting red etythemas (Fig. I), which are characteristic of L. longipalpis from Brazil and Colombia3’. This was particularly interesting because, in Costa Rica, infections with L. chagasi invariably result in a non-ulcerative form of cutaneous leishmaniasis32, while in South America infections are almost exclusively associated with viscera leishmaniasisaa. Whereas parasite isolates from cutaneous cases proved indistinguishable from isolates made from viscera infections, we showed that the two forms of the disease are transmitted by different sandfly species13. We subsequently showed that saliva of Costa Rican L longipalpis has very little vasodilatory activity but strongly enhances cutaneous leishmaniasis in mice. Flies from Brazil and Colombia displayed much higher vasodilatory activity but reduced capacity to enhance cutaneous proliferationa4. Although it remains speculative, we propose that at least some of the variability in the clinical presentations of L chagasi infections in Latin America may be due to the different composition of the saliva of their vectors. If confirmed, this will be the first demonstratron of the importance of genetic variability in vector species to the epidemiology of leishmaniasis and the first example, for any vector-borne disease, of a vector modulating the pathology of the par-a site it transmits. Variability in clinical manifestations is not unique to L chagas/ infections. In North Africa and Southern Europe, L. infanturn (a parasite arguably identical with L chagasr] causes visceral infections in some areas, but primarily cutaneous disease in other geographical region&36. Cutaneous lesions due to L. donovanl, the causative agent of kala-azar in Africa, have also been reportedaT. In the Mediterranean region, a number of Phlebotomus (Larroussit~s) spp are known to transmit L. infanturn and in East Africa P. (Synphlebotomus) spp are the possible vectors of L. donovania*. Data on the saliva of local vectors and their distribution in relation to clinical manifestations and parasite strains may be crucial for understanding the epidemiology of the diseases they transmit. Parasitology Jodoy, vol I I, no 4, 1995
Fig. I. Appearance of erythemas produced by the bites of three sibling species of the sandfly lutzomyio longipalpis (photographed 12 h after feeding): Brazil (Minas Gerais) (a), Colombia (Tolima) (b), Costa Rica (Guanacaste) (c,d). (Reproduced, with permission, from Ref. 34.)
Different clinical manifestations of seemingly identical parasites in separate geographic regions are also known within the L. braziliensis complex. L. b. panomensls and L. b. braziliensls normally produce a cutaneous sore at the site of the sandfly bite and a certain percentage of untreated cases go on to develop secondary chronic infections in the mucosal membranes of the nose and pharynx (=mucocutaneous leishmaniasis). Of those Infected with L. braziliensis, a large percentage develop mucocutaneous manifestations in Brazil, Bolivia and Colombia (2558 I%). In Central America (Costa Rica, Nicaragua, Panama), the percentages are significantly lower (2-5%; compiled from references cited by Grimaldi, et a/.33). There are I5 proven or suspected vectors of L. b. braziliensis and nine suspected vectors of L. b. par-amens/s In Latin America3*. While some of these differences are certainly attributable to host factors and parasite strains, it is not inconceivable that genetlc differences among vectors
also contribute to this complex epidemiological scenario. Research on genetic variability in natural populations of Phlebotomine sandflies has been largely focused on the resolution of taxonomic problems. Distinguishing vector from non-vector species is important, but these efforts should be extended beyond the description of taxonomrc character;. Studies of sandfly genetics, in topics such as vectorparasite interactions, reproductive biology and population genetics are sorely lacking. Exciting research in several areas, including variability in sandfly sex pheromones12J9 and the relationship between sandfly saliva and host immunological response to Leishmanio parasites40m42 suggest promising topics for future research in genetics. Finally, detailed studies of the distribution of polymorphisms at the nucleotide level will provide descriptions of the genetic structure of sandfly populations with a degree of resolution not previously possible. Extending our knowledge of sandfly genetics will 153
Focus improve
of the role
our understanding
of vector
species
in the
epidemiology
of the diseases they transmit provide
information
velopment
of new
and should
critical to the strategies
aimed
deat
their control.
Acknowledgements Thus workwassupported by grant ID-9 IO43 from the UNDPIWotid Bank/WHO/Special Programme for Research and Training in Tropical Diseases (TDR) and from the MacArthur Foundation program on the Molecular Biology of Parasite Vectors. We thank Robert B. Tesh and Richard D. Ward and three anonymous reviewerj for their comments on an earlier version of this paper. References
I 2 3 4 5 6 7 8 9 IO
Ward, R.D. (I 986) Montpellrer Nlbtoekkuer (Coil. Int. CNRS/INSER 1984) IMEEE, 325-329 Ryan, L. et al. (I 986) Acta Tropica 43, 85 -89 Kamhawi, 5. et al. (I 987) Med. Vet Entomoi. I, 97-102 Ready, PD. Smith, D.F. and Killick-Kendnck R ( 1988) Med. Vet Entomoi 2. 109--I I6 Ready, P.D. et al. (I 99 I) Mem inst Oswaido Cruz 86,4 I-49 Adamson. RE. et (71 (I 99 I ) Parassltologra 33 (Suppl. I). 45-53 Adamson, R.E. et al. (1993) Med. Vet Entomol. 7. 203 --207 Rogo, L.M.. Khamala. C.P.M. and Mutinga, M.J. ( 1988) Blochem. Syst Ecol. I 6, 655 - 659 Le Pant. F. et al. (1985) CR Acad. Scr Pans 300.479-482 Calllard. T et 01. (1986) /_ Med. Entomol.
23,489 492 I I Bonnefoy, S. et al. (I 986) Cahlers ORSTOM Entomol. Med. Parasltol. 24, 2 I3 -2 I7 I2 Ward, R.D. et al. (I 988) In Biosystematlcs of Haematophagus Insects (SewIce. M.W.. ed.). pp 257-269. Systematlcs Association Special Vol. 37, Clarendon Press 13 Lanzaro, G.C. et al. (I 993) Am. ] Trap. Med. Hyg. 48, 839-847 14 Ready, P.D. and Smith, D.F. (I 989) In Lershma&s: The Current Status anb New Strategies for Control (Hart, D.T.. ed.). pp 965-972. Plenum Press I5 Krwtzer, R.D. et a\. (I 990) 1. Med. Entomol 27. l-8 I6 Perrottl, E. et al. (199 I) Parassitologia 33 (Suppl. I ), 463--469 I7 Kassem, H.A. et al. (I 990) 1. Med. Entomoi. 27,592-60 I RD., Pasteur, N. and RIOUX, J.A. I8 Ward, (I 98 I) Ann Trap. Med. Parasrtol. 75. 235-245 I9 Mery. A. et al. (1982) Bfochem Syst Ecol. IO, 83 -90 20 Nevo, E. ( 1978) Theor. Popul. Bioi. I 3. I 2 I- I77 2 I Petersen. J.L. ( 1982) in New Approaches to the Identificat!on of Parasrtes and Their Vectors (Newton, B.N. and Michal, F., eds), pp 349-362. TDR Series No. 5 22 Zhang, L-M. and Leng. Y-J. (1991) Parawtologia 33 (Suppl. I). 54 I-550 23 Nei. M. ( 1972) Am. Nat 106.283 -292 24 Kreutzer, R.D. et al. (I 987) J. Med. Entomol.
24, 609-612 25 Kreutzer, R.D. et al. (I 988) 1. Am. Mosq. Control Assoc. 4, 453-455 26 Bhat, U.K.M. and Modi, G.B. (1976) Curr Ser. 45,265 -266 27 Trolano, G. ( 1982) Parassitologra 24, 23 l-236 28 White. G.B. and Killick-Kendrick. R (1975) J. Entomal. 50, I 87- I96 29 Wu. W-K. and Tesh. R.B. (1990) Am. J. Trop.
Med. Hyg. 42, 320-328 30 Wu. W-K and Tesh, R.B. (1990) Am. _/. Trap. Med. Hyg. 42,329-334 3 I Rlbelro. J.M.C. et al. (1989) Science 243, 212-214 32 Zeledon, R. et al. (I 989) Trans. R. Sot. Trop. Med. Hyg. 83, 786 R.B. and 33 Gtimaldi, G., Tesh. McMahon-Pratt, D. (I 989) Am. /. Trap. Med. Hyg. 41,687-725 34 Warbure. A. et al. II 994) Trans. R. Phil. Sot. 345,267-267 ’ ’ 35 Ben-Ismall, R. et al. (I 992) Trans. R Sot. Trap. Med. Hyg, 86,508&5 IO 36 Gramiccla, M. et ai. ( 199 I) Trans. R. Sot. Trap. Med. Hyg. 85,370-37 I 37 Abdalla, R.E. (I 982) Ann. Trap. Med. Parasitol. 76,299 -307 38 Klllick-Kendnck R. (I 990) Med. Vet Entomol. 4. l-24 39 Ward, R.D. et al. ( 1987) in Leishmaniasis: The Current Status and New Strategies for Control (Hart, D.T.. ed.), pp 235-243, Plenum Press 40 Titus, R.G. and Ribeiro, J.M.C. (1988) Soence 239, l306- I308 41 Theodos, C.M., Ribelro. J.M.C. and Titus, R.G. ( I99 I ) Infect Immun. 59, I592- I598 42 Theodos. CM and Titus, R.G. (I 993) Parasite Immunol. 15.48 I-487
Gregory Lonraro Malaria National
Research,
is at
Notional
the Loborutory of Institutes
ofHealth,
and infectious Dlseoses, BuJdrng 4, Room 126, Bethesda,
MD
Institute
20892-0425,
of
Allergy
USA.
Alon
Wartxq
IS
of Parasitology, Hebrew Unwersity-HodassahMedical School,Ein Kerem, /en&em 9 1010, Isroel. Tel: +I 301 496 1700, Fax: + I 301 402 0079, e-mail: [email protected] at
the
Deportment
Fit for Fertilization: Mating in Malaria Parasites L.C. Ranford-Cartwright The human malona paroslte Plasmodium falciparum has on obligate sexual phase in its life cycle. Male and femoie gametes must
mate
in the
mosquito
mIdgut
for
transmission to occur. When mosqu/toes ingest a mixture of two parasrte clones, the inheritance of nuclear genes suggests that mating between gametes IS random. Both cross-fertilization (between unIIke male ond female gametes) and selfing OCCUT. However, ,t has been suggested that the Inheritance of mrtochondrial markers lndicotes non-random mating. An altemotlve hypothesis, which IS presented here by Lisa Ronford-Cartwright, 1s that mating IS random, but differences ip the relative fitnesses of the gametocytes can expbn the inheritance patterns observed. Malaria parasites are haplold, except for a brief diploid stage after rJgote for154
mation in the mosquito gut and prior to the formation of the oocyst stage. An oocyst contains the haploid meiotic products (sporozoites) from a single fertilization event. Recently it has become possible to analyse this stage, allowing the inheritance of both nuclear and extra-chromosomal genes to be studied’ 4. Crossing Experiments and Inheritance of Nuclear Markers Genetic crosses have been made in the laboratory by allowing mosquitoes to feed on a mixture of gametocytes from two different cloned Plasmodium falcrparum Ilnes5 (Fig. I). Analyses of such crossing experiments, involving the clones 3D7 and HB3, showed that zygotes both heterozygous and homo-
zygous for two unlinked nuclear genes (MSPI and MSP2) were producedi,3. Homozygous oocysts are the products of matings between gametes from the same parasite clone (self-fertilization), and heterozygous oocysts the result of matings between gametes from different clones (cross-fertilization). The proportions of the three types of zygotes (homozygous for the 3D7 allele: homozygous for the HB3 allele; heterozygous containing both 3D7 and HB3 alleles) were found to be in accordance with expectations if matings between all gametes are random3. Random mating has also been suggested to occur between gametes of another human malaria parasite, Plasmodium v&ax, from studies on mosquitoes fed on humans naturally Infected with this parasite2.