4. Discussion
In Morocco, ACL due to L. tropica is transmitted by P. sergenti which has a large geographic distribution probably related to the wide ecological plasticity of this vector (Rioux et al.,1986; Ramaoui et al., 2008; Boussaa et al., 2009). CL due to L. tropica is an emerging disease even though the geographical extension of the vector is greater than that of the parasitic protozoan, and the identification of factors for parasite expansion is essential for effective disease control. P. sergenti density and genetic characteristics were investigated as determining factors for the existence of ACL transmission. Comparative intradomiciliary and peridomiciliary sand fly captures in the ACL endemic locality of El Borouj and the undamaged locality of Sidi Hajjaj were made using CDC light traps and sticky papers.
P. sergenti density in the ACL free locality was lower than that of the endemic locality, both peridomiciliary and within households. Interestingly, P. sergenti was the most abundant and densest species within households in Sidi Hajjaj, however it was the fourth species outdoors, after S. minuta and the L. infantumvectors, P. perniciosus and P. longicuspis (Table 1). The relative abundance of P. sergenti males and females varied between the trapping methods as males were more abundant in the sticky papers. In both localities, the female P. sergenti density was higher in the intradomiciliary June captures and all gonotrophic cycle categories, non-fed, fed and gravid females, were found (Table 2).
Although to date no ACL cases have been diagnosed in Sidi Hajjaj, these sand fly density figures seem sufficient for the maintenance of L. tropica transmission (Rioux et al., 1986; Ramaoui et al.,2008; Barón et al., 2013) and would make this locality susceptible to the establishment of an ACL transmission cycle. Over the last few decades, L. tropica foci have spread to several regions of Morocco including those where CL caused by L. major orL. infantum has been reported, which shows the changing geographical patterns of this species (Baghad et al., 2020). The growing mobility of humans from endemic to non-endemic cities raises the possibility of emerging foci in areas where P. sergentipopulations are well established. Kholoud et al. in 2020 suggested that ACL dissemination in Morocco is associated to an increase in human travel and local tourism linked to economic expansion and infrastructure development as shown by the synchronized occurrence of new ACL foci with the construction of new motorways. However, the factors underlying the spatio-temporal transmission dynamics of leishmaniasis are not well understood, and the epidemiological picture is not as simple as deduced from the previous statement.
The molecular characterization of P. sergenti populations in both localities using the PCR-RFLP technique of the cytochrome b mitochondrial gene, has allowed us to find that the main P. sergenti mitochondrial lineage in El Borouj is Lineage IV (97.6%) while the remaining 2.4% belongs to lineage II. In contrast, lineage II was the most abundant in Sidi Hajjaj (80%) followed by 20% lineage I specimens. Therefore, a different main P. sergenti mitochondrial lineage has been highlighted in each one of the 2 localities under study. Phlebotomus sergenti is characterised by high genetic diversity and classified in at least twenty haplotypes in four mitochondrial lineages (Yahia et al., 2004; Barón et al.,2008).
In El Borouj, L. tropica DNA was detected in 5 out of 184 (2.7%) female P. sergenti (Gijón-Robles et al., 2018) in captures made throughout 2014 (4+/72: 5.6% and up to 18,000 parasites/µg DNA; data not shown) and 2015 (1+/112: 0.9%). The five positive L. tropica females belonged to the most prevalent P. sergentilineage in El Borouj, lineage IV. This is the first time that the mitochondrial lineage of a P. sergenti specimen that is acting as a vector for L. tropica has been identified. A local increase in the abundance of this P. sergenti lineage that seem transmitsL. tropica more efficiently, could explain the emergence of ACL in El Borouj and its absence in Sidi Hajjaj.
Lineage I is over-represented in southwestern Europe (Merino-Espinosaet al., 2016) and this is the first time that its presence in Morocco is reported. No autochthonous ACL cases have been detected in the Iberian Peninsula, despite P. sergenti being commonly found at sufficient densities to act as a vector, and the existence of 2 mitochondrial lineages, one of them, held in common with Morocco (Lineage III) (Barón et al., 2008, 2013; Merino-Espinosa et al., 2016).
The existence of differential ecological traits between P. sergenti mitochondrial lineages has been pointed out: Merino-Espinosa et al. in 2016 found that Lineage I appear to have adaptive advantages represented by a wider tolerance to temperature and altitude changes, that would make it better suited to leading geographical expansion into the rest of Europe. Similarly, there are bioclimatic differences between El Borouj and Sidi Hajjaj (Table 4) that could explain the over representation of P. sergenti lineage IV in El Borouj which is warmer and drier, and its absence in Sidi Hajjaj.