Malformations of the genitalia in male Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae)

Abstract

Phlebotomus papatasi (Scopoli, 1786) (Diptera: Psychodidae) is a major vector of Leishmania major (Kinetoplastida: Trypanosomatidae), a causative agent of zoonotic cutaneous leishmaniasis. Morphological characters of sand fly genitalia are key indicators for species identification. Various anomalies affecting male genitalia have been previously described. We take advantage of a large sand flies survey conducted in 32 stations in Central and Southern Morocco to systematically quantify the prevalence and spatial distribution of malformations affecting the genitalia of P. papatasi. Among 597 examined males, 122 were abnormal (20.4%). Malformations were widespread and largely concerned the number of spines in the lateral lobes and in the styles. Asymmetrical anomalies in lateral lobes were common. Correspondence analysis of our results highlighted the symmetrical anomalies observed in the lateral lobes, and abnormal styles of the male genitalia were found to be associated with environmental disturbances since they were prevalent in sewage dumps. Female phlebotomine sand flies are involved in the transmission of arboviruses and Leishmania parasitic protozoans. Phlebotomus (Phlebotomus) papatasi (Scopoli, 1786) is a known vector of Leishmania major Yakimoff and Schokhor (Kinetoplastida: Trypanosomatidae) (Killick-Kendrick 1990), a causative agent of zoonotic cutaneous leishmaniasis in several countries, including Saudi Arabia (Killick-Kendrick et al. 1985), Morocco (Rioux et al. 1986), Tunisia (Ben Ismail et al. 1987), and Iran (Yaghoobi-Ershadi et al. 1995). P. papatasi has a large geographical distribution extending from India, the Middle East, and the Mediterranean Basin (Lewis 1982). In Morocco, the presence of P. papatasi was reported as early as 1916 (Delanoë 1916) and this species is very common (Bailly-Choumara et al. 1971, Rioux et al. 1984, Guernaoui 2006). P. papatasi has a significant ecological plasticity and can be found in various biotopes, including houses, animal sheds, and caves (Guernaoui 2006). The density of P. papatasi was higher in wild dumps and areas of sewage near large cities. In these particular environments, P. papatasi constituted more than 90% of sand flies (Guernaoui and Boumezzough, in press). Moreover, this vector adapts very well to artificial habitats (Kamhawi et al. 1991) and its density is more important in disturbed ecosystems (Wasserberg et al. 2003). In these habitats, P. papatasi males showed morphological abnormalities in their genitalia (unpublished observations). Morphological anomalies in the male genitalia of several New and Old World phlebotomine sand flies have been reported (Abonnec et al. 1971, Carzorla et al. 1991, Dedet et al. 1984, Marcondes 1999, Barreto et al. 2000). Their identification is of systematic interest because they affect the elements used in the specific species diagnosis of sand flies, and in the case of some bilateral anomalies, they can be at the cause of misidentifications. We report a systematic description of anomalies found in male genitalia and ascertain their prevalence and their spatial distribution in different Moroccan populations. We use multivariate analysis to examine the possible association between anomalies and environmental disturbances due to human activity. Sand flies were collected between April and December, 2000 to 2006 (Guernaoui 2006, Ramaoui et al. 2008) in 32 stations of Central and Southern Morocco (Table 1). Geographical localization of representative examples of stations is shown in Figure 1. The area of study is representative of the different bioclimates known to support the growth of P. papatasi. The area of Erfoud (Errachidia) is characterized by a Saharan bioclimate, the area of Souss (Agadir) by a per-arid to arid bioclimates, Chichaoua province (Imi'Ntanout) and Al-Haouz area (Marrakech) by arid to semi-arid bioclimates, and the area of Demnate (Central Hight-Atlas Mountains) by a sub-humid bioclimate. Map showing the study area in Morocco. Adult sand flies were collected in stations in urban, rural, and natural environments. Two strikingly different environments that delineate two categories of stations were defined. Stations in category (A) include areas of application of sewage, dumps near big cities, and ruins serving as dumps for household waste in villages with poor sanitary conditions. In contrast, stations classified in category (B) encompassed natural ecosystems and villages in rural areas with low population density. Representative examples of the two categories of stations are shown in Figures 2a and 2b. Examples of the environmental conditions found in the stations. Stations in category A: A-1 dump and area of application of sewage near Marrakech city, A-2 dump near Agadir city. Stations in category B: B-1 village in the province of Chichaoua, B-2 village in Al-Haouz province. Sand flies were caught using CDC light traps and/or sticky traps. CDC light traps were used inside houses and the sticky traps were set in and around human dwellings and also in natural environments. Collected specimens were preserved in alcohol and were processed according to the methods described by Abonnenc (1972). Females were identified by examination of the pharyngeal armatures and spermathecae morphology. The external male genitalia were examined. Particular attention was given to the presence of potential malformations in those body parts used for the species diagnosis. The number of abnormal males and of males without abnormalities in both categories of stations A and B were compared using a chi-square test. Potential associations among different malformation and station categories were analyzed with ADE 4 software (Thioulouse et al. 1997). To adjust qualitative and semi-quantitative ecological data, correspondence analysis was performed (Benzékri 1982, Legendre and Legendre 1998). This analysis allowed a global analysis by preserving maximum data, particularly those that were qualitative. For each locality, the presence or absence of the various malformation types was ascertained and the quality of the surrounding environment was noted as being in category A (dumps and areas of application of sewage) or in category B (wild sites and villages). Two series of analysis were performed. First, we analyzed the potential relationship between anomalies. Secondly, the relationship between stations was ascertained. Such analysis demonstrated the potential association between anomalies and stations. The position of each type of anomaly with respect to the environment in the two categories of stations was examined. Finally, an inter-group (permutation) test (Thioulouse et al. 1997) was performed to examine the robustness of the association between the typology of anomalies and that of the station categories. Conventional characteristics of the male genitalia of P. papatasi are illustrated in Figure 3a. The length of the coxite and of the style joined together exceeds the thorax length. The style is long and spindly. It carries five spines: three apical and two subterminal. The coxite presents a small lobe in its base furnished with short setae and in its subterminal part a dozen well-differentiated long setae. The paramere is trefoil and the lateral lobe carries two spines at its distal end. P. papatasi genitalia (©IRD/N. Rahola): a) without anomalies; b) Asymmetrical lobes with a supernumerary spine; c) Asymmetrical lobes with a missing spine; d) Style with a supernumerary spine; e) Style with a missing spine; f) Style with a missing spine and asymmetrical lobes with a supernumerary spine. Females of P. papatasi were identified morphologically. No morphological variations were observed among 322 females examined, although the presence of malformations in other parts of the body cannot be ruled out. For males, among 597 specimens, 20.4% bore malformations (122 specimens). The following anomalies, concerning specifically the number of spines in lateral lobes and in styles, were observed in P. papatasi genitalia: Symmetrical or asymmetrical supernumerary spine in the lateral lobes (Figure 3b) Symmetrical or asymmetrical missing spine in the lateral lobes (Figure 3c) Style with a supernumerary spine (six spines) (Figure 3d) Style with a missing spine (four spines) (Figure 3e). Various malformations that affect both the styles and the lateral lobes were found in four specimens (Figure 3f). The variations in the morphology of male genitalia were previously reported. Abonnenc et al. (1971) cited anomalies from American and European phlebotomine sand flies. They showed that some specimens of P. papatasi bore styles with a supernumerary spine. Later, Dedet et al. (1984) described anomalies in African sand flies affecting the style and the lateral lobes. These malformations affected about 1.7% of the P. papatasi collected in a station from Algeria and were reported to be exceptional in P. papatasi collected in Albania (Adhami et al. 1991). In contrast, 30% of the P. papatasi collected in a particular locality in Egypt presented an abnormal morphology (Kassem et al. 1988). However, where anomalies were recorded in a single population (single station), we found that the majority of the examined populations had at least one type of malformation (Table 1). We also found that the prevalence of malformations was relatively high in some examined populations since the prevalence of abnormal males exceeded 30% in sand flies collected at Demnate (48%), Wiskdou (42%), and Lekhmis or Douar Harmel (31%). Regarding P. papatasi populations, the morphological inventory of aberrant styles and lateral lobes showed that the prevalence of each type of anomaly differed significantly (Table 1). Anomalies affecting lateral lobes were generally more frequent (95%) than those affecting the styles (5%). Abnormal styles were found to be exclusively asymmetrical. This was not the case in lateral lobes where both symmetrical and asymmetrical anomalies were recorded. In lateral lobes, asymmetrical anomalies were more prevalent (40% and 35% for missing spine and supernumerary spine, respectively) than symmetrical ones (12.5% and 7.5% for supernumerary spine and missing spine, respectively). When considering the distribution of malformations in the prospected stations, we observed that asymmetrical anomalies affecting lateral lobes were common, as an asymmetrical supernumerary spine and asymmetrical missing spine were reported in 54.8% and 48.4% of the studied stations, respectively. Symmetrical lateral lobes anomalies were found only in 16.1% (missed spine) and 25.8% (supernumerary spine) of the stations. The frequency of malformations affecting the style were relatively low, only 9.7% of the prospected stations showed males with asymmetrical supernumerary spine and 6.5% of the stations presented males with an asymmetrical missing spine. The cause of these malformations in sand fly genitalia remains unclear. It has been hypothesized that some of them could correspond to the segregation of a recessive character under a selective pressure of unknown nature, or could be the result of injuries received by the insect during the course of its ontogeny (Dedet et al. 1984, Kassem et al. 1988). The action of chlorfluazuron, an insect growth inhibitor, added in the larval diet was shown to cause morphological abnormalities of adult Lutzomyia longipalpis (Quesada and Montoya-Lerma 1994). It is well known that morphological characteristics of living organisms can be significantly affected by environmental conditions during their ontogenetic development, especially for sand flies, known for their inability to disperse (Killick-Kendrick et al. 1984). We thus hypothesized that in sand flies, malformations of the male genitalia were associated with some specific environment. Furthermore, a careful examination of our data pinpointed that two groups of stations or populations can be distinguished. The first group corresponds to populations with one to two types of malformations and a prevalence of abnormal males ranging from 0% to 20%, and a second group including populations of males bearing three to four types of malformations with a prevalence of 15% to 48%. In addition, we compared the number of males in stations in category A and in stations in category B using a chi-square test. The high prevalence of malformations was related to highly disturbed environments (dumps and areas of application of sewage) (P< 0.0001). We then used the multivariate analysis to examine the association between different abnormality types and the relation between each anomaly type and the station categories. Correspondence analysis (Figure 4) shows a distribution of genitalia anomalies of P. papatasi in the factorial plan (II-III) (Figure 4–1). This plan accounts for 65% of the total inertia. Figure 4–2 shows the discrimination of stations according to their environment that falls in the same factorial plan. Stations are gathered into two categories, A or B, according to the state of their environment that also distinguishes the types of malformation. These two categories are clearly distinguished by the factorial axis II. On the left of the axis, we found asymmetrical anomalies found in the lateral lobes, which would rather be associated with the B environment. In this category, we found stations not highly disturbed (salvage sites, villages) (Figure 2b). Moreover, these anomalies are common and largely widespread in the studied populations. Conversely, on the right of axis II, we found symmetrical anomalies present in the lateral lobes and abnormal styles which would be rather associated with the A environment that regroup stations, including public dumps near big cities like Marrakech (Harbil) and Agadir, areas of application of sewage (Azzouzia), and some villages with ruins serving as dumps for household waste with poor sanitary conditions (Figure 2a). Altogether, our results show a typology of the environmental quality that strongly correlates with the distribution of malformations (P<0.01). Correspondence analysis Anomalies- Stations: Distribution of anomalies* (1) and stations** (2) in the factorial plan (II-III). *List of anomalies: 1AL: Asymmetrical Lobes with a missing spine; 3AL: Asymmetrical Lobes with a supernumerary spine; 1SL: Symmetrical Lobes with missing spines; 3SL: Symmetrical Lobes with supernumerary spines; AbS: Abnormal Styles (with a missing or a supernumerary spine). **Stations are grouped into two categories (A: dumps and areas of application of sewage; B: wild sites and villages). The typology anomalies- environment is statistically significant (P<0.01). In conclusion, our work clearly demonstrated that phenotypic variation in the genitalia of male P. papatasi is associated with some specific environments. Asymmetrical anomalies in lateral lobes are ordinary and affect a majority of the examined populations, but symmetrical anomalies in lateral lobes and abnormal styles strongly correlate with human-made disturbances since they are prevalent in wild dumps and areas of application of sewage. However, the population structure of P. papatasi has yet to be understood. The genetics and the possible importance of these anomalies have to be evaluated. We thank Dr. Didier Fontenille (UR 016, IRD) for reviewing the manuscript.