Species diversity and vector competence of sand flies in an urban cycle of cutaneous leishmaniasis in Shiraz, Southwestern Iran

INTRODUCTION

Cutaneous leishmaniasis (CL) remains a formidable global health challenge, with transmission dynamics intricately linked to the complex ecology of phlebotomine sand fly vectors.^[1] The distribution and burden of this disease are largely dictated by the presence of competent vector species, their population dynamics, and their interaction with susceptible hosts within specific environmental niches.^[2] Understanding these entomological parameters – species composition, seasonal abundance, and infection rates – is therefore fundamental to deciphering transmission cycles and designing effective, location-specific control programs.^[3]

In urban and peri-urban landscapes, transmission ecology undergoes significant modification.^[4] Urbanization alters microhabitats, creates novel breeding sites, and concentrates human populations, potentially shifting vector species dominance, prolonging seasonal activity, and facilitating new parasite-vector associations.^[5] In the Middle East, including Iran, such environments have seen the stabilization of anthroponotic cycles, particularly those involving Leishmania tropica, yet the precise entomological drivers underpinning these urban foci are often inadequately characterized.^[6] While Phlebotomus sergenti is recognized as a primary vector for L. tropica, and P. papatasi for Leishmania major, their relative abundance, seasonal phenology, and realized vector competence can vary dramatically between geographic locations and habitat types, influencing local disease epidemiology.^[7,8]

A critical knowledge gap persists regarding the integrated entomological profile of CL vectors within historic urban centers, where unique architectural and socioecological conditions prevail.^[9] Comprehensive data on local sand fly species diversity is essential, as the presence of secondary or suspected vector species can complicate transmission networks.^[10,11] Perhaps most important is the direct assessment of natural infection rates within vector populations, which provides definitive evidence of a species’ role in transmission and can distinguish between active zoonotic, anthroponotic, or mixed cycles.^[12,13]

In southern Iran, a region endemic for both anthroponotic cutaneous leishmaniasis (ACL) and zoonotic cutaneous leishmaniasis (ZCL), historic cities such as Shiraz represent significant and persistent foci of human disease.^[14] Previous studies have indicated the involvement of P. sergenti, but a holistic analysis linking detailed species inventory, precise seasonal fluctuation, and molecular evidence of parasite infection within the context of an urban environment is lacking. Such an integrated entomological assessment is necessary to move beyond general assumptions and provide evidence-based insights for targeted intervention.

This study was therefore designed to elucidate the entomological foundations of CL transmission in the historic urban landscape of Shiraz. Specifically, we aimed to characterize the species diversity and relative abundance of phlebotomine sand flies and evaluate the vector competence of the predominant species through molecular detection of Leishmania DNA. By synthesizing these data, this investigation seeks to clarify the vectorial capacity driving the urban cycle of CL in this important endemic focus.

This study comprehensively evaluates the CL transmission cycle in Shiraz. Using a multidisciplinary approach to characterize the faunistic distribution and infection rates of sand fly vectors across urban microhabitats.

MATERIALS AND METHODS

RESULTS

Sand fly collection and species composition

A total of 435 sand fly specimens were collected during the 2024 survey period using the integrated multi-method approach. Standardized sticky trapping accounted for the majority of captures (n = 390, 89.7%), providing the core dataset for seasonal and spatial analysis. The complementary active methods – CDC light traps and manual aspiration – contributed an additional 45 specimens (10.3%), enhancing the collection of phototactic species and flies from cryptic resting sites.

Morphological identification of all specimens revealed the presence of four species belonging to two genera [Table 1]. The genus Phlebotomus was dominant, comprising 88.5% (n = 385) of the total fauna. Within this genus, P. sergenti was the most abundant species, representing 53.6% of all sand flies collected. Phlebotomus papatasi was the second most frequent species (34.0%). Specimens from the genus Sergentomyia were less common, with Sergentomyia tiberiadis and S. clydei accounting for 7.1% and 5.3% of the total, respectively [Figures 1 and 2].

Table 1: Species composition and relative abundance of sand flies collected in the historic urban district of Shiraz, 2024

Species	Number	Percentage
Phlebotomus sergenti	233	53.6
Phlebotomus papatasi	148	34.0
Sergentomyia tiberiadis	31	7.1
Sergentomyia clydei	23	5.3
Total	435	100.0

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Figure 1:

Sand flies caught in Shiraz from southwestern Iran during 2024; (A) Male genitalia of Phlebotomus sergenti, (B) Female spermatheca of P. sergenti, (C) Male genitalia of Phlebotomus papatasi, (D) Female spermatheca of P. papatasi.

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Figure 2:

Sand flies caught in Shiraz from southwestern Iran during 2024; (A) Ciborium teeth of Sergentomyia tiberiadis (yellow arrow), (b) Female spermatheca of S. tiberiadis (black arrow), (C) Ciborium teeth of Sergentomyia clydei (black arrow), (D) Female spermatheca of S. clydei (black arrow).

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Molecular detection of Leishmania and vector infection rates

A subset of 112 female sand flies, encompassing all collected species, was analyzed for natural Leishmania infection through nested PCR. Infection was detected exclusively in the predominant vector species, P. sergenti. Out of 52 female P. sergenti tested, 14.3% (n = 7) were found positive for Leishmania tropica, as indicated by the 760 bp amplification product [Figure 3]. No infections with Leishmania major (650 bp band) were detected in any P. sergenti specimens.

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Figure 3:

Representative agarose gel electrophoresis of nested polymerase chain reaction products for Leishmania detection in sand flies. Lanes: M, 100-bp DNA ladder; 1, Leishmania major positive control (650 bp); negative control; 6, Leishmania tropica positive Phlebotomus sergenti field samples (760 bp); Lanes 2–5, negative Phlebotomus papatasi field samples; Lanes 7–14, negative P. sergenti field samples.

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All tested specimens of P. papatasi (n = 41), S. tiberiadis (n = 12), and S. clydei (n = 7) were negative for both Leishmania species. The overall infection rate within the total tested female sand fly population was 6.25% (7/112).

DISCUSSION

This integrated entomological investigation provides a detailed characterization of the sand fly fauna implicated in the urban transmission of CL in Shiraz. The application of complementary collection methods offers a more comprehensive profile of the local vector community than single-method surveys typically afford, while molecular screening delivers direct evidence of vector–parasite associations.

Our findings confirm P. sergenti as the predominant and competent vector of L. tropica in this historic urban landscape. Its numerical dominance (53.6% of total captures) aligns with its recognized role as the primary vector of anthroponotic CL in other Iranian cities such as Isfahan and Kerman.^[20,21] The significant outdoor abundance of sand flies, particularly P. sergenti, underscores a critical ecological trait. This exophilic tendency, documented in other urban foci,^[3] suggests that peridomestic environments – courtyards, alleyways, and external wall surfaces – serve as major resting and possibly breeding sites. This has direct implications for control, indicating that interventions focused solely on indoor residual spraying may be insufficient. Outdoor space spraying or targeted environmental management, though logistically challenging in dense urban settings, should be considered during peak transmission months.^[15]

The multi-method collection strategy proved valuable in capturing different facets of sand fly behavior. While sticky traps provided a standardized measure of seasonal abundance, the supplementary use of CDC light traps and aspiration helped sample species and physiological states (e.g., host-seeking females) that may be underrepresented by passive traps alone. This methodological triangulation strengthens the reliability of the species inventory and abundance estimates, a point emphasized in recent guidelines for vector surveillance.^[22]

The distinct unimodal seasonal activity, peaking in the hot, dry summer months (April–October), is consistent with the known temperature-dependent biology of Phlebotomus species in Iran’s semi-arid climate.^[8] The correlation between rising temperatures and increased sand fly activity creates a predictable, narrow window of high transmission risk. This seasonality provides a clear temporal target for intensifying vector control measures, such as public awareness campaigns and the strategic application of insecticides, ideally just before and during the peak activity period.^[11]

The molecular detection of L. tropica in 14.3% of female P. sergenti is the most critical finding, confirming its vector competence and active participation in the parasite’s transmission cycle within the study area. This infection rate falls within the range reported for P. sergenti in other L. tropica foci in the Middle East.^[8] The absence of L. major in the tested P. papatasi population, despite its substantial presence (34% of the fauna), is noteworthy. This suggests that while P. papatasi – the classic vector of rural zoonotic L. major cycles – is well-established in urban Shiraz, but it may not be actively transmitting L. major within this specific historic district. This could indicate a spillover of the vector from surrounding rural areas without the concomitant establishment of the zoonotic parasite cycle, or a very low, undetected level of enzootic transmission. The complete lack of Leishmania detection in Sergentomyia species, consistent with their generally accepted role as non-vectors of mammalian leishmaniasis in the Old World, further focuses attention on Phlebotomus species.^[23,10]

Some limitations of this entomological study warrant consideration. The overall sand fly density, though thoroughly sampled, was moderate. This may reflect the true urban ecology of the site or the efficiency of the chosen collection methods. Future studies could benefit from incorporating additional techniques, like animal-baited traps, to better assess host-seeking rates. Furthermore, while the sample size for molecular analysis was sufficient to demonstrate infection, larger-scale screening across multiple seasons would provide more robust estimates of temporal variation in infection rates.

Data availability: The data supporting the findings of this study are available within the article.

CONCLUSION

This study delineates the entomological backbone of urban CL in Shiraz. It establishes P. sergenti as the key vector species, highlights its exophilic behavior and summer peak activity, and provides molecular confirmation of its role in transmitting L. tropica. These findings shift the intervention paradigm from a general, broad-based approach to a more precise strategy. Effective control should prioritize P. sergenti through integrated methods that target both indoor and outdoor habitats, with efforts concentrated during the predictable summer peak. This vector-centric evidence forms an essential foundation for public health authorities aiming to disrupt transmission in this and similar historic urban endemic foci.